Carbon Nanotubes For Delivery of Small Molecule Drugs

Advanced Drug Delivery Reviews 65 (2013) 19642015
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Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr
Carbon nanotubes for delivery of small molecule drugs

Bin Sheng Wong a,, Sia Lee Yoong b, Anna Jagusiak c, Tomasz Panczyk d, Han Kiat Ho a, Wee Han Ang e, Giorgia Pastorin a,b,
a
Department of Pharmacy, National University of Singapore, S4 Science Drive 4, Singapore 117543, Singapore NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of Singapore, Centre for Life Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456, Singapore Chair of Medical Biochemistry, Jagiellonian University Medical College, ul. Kopernika 7, 31034 Cracow, Poland d Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30239 Cracow, Poland e Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
b c
a r t i c l e
i n f o
a b s t r a c t
In the realm of drug delivery, carbon nanotubes (CNTs) have gained tremendous attention as promising nanocarriers, owing to their distinct characteristics, such as high surface area, enhanced cellular uptake and the possibility to be easily conjugated with many therapeutics, including both small molecules and biologics, displaying superior efcacy, enhanced specicity and diminished side effects. While most CNT-based drug delivery system (DDS) had been engineered to combat cancers, there are also emerging reports that employ CNTs as either the main carrier or adjunct material for the delivery of various non-anticancer drugs. In this review, the delivery of small molecule drugs is expounded, with special attention paid to the current progress of in vitro and in vivo research involving CNT-based DDSs, before nally concluding with some consideration on inevitable complications that hamper successful disease intervention with CNTs. 2013 Elsevier B.V. All rights reserved.
Article history: Accepted 5 August 2013 Available online 14 August 2013 Keywords: Carbon nanotubes Drug delivery Small molecule drugs Anticancer drugs Non-anticancer drugs
Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . Delivery of anticancer drugs with carbon nanotubes 2.1. Topoisomerase inhibitors . . . . . . . . . 2.1.1. Topoisomerase I inhibitors . . . . 2.1.2. Topoisomerase II inhibitors . . . 2.1.3. Anthracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1965 1991 1991 1991 1991 1992
Abbreviations: AAS, Atomic absorption spectroscopy; AMB, Amphotericin B; BBB, Blood brain barrier; BCEC, Brain capillary endothelial cells; BSA, Bovine serum albumin; CDDP, Cisplatin; Ce6, Chlorin e6; CEA, Carcinoembryonic antigen; CHI, Chitosan; CNF, Carbon nanober; CNTs, Carbon nanotubes; CP, Carboplatin; CPT, Camptothecin; CT, Catechin; DAU, Daunorubicin; dC, 2,2-Diuoro-2-deoxycytidine; DDS, Drug delivery system; DEX, Dexamethasone; DMAAM, N-dimethylacrylamide; DNA, Deoxyribonucleic acid; DOX, Doxorubicin; DSPE-mPEG 2000, 1,2-Distearoyl-phosphatidylethanolamine-methoxy-polyethylene glycol conjugate-2000; DTX, Docetaxel; DWCNTs, Double-walled CNTs; EAT, Ehlrich ascites tumor; EC, Ethyl cellulose; EDBE, 2,2-(Ethylene dioxy) bis(ethylene amine); EDX, Energy dispersive X-ray analysis; EGF, Epidermal growth factor; EGFR, EGF receptors; EPC, Endothelial progenital cell; EPI, Epirubicin; EPR, Enhanced permeability and retention; ER, ES receptor; ES, Estradiol; FA, Folic acid; FITC, Fluorescein isothiocyanate; FR, FA receptor; FTIR, Fourier transform infrared spectroscopy; GelCT, Gelatincatechin; GEM, Gemcitabine; GNP, Gold NP; HA, Hyaluronic acid; HCPT, 10-Hydroxycamptothecin; HET-CAM, Hen's egg test-chorioallantoic membrane; HMM, Hexamethylmelamine; HMME, Hematoporphyrin monomethyl ether; HR, Hyaluronan receptor; HUVEC, Human umbilical vein endothelial cells; ICP-OES, Inductively coupled plasma optical emission spectroscopy; LcL, Luciola cruciate luciferase; LRP, Lipoprotein receptor-related protein; mACs, Magnetic activated carbon particles; MAPK, Mitogen-activated protein kinase; MDR, Multidrug resistance; MTX, Methotrexate; MWCNTs, Multi-walled CNTs; NIPAM, N-isopropylacrylamide; NIR, Near infrared; NP, Nanoparticles; NSAID, Non-steroidal anti-inammatory drugs; ODT-f-GNP, 1-Octadecanethiol functionalized GNP; P-gp, P-glycoprotein; PAA, Poly(acrylic acid); PAMAM, Poly(amidoamine); PBS, Phosphate buffered saline; PCA, Polycitric acid; PDM, Polyamholyte poly [2-(dimethylamino) ethyl methacrylate]-co-(methacrylic acid); PDT, Photodynamic therapy; PEG, Polyethylene glycol; PEG PSS, Poly (ethylene glycol-b-propylene sulde); PEI, Polyethylenimine; PEO, Poly-ethylene oxide; PK, Pharmacokinetic; PL, Phospholipid; PLA, Poly(lactide); PSS, Poly(sodium 4-styrene sulfonate); Pt, Platinum; PTT, Photothermal therapy; PTX, Paclitaxel; PVA, Poly(vinyl alcohol); QD, Quantum Dot; RES, Reticuloendothelial system; RF, Radiofrequency; Rh, Rhodamine; ROS, Reactive oxygen species; SCID, Severe combined immunodecient; SD, Sprague Dawley; SEM, Scanning electron microscopy; siRNA, Small interference ribonucleic acids; SWCNTs, Single-walled CNTs; TEM, Transmission electron microscopy; TPGS, Tocopheryl PEG succinate; Trf, Transferrins; US-CNTs, Ultra-short CNTs; UVvis, Ultravioletvisible; XPS, X-ray photoelectron spectroscopy. This review is part of the Advanced Drug Delivery Reviews theme issue on Carbon nanotubes in medicine and biology Therapy and diagnostics. Corresponding author. Tel.: +65 6516 1876; fax: +65 6779 1554. Correspondence to: G. Pastorin, Department of Pharmacy, National University of Singapore, S4 Science Drive 4, Singapore 117543, Singapore. Tel.: +65 6516 1876; fax: +65 6779 1554. E-mail addresses: wongbs@nus.edu.sg (B.S. Wong), phapg@nus.edu.sg (G. Pastorin). 0169-409X/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.addr.2013.08.005
B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015
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Platinum-based drugs . . . . . . . . . . . . . . . . Antimetabolites . . . . . . . . . . . . . . . . . . . 2.3.1. Antifolates . . . . . . . . . . . . . . . . . 2.3.2. Purine/pyrimidine antagonists . . . . . . . . 2.4. Antimicrotubules . . . . . . . . . . . . . . . . . . 2.5. Other anticancer drugs . . . . . . . . . . . . . . . 3. Delivery of non-anticancer drugs with carbon nanotubes . . . 3.1. Antimicrobials . . . . . . . . . . . . . . . . . . . 3.2. Anti-inammatories . . . . . . . . . . . . . . . . . 3.3. Antihypertensives . . . . . . . . . . . . . . . . . . 3.4. Antioxidants . . . . . . . . . . . . . . . . . . . . 3.5. Other non-anticancer drugs . . . . . . . . . . . . . 4. Progress of in vivo research on CNT-based drug delivery systems 4.1. Anticancer drugs . . . . . . . . . . . . . . . . . . 4.1.1. Topoisomerase inhibitors . . . . . . . . . . 4.1.2. Platinum-based drugs . . . . . . . . . . . . 4.1.3. Antimetabolites . . . . . . . . . . . . . . 4.1.4. Antimicrotubules . . . . . . . . . . . . . . 4.1.5. Other anticancer drugs . . . . . . . . . . . 4.2. Non-anticancer drugs . . . . . . . . . . . . . . . . 5. Concerns regarding CNT-based drug delivery systems . . . . . 6. Conclusion & future perspectives . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. 2.3.
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1. Introduction Following the discovery of their presence in the insoluble soot of arcburned graphite rods in 1991 by Japanese physicist Sumio Iijima, carbon nanotubes (CNTs) had since gained tremendous attention as a versatile nanomaterial with abundant applications [1]. First of all, with exceptionally high tensile strength and elastic modulus, CNTs represent one of the strongest and stiffest materials to be discovered [2,3]. CNTs are also excellent thermal [4,5] and electrical conductors [6,7], with additional abilities to absorb optical intensity [8], photoluminesce [9] and generate strong Raman signals [10] that enable their facile and nondestructive characterization. Equipped with all these tunable distinctive features, CNTs have been investigated and applied successfully to create novel and functional microelectronics, energy storage devices, lled composites, nanoprobes, sensors and templates [11]. In terms of biomedical applications, CNTs have also demonstrated immense potentials, particularly in the areas of tissue engineering, thermal ablation and drug delivery [12,13]. As scaffolding materials, CNTs are able to support the growth of bone cells [14,15], neurons [16,17] and cardiomyocytes [18], and even direct or promote the differentiation of stem cells into specic lineages, such as from human mesenchymal stem cells into bone cells [1921]. The ability for CNTs, especially SWCNTs, to absorb and convert electromagnetic radiation, specically near infrared (NIR), into heat or sound energy has been exploited for successful photothermal therapy (PTT) or photoacoustic therapy against cancer cells [2225]. Regarding their application in the delivery of therapeutic agents, CNTs have also been popularly employed as carriers for controlled and targeted drug delivery to improve the pharmacological activity of bioactive molecules and simultaneously diminish their undesirable systemic side effects. Indeed, various therapeutic agents, ranging from small molecules such as chemotherapeutic drugs [2632], antimicrobials [33,34] and anti-inammatory agents [35], to more complex biologics like peptidebased vaccines [36,37], antibodies [38] and small interference ribonucleic acids (siRNA) [39], have been successfully delivered with CNTs using a multitude of strategies, demonstrating superior efcacy and reduced toxicity. In fact, CNTs possess many intriguing features that make them attractive drug delivery carriers. Firstly, nanocarriers, including nanoparticles (NP), liposomes, and CNTs, experience the enhanced permeability and retention (EPR) effect, i.e. they exhibit higher accumulation in tumor tissues
as compared to normal tissues due to poorly formed blood and lymphatic vessels that supply rapidly proliferating tumors [40]. The EPR effect enables CNTs to transport chemotherapeutic agents preferentially to tumor sites [41]. Secondly, the needle-like shape of CNTs facilitates transmembrane penetration and intracellular accumulation of drugs via the nanoneedle mechanism that is independent of additional CNT functionalization and cell types [42]. Aside from direct translocation through cellular membranes, CNTs have also been shown to enter cells via energy-dependent endocytic pathways [43]. Thirdly, as a platform for drug attachment, CNTs, owing to their high aspect ratios and surface areas, display extraordinary ability for drug loading onto the surface or within the interior core of CNTs via both covalent and non-covalent interactions [44]. To further augment the efcacy of CNT-based drug delivery system (DDS), targeting molecules, such as folic acid (FA) [45], antibodies [46] and even magnetic NP [47] can be further incorporated onto the drug-loaded CNTs (covalently or non-covalently) to confer either active targeting capabilities via receptor-mediated endocytosis or local nanocarrier accumulation induced by external magnetic eld. In addition, imaging tags like radioactive nuclides [48] and uorescence probes [46] can also be conjugated with CNTs to observe their intracellular trafcking and biodistribution in vitro and in vivo easily and noninvasively. Coupled with the NIR absorption capability of CNTs, multimodal DDSs can also be created by combining NIR-induced PTT or drug release with conventional drug molecules or biologics [49,50]. Despite the above-mentioned advantages of CNTs for the purpose of drug delivery, such as high aspect ratio, functionalizable surface, fast cellular uptake, etc., the issues of toxicity surrounding the biomedical applications of CNTs still remain controversial to this date, with studies demonstrating conicting results regarding their safety proles [51,52]. As a result, despite various successful attempts of delivering drugs with CNTs in vitro and, to a lower degree, in vivo, CNT-based DDSs are still considered far from being accepted for use in actual clinical settings. Having said that, some preliminary understandings regarding the toxicity of CNTs have been unveiled. In general, CNTs with non-functionalized hydrophobic surfaces and high degree of residual heavy metal contamination tend to be more cytotoxic [53,54]. The problem of heavy metal contamination can be easily rectied by purication [48], while the issues of poor aqueous dispersibility and high aggregation tendency of pristine CNTs can be resolved by appropriate surface functionalization [55]. Functionalization of CNTs can be achieved by either non-covalently
1966
Table 1 Summary of the CNT-based DDS described in this review. Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro Anticancer drugs Topoisomerase I inhibitors HCPT MWCNTs covalently functionalized with diaminotriethylene glycol spacers In vivo Ref
Covalent conjugation via ester linkage
lEsterases
Uptake & cytotoxicity in MKN-28
[48] Biodistribution, efcacy & toxicity in hepatic H22 tumor bearing ICR mice
CPT
Oxidized MWCNTs functionalized with PVA
Physical adsorption
Cytotoxicity in MDA-MB231 & A-5RT3
[64] B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015
CPT
Oxidized MWCNTs coated with Pluronic P123
Physical adsorption
Uptake & cytotoxicity in HeLa
[65]
Irinotecan
MWCNTs with open tips
Physical encapsulation
Acidic pH
[66]
Topoisomerase II inhibitors Etoposide Carboxyl SWCNTs functionalized with CHI & EGF
Physical adsorption
Acidic pH due to CHI disruption
EGF against EGFR
Uptake & cytotoxicity in A549
[67]
Anthracyclines DOX
SWCNTs functionalized with PEG with cyclic RGD
Physical adsorption
Acidic pH CNT diameter
Cyclic RGD against integrin v3
Uptake & cytotoxicity in U87MG & MCF-7
[26]
DOX
MWCNTs dispersed with Pluronic F127
Physical adsorption
Cytotoxicity in MCF-7
DOX
SWCNTs functionalized with branched PEG
Physical adsorption
PK, biodistribution, efcacy & [57] toxicity in SCID mice with Raji lymphoma xenografts
DOX
Oxidized SWCNTs functionalized with anti-CEA antibody & uorescein using BSA as multifunctional linker
Physical adsorption
Antibody against CEA
Uptake in WiDr
[46]
(continued on next page)
1967
1968
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro DOX SWCNTs functionalized with polysaccharide coating (CHI &/or sodium alginate) & FA Physical adsorption Acidic pH CHI & sodium alginate ratio FA against FR Uptake & cytotoxicity in HeLa In vivo [45] Ref
DOX
SWCNTs covalently linked to P-gp antibody labeled with FITC
Physical adsorption
NIR
Antibody against P-gp
Uptake & cytotoxicity in K562 sensitive & resistant cell lines
[50]
DOX
SWCNTs functionalized with FA-conjugated CHI
Physical adsorption
Acidic pH disruption of CHI
FA against FR
[86]
DOX
MWCNTs dispersed with PEG-PSS labeled with FITC
Physical adsorption
Uptake in HeLa. Cytotoxicity in MDA-MB435
[87]
DOX
MWCNTs functionalized with FA-hexamethylenediamine conjugate & iron NP.
Physical adsorption
NIR
FA against FR Iron oxide NP for magnetic targeting
[85]
DOX
Oxidized MWCNTs
Physical adsorption
Acidic pH Serum protein Shorter loading time
[75]
DOX
SWCNTs functionalized with branched PEG 2500-NH2 & FA
Physical adsorption
Acidic pH Serum protein
FA against FR
[71]
DOX
PAA grafted MWCNTs functionalized with FA & iron NP
Physical adsorption
Acidic pH Iron oxide NP for magnetic targeting
FA against FR
Uptake in U87, cytotoxicity in U87 & 3 T3
[76]
DOX
EDBE-conjugated MWCNTs covalently functionalized Physical adsorption with HA
Acidic pH
HA against HR
Biodistribution in EAT bearing mice Efcacy in chemicallyinduced breast cancer bearing SD rats Toxicity in mice & rats
[73]
1969
1970
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro DOX Oxidized MWCNTs functionalized with multi-branched GNP& PEG methyl ether thiol Physical adsorption Acidic pH Uptake in A549 In vivo [77] Ref
DOX
Iron NP-lled PSS modied CNTs conjugated with poly(allylamine)-functionalized SiO2-coated CdTe QDs linked to transferrin
Physical adsorption
Acidic pH
Transferrin against Iron NP for magnetic targeting
Uptake in HeLa & HEK 293 Cytotoxicity in HeLa
[47]
DOX
CHI-coated SWCNTs covalently functionalized with FITC
Physical adsorption
Acidic pH
Uptake in EPC
[78]
DOX
CHI-coated SWNCTs chemically attached with FA
Physical adsorption
Acidic pH
FA against FR
Cytotoxicity in SMMC-7721
[79] Efcacy & toxicity in nude BALC/c mice inoculated subcutaneously with SMMC7721
DOX
PEGylated oxidized MWCNTs modied with angiopep 2
Physical adsorption
Acidic pH
Angiopep 2 peptide against LRP receptors
Uptake & cytotoxicity in C6 & BCED
[80] Biodistribution, efcacy & toxicity in glioma bearing BALB/c mice injected with C6 into right striatum
DOX
CNTs coated with zipper comprising PEI & PVA via hydrogen bonding
Physical adsorption
Heat
Uptake in breast adenocarcinoma Cytotoxicity in lung broblast, breast adenocarcinoma, HeLa, adult & neonatal HDF
[88]
DOX
Amine-MWCNTs conjugated covalently with DEX mesylate
Physical adsorption
Acidic pH
DEX mesylate for nuclear targeting
[82]
DOX
SWCNTs non-covalently functionalized with FAterminated methoxy-PEG
Physical adsorption
Acidic pH
FA against FR
Uptake & cytotoxicity in HeLa & 3 T3
[81]
DOX
MWCNTs linked with EDBE conjugated with FA, HA or -estradiol-17-hemisuccinate
Physical adsorption
Acidic pH
FA against FR HA against HR ES against ER
Uptake & cytotoxicity in A549, HeLa & MCF-7
[84]
(continued on next page) 1971
1972
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro In vivo Ref
DOX
MWCNTs covalently functionalized with amineterminated PAMAM dendrimers modied with FITC & FA
Physical adsorption
Acidic pH
FA against FR
Uptake & cytotoxicity in high & low FR expressing KB
[74]
DOX
Poloxamer 188 modied SWCNTs functionalized with AS1411 aptamer with NIR-induced hyperthermia
Physical adsorption
Acidic pH
AS1411 aptamer against nucleolin
Uptake & cytotoxicity in EC 109
[83]
DOX
SWCNTs labeled with recombinant thermostable LcL
Physical adsorption
Biodistribution in FVB mice
[93]
DOX
SWNCTs functionalized with Cremophor EL
Physical adsorption
Acidic pH
PK, biodistribution, efcacy & [72] toxicity in S180 sarcoma bearing ICR mice
DOX
PEGylated SWCNTs with non-covalently attached pyrene
Chemical conjugation onto pyrene with carbamate linker
Enzymatic cleavage of carbamate
Uptake & cytotoxicity in B16-F10
Efcacy & toxicity in C57/BL/ [99] 6 mice with subcutaneous implantation of B16-F10
DOX
PEGylated SWCNTs with hydroxinobenzoic acid linker
Physical adsorption & chemical conjugation via hydrazone bonds
Acidic pH
Uptake & cytotoxicity in HepG2 & HeLa
[100]
DOX
Isolated SWCNTs dispersed in NIPAM & DMAAM hybrid gel
Physical adsorption
Acidic pH NIR
[101]
1973
1974
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro EPI MWCNTs with or without carboxylic groups & SWCNTs Physical adsorption Acidic pH In vivo [102] Ref
DAU
SWCNTs functionalized with PL-PEG
Physical adsorption
DAU
SWCNTs functionalized with sgc8c aptamer
Physical adsorption
Acidic pH
Sgc8c aptamer against tyrosine kinase-7
Uptake & cytotoxicity in Molt-4 & U266
[103]
Pirarubicin
SWCNTs functionalized with PL-branched PEG
Covalent conjugation via ester bond
Cytotoxicity in BIU-87 & C2C-12
Efcacy & toxicity in chemically-induced SD rat bladder cancer model
[104]
Mitoxantrone
SWCNTs functionalized with branched PEG-NH2
Physical adsorption
Acidic pH
Cytotoxicity in HeLa
[71]
Platinum-based drugs SWCNTs non-covalently functionalized with PL-PEG- Chemical conjugation c,c,tNH2 [Pt(NH3)2Cl2(OEt) (O2CCH2CH2CO2H)]
Cellular reductive environment
Cytotoxicity & intracellular Pt content in Ntera-2
[29]
c,c,t-[Pt(NH3)2Cl2 (O2CCH2CH2CO2H)2]
SWCNTs non-covalently functionalized with PL-PEG- Chemical conjugation NH2. FA was attached to the remaining axial ligand on Pt (IV) prodrug
Cellular reductive environment
FA against FR
Uptake & cytotoxicity in JAR, KB & NTera-2
[115]
CDDP
Oxidized SWCNTs functionalized with EGF
Chemical conjugation via ester bond
EGF against EGFR
Uptake in HN13 with EGFR Biodistribution & efcacy in & EGFR-knockdown control. HN12 xenograft mice Cytotoxicity in HN13 with & without EGFR, NIH-3T3 & SAA
[116,117]
1975
1976
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro CDDP Oxidized SWCNTs covalently functionalized with EGF Chemical conjugation via ester bond & PEG5000 EGF against EGFR Cytotoxicity in HN12 In vivo Biodistribution, efcacy & toxicity in HN12 xenograft mice [199] Ref
CP
Open-ended oxidized MWCNTs
Cytotoxicity in EJ28. Cytotoxicity in PC-3, DU145, EJ28, A498.
[122,125]
CDDP
SWCNTs
Cytotoxicity in PC-3 & DU145
[124]
CDDP
Pristine MWCNTs with 1-octadecanethiol-coated GNP caps
GNP cap
[126]
CDDP
US-CNTs wrapped with Pluronic F108
Pluronic coat RF eld
Cytotoxicity & intracellular Pt content in MCF-7 & MDAMB-231 Cytotoxicity in Hep3B & HepG2
[128,131]
Pt (IV) prodrug
MWCNTs
Reductive environment (e.g. ascorbic acid)
Intracellular Pt content in A2780
[28]
Oxaliplatin
Oxidized MWCNTs covalently functionalized with PEG600
PEG coating
Cytotoxicity in HT29
[132]
Antifolates MTX
MWCNTs with 1,3-dipolar cycloaddition of azomethine ylides with orthogonally protected amino functions, tagged with FITC
Covalent conjugation
Uptake & cytotoxicity in human Jurkat T lymphocytes
[30]
MTX
Oxidized MWCNTs with 2 different cleavable linkers, Covalent conjugation tetrapeptide Gly-Leu-Phe-Gly or 6-hydroxyhexanoic ester
Proteases or esterases
Uptake & cytotoxicity in MCF-7
[136]
1977
1978
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro MTX SWCNTs covalently functionalized with Oligo-HANH2 Covalent conjugation In vivo [137] Ref
MTX
MWCNTs non-covalently functionalized with DSPEmPEG 2000
Physical adsorption
Acidic pH
[134]
MTX
Physical adsorption
Neutral pH
[84]
Purine/pyrimidine antagonists GEM PAA-grafted MWCNTs deposited with iron magnetic NP
Physical adsorption
Iron NP for magnetic targeting
Uptake & cytotoxicity in BxPC-3 & SW1990
Biodistribution & toxicity in SD rats. Efcacy & toxicity in metastatic nude BALB/c nu/ nu mice subcutaneously inoculated with BxPc-3
[31,140]
GEM
MWCNTs covalently conjugated with FA
Physical adsorption
Acidic pH
FA against FR
Biodistribution & PK in albino rats
[141]
dC
SWCNTs covalently functionalized with PEI
Physical adsorption
Endoscopic ultrasound
[142]
Antimicrotubules SB-T-1214
SWCNTs covalently functionalized with biotin & uorescein
Chemical conjugation via cleavable disulde bond
Intracellular thiols
Biotin against surface biotin receptors
Uptake & cytotoxicity in L1210FR, L1210 & W138
[155]
(continued on next page) 1979
1980
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro PTX SWCNTs adsorbed with branched PEG phospholipids Chemical conjugation via ester bond Esterases Cytotoxicity in 4T1 In vivo PK, biodistribution, efcacy & [32] toxicity in BALB/c mice subcutaneously injected with 4T1 Ref
PTX
MWCNTs functionalized with hyperbranched PCA
Esterases Acidic pH
Cytotoxicity in A549 & SKOV3
[156]
PTX
PEGylated SWCNTs & MWCNTs
Physical adsorption
pH depending of nature of CNTs
Cytotoxicity in MCF-7 & HeLa
[147]
PTX
Supramolecular complex of SWCNTs & PDM
Physical adsorption
Uptake & cytotoxicity in Caco-2
[148]
PTX
Hydroxy-functionalized MWCNTs covalently coated with PLA-PEG
Physical adsorption
Uptake & cytotoxicity in U87 Biodistribution, toxicity & inammatory responses in & HUVEC. BALB/c mice Inammatory protein expression in rat epithelial cells.
[157]
PTX & C6-ceramide
CNTs (no mention on the specic type) noncovalently functionalized with PL-PEG-NH2
Inductive heating with external alternating current or magnetic eld pulse
Synergism study between PTX & C6-ceramide in L3.6, PANC-1 & MIA PaCa-2 Uptake & cytotoxicity in L3.6.
[158]
PTX
Physical adsorption
Neutral pH
[84]
1981
1982
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro DTX SWCNTs non-covalently functionalized with PVP K30 Physical adsorption & DSPE-PEG-Maleimide linked with NGR peptide, combined with NIR-induced PTT NGR peptide against CD13 In vivo [49] Ref
Uptake & cytotoxicity in PC- PK & biodistribution in 3 healthy mice. Efcacy & toxicity in murine S180 BALB/c mice
Other anticancer drugs Tamoxifen Oxidized SWCNTs with octa(ethyleneglycol) linker
[159]
Thalidomide
PEGylated oxidized SWCNTs functionalized with cyclic RGD & Rh
Chemical conjugation
Cyclic RGD against integrin v3
Uptake in U87MG & MCF-7.
Biodistribution in wild type zebrash embryos. Targeting ability in transgenic zebrash embryos with green uorescent proteinproducing endothelial cells. Angiogenesis assay in transgenic zebrash embryo xenografted with HT1080
[165]
CT
Hybrid of non-covalent inclusion of MWCNTs to covalent complex of gelatin & CT
Physical adsorption
Cytotoxicity in HeLa
[166]
HMM
SWCNTs or DWCNTs with open ends sealed with C60 Physical encapsulation
C60 caps
[121]
Ce6
Oxidized SWCNTs wrapped with CHI
Physical adsorption
[169]
Ce6
Oxidized SWCNTs non-covalently wrapped with thrombin aptamers
Chemical conjugation onto aptamers
Thrombin
Cytotoxicity in Ramos
[170]
5-Aminolevulinic acid
PAMAM modied MWCNTs
Physical adsorption
Uptake & cytotoxicity in MGC-803
[171]
Bodipy-based PDT sensitizer
SWCNTs
Physical adsorption
[172]
1983
1984
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro HMME Amine-functionalized SWCNTs covalently linked to HA Physical adsorption HA against HR Uptake & cytotoxicity in B16-F10 In vivo Efcacy & toxicity in C57 mice subcutaneously injected with B16-F10 [173] Ref
Non-anticancer drugs Antimicrobials AMB
Ammonium-functionalized MWCNTs & SWCNTs
Uptake & cytotoxicity in Human Jurkat Lymphoma T cells Antifungal activity in Candida & Cryptococcus fungi
[34]
AMB
Oxidized MWCNTs & PEGylated SWCNTs
Antifungal activity against a collection of fungi
[33]
AMB
MWCNTs functionalized with ethylene diamine
Antileishmanial activity in intramacrophage amstigotes
Antileishmanial efcacy in Syrian Golden Hamster infected with L. donovani Toxicity in healthy BALB/c mice Antileshmanial efcacy in Syrian en Hamster (Oral administration)
[174,201]
AMB
Mannosylated MWCNTs
Physical adsorption
Mannose to target macrophages
Uptake in J774
Biodistribution & toxicity in albino rats
[175]
Dapsone
Oxidized MWCNTs
Uptake & cytotoxicity in rat peritoneal macrophages
[177]
Pazuoxacin mesilate
MWCNTs functionalized with ethylene diamine
Physical adsorption
Acidic pH
[178]
Gentamicin
Bullfrong collagen hydrogel doped with 1% w/w oxidized CNTs (Type of CNTs not specied)
Physical adsorption
Presence of CNTs
[179]
1985
1986
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro Chloroquine DWCNTs coated with PEI & plasmid encoding luciferase Physical adsorption Acidic pH Transfection ability & cytotoxicity in HeLa In vivo [180] Ref
Anti-inammatories DEX phosphate
Oxidized MWCNTs sealed with a lm of polypyrrole via electropolymerization
Polypyrrole lm Electrical stimulation
Anti-inammatory activity demonstrated in lipopolysaccharideactivated microglial cell
[35]
DEX phosphate
CHI/SWCNTs hybrid lm
Electrical stimulation Presence of CNTs
[181]
Diclofenac sodium
Spherical gelatin/MWCNTs hybrid microgel
[182]
Diclofenac sodium
Carboxymethyl guar gum/oxidized MWCNTs hybrid hydrogel
Amount of CNTs
[183]
Diclofenac sodium
MWCNTs
Physical adsorption
[188]
Ketoprofen
Electrospun bers comprising PEO & pentaerythritol triacrylate interspersed with MWCNTs
Biocompatibility in L929
[184]
Indomethacin
Osmotic pump tablet system coated with cellulose acetate membrane containing MWCNTs
Present in core tablet
Presence of CNTs
[185]
1987
1988
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro Antihypertensives Diltiazem hydrochloride Composite membrane of PVA & oxidized MWCNTs Physical encapsulation Presence of CNTs In vivo [186] B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015 Ref
Metoprolol tartrate
EC microsphere impregnated with MWCNTs
Presence of CNTs
[187]
Candesartan cilexetil Diltiazem hydrochloride
MWCNTs
Physical adsorption
[188]
Carvedilol
Pristine & oxidized MWCNTs
Physical adsorption
[189]
Antioxidants TPGS
SWCNTs & MWCNTs
Physical adsorption
[192]
Quercetin Rutin
SWCNTs (pristine, hydroxylated & carboxylated)
Physical adsorption
[193]
1989
1990
Table 1 (continued) Drug CNT system Drug loading Release control Targeting mechanism Biological studies In vitro Gallic acid Pristine MWCNTs Covalent conjugation Biocompatibility with HETCAM In vivo [194] Ref
Other non-anticancer drugs Acetylcholine SWCNTs
Physical adsorption
Lysosomal & mitochondrial damage
Efcacy & toxicity in kainic acid-induced Alzheimer's Kunming mice
[195]
Theophylline
Hybrid microspheres of alginate & CNTs (types not specied) dispersed with triblock copolymer of PEO137-b-PPO44-b-PEO137
Presence of CNTs
Cytotoxicity in L929
[196]
1991
coating CNTs with amphiphilic macromolecules like lipid, polymers and surfactants, or covalently modifying the backbone of CNTs with hydrophilic functional groups [56]. Besides improving the water dispersibility and reducing the cytotoxicity of CNTs, surface functionalization also provides extra attachment sites for additional chemical or supramolecular loading of drugs, for targeting strategies or for imaging purposes [46]. In addition, properly functionalized CNTs, specically those with polyethylene glycol (PEG), are also able to achieve prolonged circulation half-life and improved bioavailability by escaping opsonization-induced reticuloendothelial system (RES) clearance [57]. The physical dimensions of CNTs, such as length and diameter, also have some bearings on the toxicity of CNTs, where longer and thinner structures tend to inict greater cytotoxicity [58,59]. With all these modications, CNTs with improved biocompatibility and solubility have been successfully created. The ability for some of these functionalized CNTs to be cleared by renal excretion also addresses some of the pharmacokinetic (PK) safety concerns related to the elimination of CNTs following administration [6063]. In this review, the use of CNTs as carriers or adjuncts for the delivery of various small molecule drugs, including both anticancer and nonanticancer drugs, is examined, with specic focus on their loading method, release characteristics, targeting ability (if any) and resultant therapeutic efcacy and toxicity (please refer to Table 1 for a summary of all the CNT-based DDSs described in this review). Even though biologics like peptides and nucleic acids have also been delivered with CNTs, they were not encompassed in this review. As in vivo works are highly imperative for estimating the clinical feasibility of CNTs as viable DDSs, a section of this review is dedicated to the current progress of in vivo research involving CNT-based DDSs. Last but not the least, certain limitations and considerations regarding the use of CNTs for drug delivery are also discussed briey. 2. Delivery of anticancer drugs with carbon nanotubes While chemotherapy has long been employed to manage cancers, either alone or in combination with other treatment modalities like surgery and radiation, it is often associated with undesirable systemic toxicity due to non-specicity, narrow therapeutic window and development of drug resistance. Therefore, novel ways of selectively delivering anticancer drugs to tumors with improved therapeutic efcacy and reduced adverse effects are highly desired. In this section of the review, the use of CNTs for the delivery of anticancer drugs of various pharmacological classes is examined. 2.1. Topoisomerase inhibitors Topoisomerases are a group of enzymes that relieve the torsional strain of supercoiled double helical deoxyribonucleic acid (DNA) by making either single or double stranded nicks at the DNA phosphate backbone and allowing the DNA to be unwind, before eventually resealing the cleaved DNA. As failure to relieve these tensions could lead to the arrest of DNA replication and subsequently apoptosis, some chemotherapeutic agents have leveraged on this property to slow down cancer cell growth by inhibiting the activity of eukaryotic topoisomerases. These agents are collectively known as topoisomerase inhibitors. 2.1.1. Topoisomerase I inhibitors Topoisomerase I catalyzes a transient break of 1 strand of duplex DNA and allows the unbroken complementary strand to unwind through the enzyme-linked strand. After successful DNA relaxation, topoisomerase I also relegates the broken DNA. Examples of clinically used topoisomerase I inhibitors include irinotecan, topotecan and camptothecin (CPT). In an attempt to raise its water solubility and antitumor effect, a congener of CPT, namely 10-hydroxycamptothecin (HCPT), was covalently conjugated to MWCNTs via a cleavable ester bond [48]. With a HCPT
loading of 16% w/w, the conjugate remained stable in the absence of esterases in buffer solution, and released HCPT readily in fetal bovine serum after hydrolysis of ester linkages by esterases present in the serum. While the uptake study of the conjugate with additional uorescein isothiocyanate (FITC) tag in human gastric carcinoma MKN-28 cells revealed successful internalization of the CNT conjugate, no comparison was made to the uptake of free HCPT. Thus, it is not possible to conclusively assert if CNTs enhanced the cellular uptake of HCPT. Nonetheless, in vitro cytotoxicity of the HCPTCNT conjugate was observed to be higher than that of lyophilized clinical HCPT injection at equivalent HCPT concentration, with the non-HCPT loaded CNT carrier inicting only negligible killing in MKN-28 cells. Employing the technique of non-covalent supramolecular attachment, a nanocarrier comprising poly(vinyl alcohol) (PVA)-functionalized MWCNTs loaded with CPT via interactions was reported by Sahoo et al. [64]. The loading of CPT was estimated to be around 0.1 g per g of PVA-MWCNT by ultravioletvisible (UVvis) spectra. The release of CPT, however, was observed to be rather slow, achieving only around 20% cumulative release by 72 h in buffer of pH 7.4 at 37 C. While the slow release prole is indicative of a strong association between CPT and CNTs, strategies to improve or even trigger the release of CPT, if devised successfully, would be extremely useful for controlled drug release purposes. In spite of this, the construct was found to be approximately 15 fold more potent than free CPT against MDA-MB-231 human breast cancer cells by MTT assay. Similar chemo-enhancing effect was also observed in metastatic skin tumor cell line A-5RT3. In another study, CPT was supramolecularly loaded onto MWCNTs coated with tri-block copolymer Pluronic P123 via stacking interactions [65]. The formation of the supramolecular complex was veried and quantied by UVvis spectra and photoluminescence. Approximately 8 1016 of CPT molecules were estimated to be present on every mg of the coated MWCNTs. With enhanced water solubility compared to free CPT, the complex could be internalized into the cytoplasm and onto the cell membrane of human cervix adenocarcinoma HeLa cells, as indicated by the uorescence signal of CPT. However, it is not possible to ascertain if the uorescence signals observed were due to free CPT that had been released from the CNTs or CPT molecules that were still attached on the CNTs, especially since an in vitro release study of the construct was not conducted. Without a comparison with free CPT, it also could not be assessed if CNTs were able to enhance the cellular uptake of CPT. Nevertheless, the construct demonstrated signicant improvement in cell killing ability over free CPT in MTT assay against HeLa cells. Irinotecan, a more water soluble semisynthetic analog of CPT, was encapsulated into the cavity of puried MWCNTs with opened tips, achieving a loading of around 32% as determined by thermogravimetric analysis [66]. The release of irinotecan was slightly improved in mildly acidic condition (pH 6.0 versus 7.0), possibly due to increased stability and hydrophilicity of irinotecan in acidic medium. Intriguingly, further decrease of pH to 5.0 appeared to have no additional inuence on the rate of drug release. The anticancer activity of this construct was however not investigated. 2.1.2. Topoisomerase II inhibitors Unlike topoisomerase I inhibitors, which only nick at a single strand, topoisomerase II inhibitors cleave both strands of DNA, which then enables the passage of another unbroken DNA duplex through the broken points, before nally resealing the strands. Inhibitors of topoisomerase II, such as etoposide and teniposide, prevent the rejoining of the nicked strands, resulting in double strain breaks and consequently cell death. A targeted DDS comprising carboxyl SWCNTs functionalized with chitosan (CHI) and epidermal growth factor (EGF) physically loaded with etoposide was fabricated by Chen et al. [67]. In this system, CHI, a cationic polysaccharide, was non-covalently attached onto the surface of carboxyl SWCNTs to improve the water dispersibility of CNTs, and to serve as a linker for subsequent covalent conjugation of EGF against
1992
EGF receptors (EGFR)-overexpressing cancer cells. The loading capacity of etoposide was around 25 to 27% w/w via stacking and electrostatic interaction. Release of etoposide from the system was accelerated under low pH conditions of 5.5, presumably due to increased amine protonation and enhanced solubility of CHI in acidic condition. The ability for this system to release more drugs at low pH is particularly advantageous for cancer therapy, as the intracellular environment of cancerous tissues tends to be more acidic [68]. When tested on human alveolar carcinoma epithelial cell line A549, the delivery system was found to be more potent than free etoposide. Surface attachment of EGF further contributed to the cytotoxicity of etoposide by facilitating EGFmediated energy-dependent endocytosis. Interestingly, the same construct that was tagged with FITC was shown to accumulate in the nucleus of A549 cells after 3 h of incubation. However, there was no clear mention in the study if FITC was covalently or non-covalently linked to the CNT construct. Even if FITC was covalently attached to CHI, CHI was itself non-covalently attached onto the surface of CNTs. Therefore, it is not possible to conrm that all the molecules previously incorporated onto the CNT carrier were preserved throughout the experiment as a single entity. The uorescent signal observed in the nucleus is thus unable to unequivocally support the nuclear localization of the complex, as the signal could arise from dissociated FITC and not from the entire uorescently tagged CNT complex. 2.1.3. Anthracyclines Anthracyclines, such as doxorubicin (DOX), daunorubicin (DAU) and epirubicin (EPI), represent a unique class of anticancer drugs that can also inhibit topoisomerase II. However, anthracyclines differ from the other topoisomerase II inhibitors by exhibiting multiple mechanisms of action. With a at and aromatic tetracyclic ring structure, anthracyclines are able to intercalate between DNA base pairs and inhibit the synthesis of DNA. In addition, the hydroquinone moiety of anthracyclines can also be metabolized and generate iron-mediated free oxygen radicals that damage DNA and cell membranes. In spite of their high clinical effectiveness against many cancers, the use of anthracyclines is unfortunately plagued with dose limiting myelosuppression, alopecia, acute nausea and vomiting, vesicant effects and, most notably, cardiotoxicity. More effective and safer ways of delivering anthracyclines are hence of signicant research interest. Already, liposomal formulation of DOX, for instance Doxil and Myocet, have been invented and employed clinically with diminished incidence of cardiotoxicity [69,70]. By exploiting the ability for the at aromatic tetracyclic structure of DOX to establish strong and hydrophobic interactions with the also aromatic surfaces of CNTs, Liu et al. have created a novel DDS comprising PEG-functionalized SWCNTs supramolecularly attached to DOX with an ultrahigh loading capacity of around 400% [26]. Shortly after the report by Liu et al., another similar strategy of DOX delivery, this time with MWCNTs dispersed with 1% Pluronic F127, was reported by Ali-Boucetta et al., validating the results of Liu et al. and suggesting that non-covalent attachment of DOX via interactions are applicable to both SWCNTs and MWCNTs [27]. Remarkably, the MWCNTDOX complex was observed to enhance the cytotoxicity of DOX on human breast cancer cells MCF-7 signicantly. More importantly, the DOX-free carrier of MWCNTs dispersed with Pluronic alone did not depress cell viability, implying that the cytotoxicity effect observed was attributable entirely to improved DOX efcacy rather than any inherent toxicity of CNTs. A thorough understanding on the various factors that govern the non-covalent adsorption and desorption behaviors of DOX on CNTs is useful in helping researchers to devise strategies to control the loading and release of DOX from CNTs. These factors include loading and release pH, loading DOX concentration, time allocated for adsorption, diameter of CNTs, coating/functionalization on CNTs, temperature, presence of competing proteins and external radiation. With an amine group present in its structure, the physicochemical properties of DOX are highly sensitive to changes in environmental
pH. Typically, DOX remains unionized and hydrophobic in neutral and basic pH. In acidic condition, the amine on DOX can be protonated, raising its hydrophilicity and solubility. This change in hydrophobicity is an essential feature that controls the loading and release of DOX from CNTs. Several studies have consistently veried higher degree of DOX loading in basic conditions, as DOX can maintain its unionized state and associate stronger with CNTs via and hydrophobic interactions [47,7174]. Conversely, it was shown that DOX could be released more readily in acidic environment after protonation [26,45,47,7284]. This differential rate of drug release is useful in targeted delivery of DOX to cancer cells, as tumor microenvironments tend to be more acidic. In addition, as the internal pH environment of lysosomes is acidic (pH 5.5), release of DOX from CNTs can also be triggered automatically after receptor-mediated endocytosis and internalization of the CNTDOX complex into lysosomal compartments, liberating free DOX to enter nucleus and exert its cytotoxic effect. In fact, the importance of acidic pH for ensuring adequate release of DOX was demonstrated by the loss of anticancer activity against A549 with MWCNTDOX complex coincubated with ammonium chloride, as a result of lysosomal accumulation of ammonium ions [73]. While the supramolecular loading of DOX on CNTs is promoted by high pH, care however must be taken not to incubate CNTs with DOX in an environment that is too basic, as DOX can start to destabilize above pH 6 and becomes totally inactivated at pH 9 in daylight at 25 C [71]. Loading of DOX is affected by the concentration of DOX in the loading solutions. As there exist a nite surface area on CNTs to which DOX can bind, there is thus a saturated adsorption capacity for each system. In the loading of DOX onto SWCNTs functionalized with P-glycoprotein (P-gp) antibody, it was observed that the adsorption capacity of DOX initially increased with increasing DOX concentration in the loading solution, but eventually reaching a plateau with a maximum loading capacity following continual rise of DOX concentration in the loading solution. Similar adsorption isotherm prole was also observed in other studies [74,75,85]. Interestingly, in the loading of DOX onto FA conjugated magnetic MWCNTs, a linear increase in DOX loading content and a nearly constant DOX loading efciency of above 96% were observed when the ratio of DOX to MWCNTs was increased from 0.2 to 2.0 [76]. This discrepancy might be attributed to the fact that saturated adsorption capacity has not been reached. The time allocated for DOX adsorption/incubation can signicantly alter the level of DOX loading as well as its rate of release from oxidized MWCNTs [75]. As the adsorption kinetic of DOX on CNTs is slow, 10 days were required for complete saturation and equilibrium to be attained. Nevertheless, 2 h of incubation could easily accomplish a loading that is considered adequate for chemotherapy. Comparing the adsorption behaviors of DOX with 2 h and 10 days of incubation, misty layer coatings and sludge-like substances were observed on the surface of CNT samples under transmission electron microscopy (TEM) respectively, indicating stronger and more favorable adsorption of DOX on CNTs with prolonged incubation time. Desorption of DOX, on the other hand, was more favored with shorter incubation time under both neutral and acidic conditions. While the de-sorption percentage for 10 days-incubated sample was low, the total amount of DOX released was actually higher than that of 2 h-incubated sample. The diameter of CNTs is also able to inuence the binding and release of DOX from CNTs. Specically, DOX was able to bind more strongly but be released slower from MWCNTs of larger diameter, as larger tubes possess bigger and atter graphitic side walls that can facilitate more efcient interactions between DOX and CNTs [26]. The coatings on functionalized CNTs can also be manipulated to modify the loading and release efciency of DOX. CHI, being a natural cationic hydrophilic polymer, has been used to coat CNTs. CHI is stable at pH 7.4 but degrades readily in acidic pH. In one study, oxidized SWCNTs supramolecularly attached with DOX were coated with FAdecorated CHI, and it was shown that the CHI-FA conjugate coating lowered the rate of DOX release compared to the uncoated samples,
1993
by lengthening diffusion path length and forming additional hydrogen bonds between FA and DOX [86]. Degradation of CHI coating in acidic pH, as indicated by scanning electron microscopy (SEM), was suggested to be another factor contributing to the enhanced release of DOX in acidic condition in addition to DOX protonation. Sodium alginate and CHI, either alone or in combination, were coated non-covalently onto oxidized SWCNTs, and the effects of different polysaccharides coating on the loading of DOX were studied [45]. As CHI is cationic and alginate is anionic, they confer the coated SWCNTs with different zeta potential. Alginate-coated SWCNTs resulted in the highest DOX loading, as the negative charges on alginate facilitated association with cationic DOX. Conversely, low level of DOX loading was observed for CHI-coated CNTs due to mutual repulsion. The release of DOX followed an inverse relationship to the loading study, in which constructs with higher drug loading efciency released DOX slower. From these results, it is revealed that, in addition to stacking, electrostatic interaction also plays an important role in the adsorption of DOX on CNTs. It is possible to achieve desirable DOX loading and release prole by modulating the CHI-to-alginate ratio used for coating CNTs. The amount of adsorbed coating molecules also has some bearings on the amount of DOX that can be loaded on the surface of CNTs. Poly (ethylene glycol-b-propylene sulde) (PEG PPS), a biocompatible amphiphilic diblock copolymer, was used to disperse MWCNTs, and the amount of DOX loaded was observed to be inversely proportional to the concentration of PEG PPS used, suggesting that DOX loading was dependent on the surface area left free from PEG PPS adsorption [87]. Similar trend of increasing coating density leading to decreasing DOX binding was also observed for DOX being loaded on oxidized SWCNTs functionalized with branched PEG 2500-NH2 [71]. Notably, while PEG coatings generally reduced the extent of DOX binding to SWCNTs by 10% as compared to uncoated SWCNTs, different PEGs of varying molecular weights, irrespective if they were covalently or non-covalently conjugated to the SWCNTs, appeared to have no signicant impact on the loading of DOX. Similar negligible inuence is also observed in another study [80]. Yet in the report by Niu et al. that compared the loading and release of DOX from PEGylated SWCNTs functionalized with or without FA, while similar drug release proles were observed for both constructs, the loading efciency of the construct with FA was slightly higher than that without. This was possibly due to the carboxylic groups of FA conferring negative surface potential to the SWCNTs and enriching the electrostatic attraction between DOX and FA-PEG-SWCNT [81]. This complicated inuence of surface coating on drug loading is further evident by the contradictory observation made by Wen et al., where the loading, encapsulation efciency and release proles of DOX on multifunctional dendrimer-modied MWCNTs were similar with and without FA functionalization [74]. With all these conicting results, it is therefore important to always evaluate the extent of DOX loading and release individually for every CNT construct created with different coating and/or targeting molecules. It is also possible to manipulate the release of DOX from CNTs with temperature. Capitalizing on the unique property for 2 polymers, polyethylenimine (PEI) and PVA, to complex at low temperature via hydrogen bonding and de-complex at high temperature, a thermosensitive DOX DDS based on polymer-gated CNTs was created [88]. Oxidized CNTs were rst covalently conjugated with PEI, and then coated with PVA via hydrogen bonding complexation, forming zippers. At 40 C, the zippers opened due to weakening of hydrogen bonding between PEI and PVA, allowing DOX to penetrate through the sparse polymeric network and attach on the surface of CNTs. After cooling back to room temperature (25 C), hydrogen bonding between PEI and PVA reestablished, forming back the zippers and limiting the movement of DOX. When tested on lung broblasts, breast adenocarcinoma and HeLa cells, while free DOX demonstrated non-discriminatory killing at temperature ranging from 35 C to 40 C, the zippers DOXCNT construct was relatively non-toxic below 37 C but reaches comparable cell inhibition level as free DOX at 40 C. The ability for such delivery system to
discriminately release DOX at elevated temperature maybe advantageous for cancer treatment, as tumors tend to display higher temperature than normal healthy tissues [89]. One of the major differences between in vitro drug release studies in phosphate buffered saline (PBS) and the actual release of drugs from nanocarriers in the biological system is the presence of proteins in biological uids. Many proteins possess aromatic rings and hydrophobic cavities that can interact with the hydrophobic surface of CNTs [90]. The phenomenon of proteins binding to nanoparticles (including CNTs), known as the corona effect, has already been demonstrated by a few studies [91,92]. Inevitable protein adsorption on CNTs following in vivo administration may therefore alter the release of DOX by competitively displacing DOX from the surface of CNTs. In fact, the desorption of DOX from oxidized MWCNTs was accelerated by 2 times in release medium of neutral pH comprising of bovine serum albumin (BSA) or immunoglobulin G versus blank PBS [75]. Similar promotion of DOX release in cell culture medium was also demonstrated for PEGylated SWCNTs loaded with DOX [71]. A few studies, however, demonstrated negligible difference between the release proles of DOX from MWCNTs covalently functionalized with hyaluronic acid (HA) and SWCNTs non-covalently functionalized with FA-terminated PEG in buffer and serum, suggesting possibly the effects of protein binding on DOX release may be inuenced by the different functional groups/ coating present on the surface of CNTs [73,81]. As CNTs, especially SWCNTs, are able to absorb energy in the NIR region, NIR can hence also be used as a trigger to stimulate and enhance the release of DOX, by favoring the desorption process of DOX from SWCNTs, which is an endothermic process [50]. In another study, NIR was used to control and induce the release of DOX loaded onto FA and iron di-functionalized MWCNTs in PBS, while still maintaining a sustained release prole [85]. In addition to recognizing the different factors capable of altering the loading and release of DOX, it is also imperative to understand the uptake process and intracellular distribution of CNTDOX complexes in cancer cells. Typically, in order to visualize the intracellular distribution of CNTs, uorescence dyes, such as uorescein [46,50,71,74,76,78,80,83], Alexa-uor-647 [73,84], rhodamine (Rh) [84] or even CdTe quantum dots (QD) [47], are used to conjugate the CNTs. In a study that investigated the subcellular trafcking of CHI-coated SWCNTs conjugated with FITC and supramolecularly loaded with DOX in endothelial progenital cells (EPC), a time-dependent release of DOX was observed. DOX appeared initially as red uorescence of darker intensity due to quenching from CNT interaction near the FITC-labeled green-colored CNTs in lysosomes, and subsequently detach from the SWCNTs inside the acidic environment of lysosomes to yield free DOX with brighter red uorescence that moves into nucleus, leaving the CNT carrier in the perinuclear region without signicant exocytosis after 3 h [78]. This release and localization pattern of DOX ensuing CNTDOX complex uptake is consistent with many other studies conducted with FITC uorescent label in other cell types [47,71,74,76]. Non-uorescent molecules have also been used to label CNTs loaded with DOX for intracellular uptake study and other imaging purposes. For instance, star-shaped and multi-branched gold NP (GNP) capable of engaging with surface enhanced Raman scattering have been successfully adsorbed onto PEGylated MWCNTs with supramolecularly loaded DOX to visualize the uptake of this CNT complex in A549 cells [77]. A novel method for in vivo imaging of SWCNTs was recently reported by chemically linking recombinant thermostable Luciola cruciate luciferase (LcL) on SWCNTs carrying DOX [93]. Unlike the use of uorophores or QDs, which need external excitation source and are unable to image nonsupercial tissues, LcL engages in bioluminescence that requires no excitation source and is able to penetrate deep within biological tissues with high spatial resolution. To further enhance the therapeutic efcacy and safety prole of DOX, a plethora of strategies has been employed to enable CNTbased DDSs to specically target selective cancer cells. Different
1994
small targeting molecules have been successfully conjugated onto CNTs, and FA is one molecule that has been popularly employed against tumor cells with overexpressed FA receptors (FR). It was demonstrated in many similar studies that supramolecularly loaded CNTDOX complexes with additional FA functionalization were able to be taken up more efciently by cancer cells that overexpress FR (e.g. HeLa) via FR-mediated endocytosis, and were more cytotoxic than non-targeting CNTDOX constructs without FA [45,71,81]. While some studies have demonstrated the superiority of FA-conjugated CNT DOX complexes in inhibiting the growth of FR-expressing cancer cells versus free DOX, no comparisons were made with non-targeting constructs without FA, leaving some concerns on the true targeting ability and specicity of these FA-functionalized CNT-based DDSs [76,79]. In an interesting study, carboxyl MWCNTs that have been covalently attached to amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers modied with FITC and FA, and subsequently loaded with DOX, were able to be selectively internalized by human epithelial carcinoma KB cells with high level of FRs and caused more cytotoxicity than in KB cells with low level of FRs [74]. The same constructs but without FA attachment, on the other hand, demonstrated indiscriminately low uptake and cytotoxicity in both KB cell types. Alas, the potency of DOX delivered by the multifunctional MWCNT complex was at most equivalent to free DOX against KB cells, with free DOX demonstrating signicant higher level of cellular uptake than the construct. Similar result of FA-conjugated CNTDOX system being more effective than non-FA-conjugated construct (but less potent than free DOX) was also seen in another study, and it was presumably due to slow rate of DOX release [71]. HA is a naturally occurring glycosaminoglycan and overexpression of activated hyaluronan receptors (HR), such as CD44 and receptor for hyaluronan-mediated motility, has been detected on tumor cells to enhance cell adhesion [94]. Against A549 cells, which are known to overexpress HR, DOX-loaded oxidized MWCNTs covalently tethered to HA via 2,2-(ethylene dioxy) bis(ethylene amine) (EDBE) were around 2.4 times more cytotoxic and induced apoptosis more efciently than free DOX at equivalent DOX concentration [73]. Furthermore, HR-mediated endocytosis facilitated the internalization of the HA-functionalized construct and subsequent translocation into lysosomes. As the major molecular targets of DOX, namely topoisomerase II and DNA, are located in nucleus, it is thus postulated that higher cancer cell kill can be achieved by delivering DOX specically to nucleus. Steroids could be employed to accomplish nuclear targeting, as the complex formed between steroid and its receptors after binding in cytoplasm would be translocated to nucleus, dilating nuclear pores up to 60 nm during the process [95]. CNTs functionalized with steroid can thus exploit this nuclear translocation mechanism and deliver their cargo specically into nucleus. Estradiol (ES) is one such nuclear targeting molecule that has been explored. -Estradiol-17-hemisuccinate was covalently conjugated onto oxidized MWCNTs with a EDBE linker and subsequently loaded with DOX [84]. The uptake and intracellular distribution of this ES-conjugated construct was determined in A549, HeLa and MCF-7 cells, together with MWCNTs functionalized with other targeting molecules such as HA and FA. As expected for steroids, ESCNTs were localized mainly in nuclear and perinuclear region, unlike with HA-CNTs and FA-CNTs where no nuclear co-localization was observed. As a nuclear targeting device, ES-CNTs were more efcient in enhancing the cytotoxicity of DOX in A549 and MCF-7 lineages. The chemo-enhancing effect of ES-CNTs was also found to be dependent on cell types, as the improvement in cytotoxicity was more apparent in ER positive A549 and MCF-7 but not in ER negative HeLa cells. Besides ES, glucocorticoid like dexamethasone (DEX) mesylate has also been covalently linked to amine-modied MWCNTs to create a nuclear targeting device [82]. While the authors claimed that the DOX-loaded CNT construct with DEX mesylate was more cytotoxic, and was more internalized by A549 cells as compared to free DOX due
to ligand-receptor specic targeting, these claims were not fully substantiated by the doseresponse curves that were almost overlapping and the lack of non-DEX mesylate conjugated CNT control. To better evaluate the nuclear targeting ability of such system, techniques such as uorescent confocal microscopy to look at the degree of nuclear accumulation of DOX and/or uorescently tagged CNTs with samples with and without the additional DEX mesylate nuclear targeting moiety could also be performed. Other than small chemical molecules, biological molecules, such as peptides, antibodies and even DNA, have also been engaged to equip DOX-loaded CNT-based DDSs the ability to attack specic cancer cells. By conjugating cyclic RGD (arginineglycineaspartic acid) peptide, that can recognize integrin v3 unregulated in many tumors, on the terminal group of PEG-functionalized SWCNTs loaded with DOX, higher degree of drug uptake and cell killing were observed in integrin v3 positive U87MG human glioblastoma cancer cells compared to non-targeted construct without cyclic RGD [26]. While the IC50 values obtained for cyclic RGDPEG-SWCNTDOX were still higher than that of free DOX, the targeted-construct was found to be more selective for tumors that overexpress integrin v3, as it was relatively less cytotoxic to integrin v3 negative MCF-7. A triple functionalized SWCNT comprising DOX, a uorescent marker (uoresceine) and a monoclonal antibody capable of recognizing carcinoembryonic antigen (CEA, which is a glycoprotein expressed only in cancer cells, especially adenocarcinoma such as colon cancers), was fabricated [46]. While DOX was non-covalently attached, both uoresceine and CEA antibody were linked to the SWCNTs covalently via BSA as a hydrophilic multifunctional linker. The complex could be internalized by CEA expressing WiDr colon cancer cells. While similar construct without CEA antibody resulted in lower complex uptake, which alluded to the role of CEA antibody in facilitating cell penetration, free DOX seemed to have however equal or even superior cellular uptake to the CEA antibody-tethered construct, as qualitatively assessed by confocal microscopy. Moreover, the specicity of the targeting ability and the efcacy of this DDS were not assessed in this study, leaving doubts on whether such system is really more specic than or superior to free DOX. P-gp is a transmembrane efux pump that can be found on cancer cells to promote multidrug resistance (MDR). Targeting DOX to cancer cells with oxidized SWCNTs covalently bound to antibody against P-gp uorescently labeled with FITC showed enhanced cellular uptake by 23 fold and cytotoxicity against MDR P-gp overexpressing K562 human leukemia cells compared to free DOX and non-targeted construct without P-gp antibody at equivalent DOX concentration [50]. The targeting role of P-gp antibody was further validated by the inability for human serum albumin-functionalized SWCNTs to be taken up efciently by resistant K562. Intracellular delivery of DOX was boosted, due to the difculty for P-gp to pump out the entire CNTDOX complex. Moreover, steric hindrance presented by the interaction between P-gp and its antibody attached on the SWCNTs also prevented efcient efux of DOX. Further investigation on the construct's efcacy on non-P-gp expressing cancer or healthy cell lines, though, could be conducted to conrm that the cytotoxicity of the construct was indeed selective. Transferrins (Trf) are a group of glycoproteins involved in the transport of iron. Overexpression of Trf has been observed in many cancer cell types due to heightened iron demand for heme synthesis and rapid cell division [96]. CdTe QD-conjugated and iron NP-lled poly (sodium 4-styrene sulfonate) (PSS)-modied CNTs coated with Trf and DOX were developed as a 3-in-1 system with biologically targeting, magnetic targeting and optical imaging properties [47]. Due to its targeting ability, Trf was able to enhance the uptake of Trf-functionalized CNT construct in Trf positive HeLa but not in Trf negative HEK 293 human kidney cells. Compared to free DOX and non-Trf-conjugated construct at equivalent DOX concentration, Trf-functionalized construct was the most cytotoxic to HeLa cells, corroborating with the greater degree of internalization previously demonstrated.
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With the aim to achieve efcient targeting of DOX to brain tumors across blood brain barriers (BBB), angiopep-2, a peptide capable of binding to lipoprotein receptor-related protein (LRP) receptors that are overexpressed on both BBB and glioma [97], was covalently attached to phospholipid (PL)PEG-MWCNTs supramolecularly loaded with DOX [80]. Higher uptake of the angiopep-2 tethered construct was observed in lysosomes of both brain capillary endothelial cells (BCEC) and C6 glioma cells compared to non-targeted construct without angiopep-2 functionalization. In terms of efcacy, the construct was also more cytotoxic compared to free DOX and the non-targeted construct. Intriguingly, the non-targeted CNT construct was found to be almost a fold less active than free DOX, though the authors did not provide any explanation. Also, to further substantiate the targeting ability of angiopep-2, it would have been useful to repeat the uptake and cytotoxicity experiments on cancer and normal cell lines that do not overexpress LRP. Aptamers are single stranded DNA or RNA nanomaterials with specic 3-dimensional structures that can selectively bind to other small molecules or even an entire cell. Being a 26mer guanine rich oligonucleotide aptamer, AS1411 is capable of interacting with nucleolin, an overexpressed multifunctional protein that contributes to rapid tumor proliferation [98]. Poloxamer 188-dispersed DOXSWCNTs complex non-covalently functionalized with AS1411 aptamer could recognize nucleolin receptors found on the surface of EC-109 human esophageal cancer cells with high afnity, thereby elevating its cellular uptake and its growth inhibitory ability in a time- and dose-dependent manner [83]. The efcacy, however, was only compared against free DOX. In order to validate the targeting ability of AS1411, in vitro growth inhibition studies with non-targeted construct without AS1411 attachment and on other cell lines that do not overexpress nucleolin could be conducted. Interestingly, the therapeutic efcacy of this DDS could be further improved with NIR. NIR irradiation at 808 nm was able to increase the cytotoxicity of the conjugate in a time- and dose-dependent manner. Incorporation of iron NP to CNTs can confer magnetic property to the CNTs, enabling one to utilize external magnetic eld to guide the magnetic CNTs to specic cells or tissues. With this aim, a dual targeted oxidized MWCNTs-based nanocarrier di-functionalized with FA and iron NP was created for DOX delivery [85]. This system was amendable to targeting using external magnetic eld, by enriching its local concentration in the tumor extracellular environment. To assess the effect of magnetic targeting, the FA-DOX magnetic MWCNTs construct was incubated with HeLa cells for 8 h in the presence or absence of external magnetic eld, followed by replacement of culture medium to simulate in vivo drug clearance. The cytotoxicity of the magnetic construct was enhanced by 23 fold with external magnetic eld, and this was estimated to be around 6 fold higher than that of free DOX. Another similar construct comprising poly(acrylic acid) (PAA)-grafted MWCNTs functionalized with FA and iron oxide magnetic NP also achieved greater killing of U87 human glioblastoma with external magnetic eld [76]. In this study, the effect of magnetic targeting was observed by creating 2 separate non-overlapping circular U87 growth zones in each well of a 6 well plate. After treatment with either free DOX or the magnetic CNT construct, a magnet was placed below only 1 growth zone and the 2 separate zones were observed microscopically for cell growth. While no difference in cell death was observed in the 2 growth zones treated with free DOX, no cells were found in the magnetically targeted zone treated with the magnetic complex. Conversely, cells in the non-magnetically targeted zone continued to grow beyond the original circular boundaries, revealing the ability for external magnetic eld to concentrate the magnetic nanocarrier within a xed location. Similarly, the uptake and cytotoxicity of the 3-in-1 system of QDs-conjugated magnetic CNTs loaded with Trf and DOX created by Chen et al. in HeLa cells were also further improved by enriching the concentration of the CNT construct in a conned area using external magnetic eld [47]. Interestingly, in this magnetic CNT DDS, iron NPs were specically encapsulated within the interior of CNTs so as to
protect the NPs from agglomeration, enhance their chemical stability, free up the external surface of CNTs for improved DOX binding and lastly minimize magnetic-induced uorescence quenching of the QDs conjugated on the CNT exterior. While the CNTDOX DDSs discussed so far have all been developed based on the supramolecular interaction between DOX and CNTs, there are also studies that adopt other methods of loading DOX onto CNTs. In one study, DOX was rst covalently linked to a pyrene via an enzymatically cleavable carbamate bond, and the pyreneDOX complex was subsequently irreversibly attached onto PEG-SWCNTs via strong hydrophobic and interactions [99]. In vitro release demonstrated efcient liberation of DOX in cancer cell lysate, as a result of enzymatic cleavage by carboxylesterase, but inefcient hydrolysis in pH 7.4 buffer. The absence of pyrene in the dissolution medium suggested that any DOX measured was not due to pyreneDOX complex desorption but carbamate hydrolysis. Even though the construct was able to be internalized into lysosomes and induce time- and concentration-dependent cell death in vitro against B16-F10 melanoma cells, it was less potent as compared to free DOX especially at lower drug concentration. Furthermore, while the authors claimed that DOX was attached to SWCNTs via pyrene linkers, they have neglected the possibility of direct interaction between DOX and SWCNTs. Having said that, being a universal linker, the use of pyrene actually offers the possibility to connect CNTs with other drugs, targeting or tracking molecules that are otherwise unable to associate with CNTs non-covalently. Taking into consideration the natural tendency for DOX to associate with the surface of CNTs, Gu et al. attempted to further enhance the loading, release and activity of DOX by covalently attaching DOX via a hydrazine linker onto PEGylated SWCNTs [100]. Loading of DOX was improved from 160 to 220% compared to PEGylated CNTs that were only supramolecularly attached with DOX, as DOX was now able to associate with CNTs via 2 different interactions (i.e. covalent and noncovalent). The release of DOX could be further accelerated at low pH as hydrazine bonds are cleavable under acidic condition. As a result of enhanced drug loading and release efciency, the construct was able to be better uptaken and it exerted greater cytotoxicity in both human hepatocellular carcinoma HepG2 and HeLa cells at equivalent DOX dosage compared to supramolecular-only DOXPEGylated CNT complex. However, no comparison was made to free DOX. Instead of serving as a drug carrier, CNTs can also be used as an adjunct to modulate the loading and release of DOX from another parent DDS. For instance, SWCNTs were incorporated as isolated bers in a hybrid gel system of N-isopropylacrylamide (NIPAM) and N-dimethylacrylamide (DMAAM), acting as a molecular reservoir to store DOX in basic condition and release the drug in acidic environment [101]. This composite gel SWCNT system could also respond to NIR irradiation and released the encapsulated DOX cargo by inducing rapid volume phase transition of the gel between shrinkage and swelling. Aside from DOX, other members of the anthracyclines drug class have also exhibited promising potentials to be amendable for CNT delivery. Since all anthracyclines share a similar planar aromatic tetracyclic core structure, it is of no surprise that the other members of this drug class can also be supramolecularly attached onto CNTs via interactions like DOX. With the aim to investigate the adsorption behavior of EPI hydrochloride on MWCNTs, Chen et al. had identied several key factors that inuence the loading of EPI [102]. Adsorption of EPI was favored by high pH, low temperature, large overall surface area and smaller CNT diameter. Functionalizing MWCNTs with carboxylic acids also improved EPI loading, by increasing free surface area through reducing CNT aggregation and forming additional hydrogen bonds between EPI and CNTs. Specically regarding the inuence of diameter, where the authors observed more rapid EPI adsorption and higher drug loading for CNTs with lower diameter, this observation is inconsistent with the one made by Liu et al., who demonstrated instead stronger binding and lower rate of DOX release from SWCNTs with higher diameter due
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to atter graphitic side walls that promote stronger stacking [26]. More investigations are therefore needed to resolve this discrepancy to fully understand the impact of CNT diameter on the loading and release of anthracyclines. In their rst attempt of demonstrating DOX attachment on CNTs, Liu et al. had also applied the same supramolecular binding strategy to other aromatic molecules like DAU, albeit with different degree of loading than DOX [26]. In another paper, DAU was non-covalently loaded onto SWNCTs functionalized with sgc8c aptamer, a three-dimensional single stranded DNA structure capable of targeting leukemia biomarker protein tyrosine kinase-7, with high loading efciency of 157% w/w and a similar pH dependent release prole as DOX [103]. At equivalent DAU concentration, this DDS was found to have higher uptake and greater selectivity towards Molt-4 (target cell line, acute lymphoblastic leukemia T cells) than U266 (non-target cell line, B lymphocyte human myeloma) compared to free DAU. However, the construct was only as effective as free DAU against Molt-4. The study also lacks a non-targeted control without aptamer functionalization. Nonetheless, the targeting role of aptamer was alluded by the inability for the construct to induce signicant cell death upon co-incubation with an antisense of sgc8c aptamer, indicating also the potential for concomitant administration of the antisense sequence to be an effective antidote for this DDS. Pirarubicin was covalently attached to PL-branched PEG functionalized SWCNTs via a cleavable ester bond. The complex demonstrated superior efcacy in vitro against human bladder cancer cells BIU-87 to free pirarubicin [104]. However, no in vitro release study was performed. Moreover, the authors made no mention about the extent of pirarubicin loading and the possibility of additional unintentional supramolecular attachment of pirarubicin on the CNT surface. There was also no indication if the doses of pirarubicin used in the in vitro study were standardized to the concentration of free pirarubicin. While technically not an anthracycline, mitoxantrone is an anthracenedione that bears similar structure and mechanisms of action as the other anthracyclines. Loading of mitoxantrone has been attempted on PEGylated SWCNTs and its loading and release patterns were almost identical to that of DOX, namely, the loading of mitoxantrone was promoted at higher pH while its release was favored at lower pH [71]. In terms of cytotoxicity, the mitoxantroneloaded SWCNTs construct was found to be around one fold less potent than that of free mitoxantrone in HeLa cells, presumably due to slow rate of drug release. 2.2. Platinum-based drugs Platinum (Pt) based compounds constitute an effective class of anticancer agents for a wide array of malignancies [105], by chelating DNA and forming intrastrand adducts that affect key cellular processes, like transcription and replication, and ultimately triggering apoptosis [106,107]. While highly effective, the use of Pt based drugs is unfortunately limited by severe dose limiting nephrotoxicity, neurotoxicity and myelosuppression, arising from pre-mature aquation and nonspecic target interactions [108,109]. As a result, sub-lethal doses of Pt compounds are often used clinically, which consequently promote the development of resistance [110]. Particularly for active Pt (II) complexes, pre-matured loss of activity is also related to poor circulation and limited tumor delivery, in addition to the presence of inactivation mechanisms in living biological systems [107]. Hence, in order to circumvent pre-matured inactivation of Pt (II) drugs, designing of more inert Pt (IV) prodrugs or combining Pt (II) drugs with drug carriers have been investigated extensively [111114]. An earlier attempt in merging Pt based anticancer agent with the use of CNTs as drug carrier has been reported jointly by Lippard and Dai's research groups, whereby a SWCNT tethered Pt (IV) prodrug conjugate was constructed and demonstrated to effectively deliver a lethal dose of cisplatin (CDDP) upon selective intracellular reduction [29]. cis,cis, trans-[Pt(NH3)2Cl2(OEt)(O2CCH2CH2CO2H)], a Pt (IV) complex, was
synthesized and covalently linked through one of its axial COOH ligands to the surface of SWCNTs which have been non-covalently functionalized with amine-ended PL-PEG chain (SWCNT-PL-PEG-NH2). This SWCNT-based longboat carried an average of 65 Pt (IV) centers per nanotube as determined by atomic absorption spectroscopy (AAS). As evidenced by a positive shift in the reduction potential of the Pt (IV) complex under pH 6.0, it is speculated by the authors that the SWCNTPt (IV) entered cells by endocytosis and the lower pH environment of endosomes served to facilitate Pt release by reductively cleaving the axial ligands by which the Pt (IV) complex was covalently linked to the SWCNTs. Remarkably, in testicular carcinoma cells NTera-2, the conjugate showed more than 25-fold enhancement in cytotoxicity as compared to the relatively inactive Pt (IV) prodrug, while a 2.5-fold improvement in efcacy was observed with respect to CDDP as assessed by MTT assay. Pt concentration of the cytoplasmic and nuclear fractions of cells treated with the SWCNTPt (IV) conjugate were 68 and 2 times higher than those of cells incubated with free Pt (IV) prodrug and CDDP alone, respectively. A further development of the above-mentioned SWCNTPt (IV) conjugate was proposed by the same research group by further conjugating the remaining axial ligand of the Pt (IV) complex with a targeting molecule, FA, forming Pt (IV)-FA. The synthesized complexes were then tethered in the same way as reported previously to SWCNTPL-PEGNH2 [115]. The authors demonstrated the selectivity of SWCNTPt (IV)-FA by performing efcacy studies in a panel of cell lines encompassing FR overexpressing human choriocarcinoma JAR and human nasopharyngeal carcinoma cells KB, and non-overexpressing control NTera-2. Both FA containing constructs, SWCNTPt (IV)-FA and Pt (IV)-FA, exhibited superior cytotoxicity to CDDP. Indeed, their respective IC50 values obtained from MTT assay were reported to be 0.01 M, 0.086 M, and 0.15 M in KB cells, indicating that FA was able to enhance the cytotoxicity of the Pt (IV) complex in FR positive cells, and that such enhancement could be further potentiated by 8-fold with the use of SWCNTs as drug carrier. Moreover, when KB cells were incubated with 1 M of SWCNTPt (IV)FA, their nuclear fraction showed a considerable amount of Pt. Conversely, in Pt (IV)-FA treated cells, Pt content could not be detected, showing that SWCNTs were able to improve Pt nuclear uptake. Additional proof of FR selectivity is provided with the nding that the IC50 of SWCNTPt (IV)FA and CDDP did not differ much (0.048 M versus 0.044 M) in FR negative NTera-2 cells. Nevertheless, while the IC50 of SWCNTPt (IV)-FA in JAR cells was reported to be as low as 0.019 M, comparisons for FR selectivity were not substantiated as there was a lack of cytotoxicity data from Pt (IV)-FA and CDDP treatment on JAR cells. Additionally, the authors also reported that, when SWCNTPt (IV)-FA was co-tethered with a uorophore, the presence of uorescent SWCNTs in endosomes was found to be greater in KB cells than NTera-2 cells, further conrming the selectivity of this construct. Interestingly, monoclonal antibody specic for CDDP intrastrand 1,2-d(GpG) cross-links was employed in an immuno-uorescence study to probe if the Pt released from SWCNTPt (IV)-FA formed adduct with nuclear DNA in KB cells. Presence of the cross-links was detected in the nucleus by antibody-derived green uorescence with SWCNTPt (IV)-FA, albeit the uorescence intensity was not that much different as compared to CDDP-treated KB cells. Another study regarding targeted delivery of CDDP using SWCNTs was reported by Bhirde et al., in which oxidized SWCNTs were functionalized concomitantly with EGF and either CDDP or QD via direct covalent amide linkage, yielding SWCNTCDDPEGF or SWCNTQDEGF [116]. A series of imaging techniques of SWCNTQDEGF incubated with EGFR-expressing head and neck squamous carcinoma cells HN13 was performed to validate the ability for the CNT carrier to selectively target EGFR. Notably, EGFR in HN13 cells was knocked down by siRNA to provide an EGFR-negative control. Both confocal microscopy and TEM showed that the uptake of SWCNTQDEGF in HN13 was signicantly greater than the EGFR-siRNA transfected HN13 counterpart. Selective in vitro efcacy against EGFR expressing cells was again
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illustrated in HN13 cells and its EGFR depleted control. While 10 M CDDP treatments seemed non-detrimental, HN13 treated with 1.3 M SWCNTCDDPEGF exhibited hindered cell growth and HN13 pretreated with EGFR-siRNA were minimally affected. Furthermore, additional cell lines NIH-3T3 broblasts, which express less EGFR than HN13, and its EGFR overexpressing counterparts SAA cells, were also included in the cell proliferation study. It was shown that 70% and 90% growth inhibition occurred exclusively in 1.3 M SWCNTCDDPEGF treated HN13 and SAA cells but not in NIH-3T3 cells, whereas growth inhibition was absent with 10 M CDDP treatment across all cell lines, indicating that SWCNTCDDPEGF with 1.3 M equivalent of CDDP were more effective in killing cells than 10 M free CDDP. Further investigations on the SWCNTCDDPEGF performed by the same research group were more inclined towards physical characterization using scanning TEM (STEM) for atomic scale visualization and quantitation of single Pt-based CDDP molecules attached to the SWCNTs [117]. Z-contrast scanning TEM technique was reported to be useful for imaging SWCNTs with covalently attached organic molecules containing single atom of medium-to-high atomic number [118] and it was also possible to quantify image intensities [119,120] to determine the number of heavy metal-based drugs or contrast agents that are associated with CNTs. In the study, intense bright dots were observed throughout the bundle, indicating the presence of attached CDDP molecules. It was estimated that there was on average 1 Pt atom per 10 nm length of SWCNTs, giving an approximation of around 1 M CDDP in the bioconjugate dispersion, which is comparable to the concentration of 1.3 M determined by UVvis absorption spectroscopy. Therefore, establishment of this technique seems to be vital as alternative for characterization and quantication of Pt content on CNT-based drug carriers. While the development of CNTs as Pt drug carriers discussed above are all based on attachment of drug cargo non-covalently or covalently on the exterior side-wall of CNTs, the interior space of CNTs represents a better alternative site for drug loading that has the potential to minimize premature deactivation of Pt-based drugs by biological nucleophiles before reaching their intended target sites [121]. Encapsulating drugs within the interior cavity of CNTs eliminates any need of forming chemical bonds between drug molecules and CNTs, meanwhile also freeing up the external surface area of CNTs for further functionalization with targeting molecule to increase the DDS's selectivity. In a pioneering study of lling CNTs with Pt based drug by Hampel et al. in 2008 [122], carboplatin (CP) was incorporated into openended MWCNTs, forming MWCNT-CP, by wet chemical approach [123], in which capillarity was the driving force for cargo to enter the interior cavity of CNTs. Encapsulation of CP was observed to be temperature-dependent, whereby 30% w/w drug loading was obtained with stirring at 90 C as compared to less than 5% with stirring at 70 C. However, no loading data were reported at temperature beyond 90 C and this temperature dependency might also be impractical for the loading of heat sensitive compounds. After conrming the presence of CP in MWCNTs with energy dispersive X-ray analysis (EDX) and electron energy loss spectroscopy, the authors further veried with X-ray photoelectron spectroscopy (XPS) that the Pt atom of CP after encapsulation still existed at a 2+ state, indicating that CP was in its intact state without being oxidized or reduced during the lling procedure. However, release of CP from the construct was only accessed qualitatively by TEM images of MWCNT-CP after immersing in cell culture medium for 0.5 and 24 h. Cytotoxicity study was performed in vitro in human bladder cancer cell line EJ28 using WST-1 assay and it was demonstrated that 20 g/mL CP decreased viability of the cells to almost 50% while MWCNT-CP (0.3 mg/mL) containing only 9 g/mL CP could lead to a further 10% drop in viability. Whereas in MWCNT-CP (0.3 mg/mL) treated cells, apoptosis induction and cell cycle arrest were contradictorily found to be at a lower degree than treatment with 20 g/mL free CP, inferring that 10% improvement in efcacy observed in WST-1 assay might be insufcient to conclude that MWCNT-CP was more effective than free CP. Notably, preliminary biocompatibility evaluation in EJ28
cells with WST-1 assay showed that the same dose of nonfunctionalized MWCNTs reduced cell viability to 80%. Surprisingly, treatment with empty MWCNTs plus 40 g/mL free CP clearly showed more profound cytotoxic effects as observed with decreased cell counts and increased apoptosis compared to 40 g/mL CP alone. Another attempt to encapsulate CDDP in CNTs was made by the same group where SWCNTs were used instead of MWCNTs [124]. Again by using wet chemistry approach to encapsulate CDDP in SWCNTs (SWCNTCDDP) with DMF as solvent, 21% w/w of drug loading was achieved. To rule out the possibility that CDDP could be adsorbed non-specically on the sidewalls of SWCNTs, Fourier transform infrared spectroscopy (FTIR) and Raman analysis were performed. Indication that CDDP was indeed encapsulated into SWCNTs was established based on the absence of typical CDDP signature FTIR and Raman characteristics on SWCNTCDDP. In vitro release study showed that CDDP was released from SWCNTs after 24 h, followed by a plateau after 72 h with a maximum release percentage of 68%, suggesting a controlled-release prole. Unfortunately, in vitro cytotoxicity evaluation of the construct demonstrated that SWCNTCDDP was far lower than free CDDP in affecting prostate cancer cells (PC-3 and DU145) viability. Despite the disappointing in vitro ndings with SWCNTCDDP, the same research group later demonstrated that CNTs encapsulated with CP were more effective than carbon nanobers (CNFs) at impairing the proliferation and clonogenic survival of tumor cells [125]. In this comparative study, CP was incorporated in CNTs and CNFs with the same method [122], with estimated loading yields of 20% and 13% w/w for CNT-CP and CNF-CP respectively. Release kinetics of CNT-CP and CNFCP differed considerably, in which CNT-CP showed controlled release of CP up to day 14 reaching a maximum cumulative release of 68%, while CNF-CP only demonstrated 10% release. In agreement with their different drug release kinetics, CNF-CP with only moderate drug release affected the proliferation of DU145 cells similarly to unloaded CNFs, whereas CNT-CP caused a dramatic reduction of cell survival to 8%. Notably, the cytotoxic activity of CNT-CP was stronger than that of free CP as demonstrated by the nding that 40% of cells treated with CP alone remained alive at the same equivalent concentration. This enhancement was also found to be reproducible in other lineages. In addition, only one-tenth of CNT-CP was needed to produce the same cytotoxic effect as free CP treatment. Moreover, colony formation assays showed that CNT-CP impaired the clonogenic survival of all four cell lines (DU145, PC-3, EJ28, A498) more effectively than CNF-CP and free CP at comparable drug concentration. Apoptosis was accessed after different treatments and it was shown that the ability for free CP to induce apoptosis was doubled when cells were incubated with CNT-CP. Coherently, a concentration of 50 g/mL of free CP was needed to induce apoptosis in 69% of cells, whereas only 5.0 g/mL CNT-CP was sufcient in inducing similar extent of apoptosis, conrming the ten times potentiation of CP with CNTs. Though the results reported in the study represent a valuable basis for detailed animal studies, no further in vivo study of CNT-CP has been reported to date. The use of MWCNTs as drug depots for Pt-based drugs was reported in a study performed by our group, in which the idea of forming CNTbased nano-bottles by capping both ends of pristine MWCNTs with GNP was rst described [126]. GNP-capped MWCNTs loaded with CDDP (capped MWCNT-CDDP) were synthesized by a rst step of nano-extraction [127] with CDDP in ethyl acetate and a second step of nano-extraction with 1-octadecanethiol functionalized GNP (ODTf-GNPs) for capping the extremities of MWCNTs. Successful CDDP encapsulation was validated by TEM and EDX analysis. It should be noted that ODT-f-GNPs did not appear to completely block the MWCNT openings but rather narrowed them. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis for Pt content in CDDP loaded uncapped and GNP capped MWCNTs revealed that 62.1% and 44.4% w/w of CDDP were encapsulated respectively. Rapid and near complete release of encapsulated CDDP from uncapped MWCNTs was demonstrated within 1 h in PBS. The presence of GNP caps,
1998
however delayed CDDP release especially in the rst h. In vitro IC50 of CDDP alone, uncapped MWCNT-CDDP, and capped MWCNT-CDDP against MCF-7 were found to be 11.7 M, 12.9 M, and 7.7 M, respectively. Lack of difference in terms of cytotoxicity between uncapped MWCNT-CDDP and free CDDP could be attributed to rapid leakage of CDDP from MWCNTs. As for the treatment with capped MWCNTCDDP, a slight leftward shift in the doseresponse curve was observed, indicating that moderate enhancement of the pharmacological actions of CDDP was conferred by encapsulation in capped MWCNTs. Nevertheless, although the idea of forming CNT bottles for drug delivery is intriguing, more research should be done to further improve the capping strategy, by for instance innovating caps that be uncapped readily to release drug cargo upon experiencing certain tumor-specic triggers (i.e. higher temperature or lower pH). Since biocompatibility issues of full length SWCNTs and MWCNTs are still unresolved (please refer to the article on toxicity of CNTs for more details), the use of potentially more bio-tolerable ultra-short CNTs (US-CNTs; 2080 nm in length and 1.4 nm in diameter) as a drug delivery platform for CDDP was recently attempted by Guven et al. [128]. US-CNTs, produced from full-length SWCNTs via a uorination and pyrolysis procedure [129], were employed to encapsulate CDDP. The authors proposed that the short and relatively uniformlengths of US-CNTs (95% 50 nm) might aid in avoiding RES trapping, enhancing cellular uptake and improving eventual elimination proles of the DDS. Indeed, one in vivo report has demonstrated that USSWCNTs are non-toxic and well tolerated by mice even at very high dose (0.5 mg/kg) [130]. To ensure that CDDP was encapsulated rather than non-specically adsorbed on the side walls of US-CNTs, the solutions from each washing step were tested for Pt content via ICP-OES and it was discovered that all surface bound CDDP could be removed with 20 washing steps of deionized water. Additionally, the authors has also prepared another US-CNT sample in which the original CDDP encapsulated constructs were dispersed via probe sonication in aqueous Pluronic-F108 surfactant solution, with anticipation that the Pluronic wrapping would slow down drug release by providing additional diffusion barriers. Indeed, with Pluronic wrapping, only 6.1% of CDDP was released into PBS at pH 7.4 in 24 h and only 16.9% over 1 week, suggesting substantial resistance provided by surfactant wrapping against complete CDDP release. Conversely, unwrapped samples showed signicant greater leakage with almost 50% of CDDP released in the rst 3 h and about 80% after 1 week. In vitro cytotoxicity investigation by MTT assay revealed that while the surfactant wrapped USCNTs alone have minor effect on cell viability, CDDP-encapsulated Pluronic-wrapped US-CNTs clearly exhibited greater cytotoxicity compared to free CDDP in two different breast cancer cell lines (MCF-7 and MDA-MB-231). This enhancement of efcacy was more apparent at 24 h of incubation than 48 h. This trend is also reected by the ndings of Pt concentration in cells, whereby cells treated with CDDPencapsulated Pluronic-wrapped US-CNTs contained signicantly more Pt than cells treated with free CDDP over the rst 24 h. Considering that clinical administration of CDDP typically involved diuresis after 68 h of infusion, this 24 h time frame of enhanced delivery could still be clinically relevant. Nevertheless, while unwrapped US-CNTs demonstrated more complete in vitro release of CDDP, the efcacy of this construct was not evaluated in parallel in this study. In a follow up of the same study, incomplete release of CDDP from Pluronic wrapped US-CNTs was addressed by using non-invasive radiofrequency (RF) eld to produce heat that can disrupt Pluronic and consequently triggers the release of CDDP [131]. After validating that Pluronic was indeed disrupted and displaced selectively by localized heat produced from RF activation, release of CDDP from the constructs was investigated. It was shown that the release of CDDP from samples pre-treated with RF increased by around 2-fold (approximately 60% release) after 1 week. As compared to previous report where only 17% of CDDP was released over a week [128], signicant improvement was observed with 30% of release from control incubation at 37 C
and 60% release from RF activated samples with elevated localized temperature of 49.5 C. For cytotoxicity study, liver cancer cells Hep3B or HepG2 were exposed to CDDP-encapsulated Pluronic-wrapped USCNTs, Pluronic-wrapped empty US-CNTs, or PBS control at IC50 for CDDP-encapsulated Pluronic-wrapped US-CNTs (2.5 M, US-CNTs 13.7 g/mL) for both cells lines in a trans-well system with or without a 3-min RF eld exposure 1, 2, or 3 times at 24 h intervals. This ensured that the nanocarriers were spatially separated from the cellular compartment and only released CDDP could diffuse through the trans-well membrane. Cell viability was assessed at 120 h when the PBS-treated control samples reached conuence. For both cell lines, after MTT assay, it was noted that even the Pluronic-wrapped US-tubes alone were slightly cytotoxic at corresponding concentration of 13.7 g/mL and this slight cytotoxicity was not enhanced by multiple RF exposures, implying that usage of US-CNTs might not fully circumvent the biocompatibility issue of CNTs. Although a general trend was observed in both cell lines that cytotoxicity was enhanced in a statistically signicant fashion with increasing RF exposures, no comparison with CDDP free drug was included. Nevertheless, the idea of using remote RF activation to trigger drug release still represents a promising strategy for selective localized destruction of cancer cells in vivo. Because CDDP is highly hydrophilic, conning CDDP within the hydrophobic cavities of CNTs is inherently challenging, as it is difcult to control the loading and release of CDDP from CNTs. As such, our group has previously reported an approach of using CNTs as macromolecular carriers to encapsulate a chemically inert Pt (IV) prodrug [28]. This unique strategy entailed the entrapment of Pt (IV) prodrug within the cavities of MWCNTs such that the drug could be stably contained by favorable hydrophobichydrophobic interactions. Upon selective chemical reduction by endogenous intracellular reducing agents such as glutathione, the hydrophobicity of the Pt (IV) prodrug would be reversed as it is reduced into its cytotoxic yet hydrophilic Pt (II) form. The reduced Pt (II) complex could no longer be trapped stably within the hydrophobic interior of CNTs and is readily released from the nanocarrier to exert its cytotoxicity. As a proof-of-concept, we engineered a highly hydrophobic Pt (IV) prodrug and demonstrated its stable entrapment within MWCNTs by nano-extraction in chloroform, forming MWCNT Pt (IV) with loading efciency of 51.7 2.0% w/w. Indeed, Pt could not be detected in the buffer solution when MWCNTPt (IV) was suspended and agitated in PBS for 4 days. To trigger Pt release, MWCNTPt (IV) was agitated in 3 mM ascorbic acid to simulate a reductive cellular environment. Release of Pt occurred at a slow rate and completed after 8 h, compared to MWCNTCDDP which unloaded Pt rapidly within 2 h, demonstrating the concept of selective Pt release arising from hydrophobicity reversal associated with Pt (IV) reduction to Pt (II). To ascertain if entrapment of Pt (IV) was amendable for drug delivery, A2780 ovarian carcinoma cells were treated with dialyzed samples [i.e. pristine MWCNTs, physical mixture of MWCNTs and Pt (IV) prodrug, or MWCNTPt (IV) complex] to simulate the exposure to bodily uids after intravenous administration. Analysis of Pt content in treated cell extracts detected signicant Pt levels in cells treated with MWCNTPt (IV), while simple mixture of MWCNTs and Pt (IV) could not withstand Pt (IV) being dialyzed before cellular internalization, leading to low cellular Pt levels. Neither in vitro nor in vivo efcacy study of MWCNTsPt (IV) has been reported. A more recent study on encapsulation of Pt based drug into CNTs was conducted with oxaliplatin, a third-generation Pt analog of the 1,2diaminocyclohexane families [132]. Oxaliplatin was incorporated into the inner cavity of PEGylated MWCNTs (MWCNT-PEG600) and control MWCNT-COOH via nano-extraction in ethanol, forming MWCNTPEG600 (Oxaliplatin) and MWCNT-COOH (Oxaliplatin) with loading of around 43.6% and 51.5% w/w, respectively. However, oxaliplatin aggregates were found on the external surface of MWCNT-PEG600 under TEM imaging, suggesting that the PEG branches on the surface of MWCNT-PEG entrapped oxaliplatin molecules. Release of oxaliplatin from MWCNTs was investigated with dialysis tubing method in PBS and
1999
the amount of oxaliplatin release was quantied by AAS analysis. Sustained release was reported in MWCNT-PEG600 (Oxaliplatin) with only 34% of oxaliplatin released into PBS within 6 h as compared to the release of oxaliplatin from MWCNT-COOH, in which 50% of incorporated drug was detected in PBS within 6 h release. Cytotoxicity of oxaliplatin, MWCNT-COOH (Oxaliplatin) and MWCNT-PEG600 (Oxaliplatin) were evaluated in HT29 human colorectal cancer cells by MTT assay. Unexpectedly, MWCNT-PEG600 (Oxaliplatin) showed the least cytotoxic effect followed by MWCNT-COOH (Oxaliplatin), with free oxaliplatin being the most potent treatment when cell viability was assessed at 12 and 24 h incubation. However, a signicant increase in cytotoxicity was observed for MWCNTs constructs with a reversed order when cell viability was assessed at 48 and 96 h incubation, indicating the possibility that the onset of action of MWCNT-PEG600 (Oxaliplatin) is slower than both controls due to its delayed release characteristics. Pt-DNA adducts formation, phosphorylated H2AX formation (important marker of ionizing radiation or genotoxic agents-induced double strand breaks [133]) and cell apoptosis assay results showed the same trend as the MTT assay. 2.3. Antimetabolites Antimetabolites embody a class of anticancer drugs that disrupts the metabolic pathway for the formation of nucleic acids in cancer cells. There are two groups of drugs that fall into this drug category, namely the antifolates and the purine/pyrimidine antagonists. 2.3.1. Antifolates Methotrexate (MTX), a FA antagonist that inhibits dihydrofolic acid reductase, is a well-known and potent antifolate, used also to treat autoimmune diseases. However, MTX suffers from poor bioavailability, low cellular uptake and toxic systemic side effects [134]. Combining MTX with functionalized CNTs has the potential to raise the bioavailability of MTX and diminish its undesirable adverse effects by delivering MTX specically to tumor cells with additional targeting molecules [135]. In the article by Pastorin et al., the authors reported the method of covalently functionalizing MWCNTs via 1,3-dipolar cycloaddition of azomethine ylides with orthogonally protected amino functions that can be selectively deprotected and subsequently modied with MTX and uorescent probe FITC [30]. Uptake of the complex was analyzed with human Jurkat T lymphocytes. It was shown that the construct accumulated in cytoplasm in a time- and dose-dependent manner. Although a rapid internalization of the construct in human Jurkat T lymphocytes was observed, its efcacy was lower than free MTX, presumably due to slow rate of drug release as a result of the presence of stable amide bonds between MTX and CNTs. Therefore, while controlled multifunctionalization of CNTs opens new perspectives in the biomedical eld, there still is a need to further modify the delivery system by utilizing cleavable linkers. Typically in other studies, disulde bonds, which are sensitive to pH changes and reducing agents, have been widely explored by researches as cleavable linkages. However, this strategy suffers from in vivo instability, as endogenous thiols can easily cleave disulde bridges during blood circulation. As such, Samori et al. have covalently linked MTX to MWCNTs with two different cleavable linkers, namely the tetrapeptide Gly-Leu-Phe-Gly (which can be selectively cleaved by proteases overexpressed in tumor cells), and the 6hydroxyhexanoic ester (which is an esterase-sensitive hydrophobic spacer commonly used in other prodrug conjugate synthesis) [136]. Interestingly, the group showed that the cytotoxic activity of the conjugates in MCF-7 cells was strongly dependent on the presence and the types of linker. Higher cytotoxic activity was observed with the protease-sensitive tetrapeptide linker than the 6-hydroxyhexanoic ester linker, where cell death was induced in 90% and 20% of MCF-7 cells, respectively, after 24 h of incubation.
In a later study, Marega et al. described the synthesis, conjugation and also diffusion-ordered spectroscopy and nuclear magnetic resonance characterization of a MTX DDS based on HASWCNTs covalent conjugate [137]. In this paper, the authors presented in details the synthesis of this new system by covalently binding MTX to the primary amino groups of HA chains functionalized on SWCNTs. The results indicated reduced mobility of the organic moieties due to covalent conjugation with CNTs. Alas, no in vitro release, uptake and efcacy studies were reported in this paper. Additional tests are thus needed to assess the possibility of using this complex as a DDS. Aside from covalent conjugation, MTX is also amendable for physical adsorption onto CNTs. A strategy of non-covalently functionalized MWCNTs with 1,2-distearoyl-phosphatidylethanolamine-methoxypolyethylene glycol conjugate-2000 (DSPE-mPEG 2000) and then entrapment of MTX has been attempted by Modi et al. [134]. The product, MWCNTmPEGMTX, was characterized for particle size, loading efciency, morphology and in vitro release. The MWCNT conjugate was found to release MTX faster in acidic medium of pH 5.8 than neutral pH 7.4. No difference in the release rate of plain MTX was observed with change in pH. The reason for the higher rate of MTX release from CNTs in acidic environment was not thoroughly explained by the authors. However, since cancer tissues tend to be acidic, it was suggested that this formulation is able to deliver and release MTX to cancer cells more effectively compared to other normal tissues. This system requires further experimental tests to assess its drug delivery potential, as no in vitro and in vivo biological studies were reported. In a recent study, Das et al. described the physical loading of different chemotherapeutic agents, including MTX, on an array of MWCNTs with different surface functionalization, including those with PEG, HA, FA and ES [84]. In this study, it was observed that, regardless of surface functionalities, the loading of MTX was estimated to be around 30% for all samples. Release of MTX was measured at two different pHs, 7.4 and 5.5. Contrary to the observation made by Modi et al. in the previous study, MTX release was higher in neutral pH (6070%) compared to acidic pH (3545%) after 24 h of incubation. This observed release prole suggests that MTX may be released and act more efciently if its carriers are localized in cytoplasm as compared to acidic lysosomes. Uptake and cytotoxicity of the various functionalized CNTs loaded with MTX were performed in A549, HeLa and MCF-7 cells. Interestingly, it was revealed that the therapeutic potential of MTX is affected by different surface functionalities. MTX, being an antifolate, exerts its antitumor activity by interfering with the biosynthetic pathway of DNA. Therefore, MTX exhibited slightly higher cytotoxicity in A549 and MCF-7 cells when it was delivered specically to nucleus, where DNA is housed, by ES-functionalized carrier, as compared to FA- and HA-CNT constructs, which were mainly localized in acidic endocytic vesicles and lysomes in cytoplasm. 2.3.2. Purine/pyrimidine antagonists Purines and pyrimidines are components of DNA, RNA and coenzymes that are required for cell proliferation. Therefore, purine and pyridimine antagonists that mimic the structures of their corresponding physiological molecules are able to hinder the synthesis of these essential metabolites and be used for cancer therapy. There are many purine and pyrimidine antimetabolites that have been approved for clinical use. Examples of purine inhibitors include mercaptopurine, thioguanine and azathioprine, while examples of pyrimidine inhibitors encompass 5-uorouracil, cytarabine and gemcitabine (GEM). The basic mechanisms of action of purine and pyrimidine antimetabolites are similar. These compounds diffuse into cells, typically with the aid of membrane transporters, and are converted to analogs of cellular nucleotides by enzymes of the purine or pyrimidine metabolic pathway. These metabolites then inhibit one or more enzymes that are essential for DNA synthesis, causing DNA damage and inducing apoptosis [138]. GEM is a low molecular weight nucleoside and an antimetabolite that inhibits cellular DNA synthesis. GEM is used clinically to treat or
2000
manage many solid tumors such as pancreatic cancer, non-small cell lung cancer, head and neck cancer, tumors of the bladder, breast, ovary, cervix and biliary tract [139]. In one of the rst attempts of delivering GEM with CNTs, Yang et al. created a magnetic CNT-based DDS of GEM by physically adsorbing GEM onto PAA-grafted MWCNTs with surface deposited iron NPs [31,140]. While the carrier was shown to lack toxicity at high concentration of 25 g/mL to the two human pancreatic cancer cell lines tested, BxPC-3 and SW1990, the GEM-loaded magnetic CNT construct could only achieve similar reduction in cell viability relative to the group treated with free GEM. Despite no improvement in in vitro efcacy demonstrated, the comparable cytotoxicity proles suggested that the carrier preparation process was at least not deleterious to the activity of GEM. Additionally, it was also shown that the construct was able to be internalized by BxPC-3 and be found close to nucleus. However, without a proper control on the uptake of free GEM, it is not possible to conclude if the use of CNTs is able to enhance the cellular internalization of GEM. In order to achieve specic tumor targeting, GEM was loaded physically onto FA-conjugated MWCNTs to target cancer cells with overexpressed surface FR [141]. With an entrapment efciency of around 80%, the release of GEM was sustained and higher in acidic lysosomal pH of 5.0 compared to pH 7.4, due to ionization of GEM which increases its hydrophilicity in acidic medium. Owing to FA functionalization, the construct also experienced increase in hydrophilicity, consequently leading to lower degree of hemolysis induction as compared to free GEM. When tested on MCF-7 cells with MTT assay, the CNT construct was observed to be slightly more cytotoxic than free GEM and in comparison with the same CNT construct without FA functionalization in a concentration-dependent manner. However, the authors have made several unfounded claims based on this result, such as the construct causing more effective apoptosis than free GEM without actually conducting any apoptosis analysis. The true targeting ability of this DDS is also not fully investigated since the intracellular uptake of constructs with and without FA conjugation and a duplicate cytotoxicity experiment on non-FR overexpressing cancer cell lines were not conducted. In a different approach, a GEM analog, 2,2-diuoro-2-deoxycytidine (dC), which differs from GEM at the 2 position, with both uorine atoms being replaced with hydrogen, was non-covalently linked to water soluble PEI functionalized SWCNTs via hydrogen bonds [142]. Loading of dC of around 18% by mass was achieved by sonicating a mixture of dC and aqueous suspension of PEI-SWCNTs. Endoscopic ultrasound was used to trigger the release of dC from the conjugate. While no drug was release without ultrasound, a marked increase in dC concentration following a zero-order release pattern was observed after the application of endoscopic ultrasound in the release medium. As these are only preliminary studies involving dC as a cheaper model drug for GEM, further experimentations are therefore needed with GEM to evaluate the efcacy and toxicity of this controlled CNT-based DDS. 2.4. Antimicrotubules Microtubules are slender, cylindrical laments found in the cytoskeletons of plant and animal cells. They are dynamic, ubiquitous protein polymers composed of the protein tubulin that oscillate between phases of elongation and shortening [143]. With their dynamic structures, microtubules can adopt different organizations depending on their cellular functions such as maintenance of cell structure, protein trafcking, chromosomal segregation, etc. [144]. During mitosis, microtubules assemble into mitotic spindles that distribute chromosomes to opposite poles of a dividing cell. Thus, as a result of their essential roles in cell division and mitosis, microtubules are pharmaceutically validated targets for anticancer chemotherapy [145]. Anticancer drugs that target microtubules, also referred to as antimicrotubules or antimitotic, can be broadly classied into two categories depending on their mechanisms of action: the rst group inhibits
tubulin polymerization and mitotic spindles formation to prevent mitosis (i.e. destabilizers), and the second group paradoxically stimulates tubulin polymerization that consequently causes cells to become clogged with excessive microtubules and triggers apoptosis (i.e. stabilizers). Examples of destabilizers include vinca alkaloids, colchicine, combretastain A4 and podophyllotoxin. Stabilizers, on the other hand, consist of taxanes and epothilones [144]. Just like most other conventional non-targeted chemotherapeutic agents, the use of antimitotic drugs is often associated with many undesirable systemic effects such as severe peripheral neuropathies, immunosuppression, myelosuppression and gastrointestinal toxicity. For this reason, on one hand scientists are constantly looking for drugs that will have milder side effects and on the other hand they are actively seeking carriers for drugs that will reduce their toxic effects [144]. Taxanes are diterpenes alkaloids produced by plants of the Taxus genus. Among them are compounds like paclitaxel (PTX), originally extracted from the Pacic Yew tree (Taxus brevifolia) in the 1960s, for the treatment of ovarian cancer and marketed under the trade name Taxol, as well as its semisynthetic analog docetaxel (DTX) derived from a precursor found in the European Yew tree (Taxus baccata). DTX is more water-soluble and exhibits improved anticancer activity than PTX. Both PTX and its analog are widely applied in chemotherapy of solid tumors, particularly those resistant to classic cytotoxic drugs [144,146]. Taxanes generally have low solubility in water compared to other anticancer drugs. Additional compounds are therefore necessary to increase the solubility, improve the bioavailability and limit the side effects of taxanes [147]. In the case of Taxol, intravenous administration is accompanied by Cremophor EL (polyethoxylated castor oil) and ethanol as co-solvents [148]. However, this has the potential to cause side effects, such as hypersensitivity, anaphylaxis, hyperlipidemia and abnormal lipoprotein patterns. Moreover, intravenous administered PTX is quickly removed from the bloodstream [149]. Oral administration is too hampered by very low bioavailability resulting from poor solubility and P-gp efux in digestive tracts [148]. These limitations have led researchers to look for carriers that could prolong the effect of taxanes while remaining undetectable by macrophages. Recent studies have involved the use of liposomes with hydrophilic PEG polymers, polymer micelles and polymer NPs [149]. A notable development is the introduction of Abraxane (PTX bound to human albumin) which has been approved for clinical use [150]. CNTs appear to be a promising carrier, capable of loading taxanes and ensuring their efcient delivery and release. Comparison between PTX and DOX reveals signicant differences in their interactions with CNTs. Unlike in DOX where there exists a planar aromatic tetracycline plane, as the aromatic rings in PTX are not aligned in a common plane, PTX is unable to interact with CNTs supramolecularly via non-covalent interactions in the same way that DOX associates with CNTs [147]. In addition to being a carrier, CNTs are also able to potentiate the activity of PTX. Arya et al. described the ability for SWCNTs to enhance the activity of PTX against lung cancers, demonstrating a reactive oxygen species (ROS)-dependent synergism between CNTs and PTX on two cell lines, A549 and NCI-H460 [151]. Various ROS inducing agents have been reported to sensitize cancer cells to conventional anticancer agents like PTX. PTX is a microtubule interfering agent which induces apoptosis and enhances the activation of mitogen-activated protein kinase (MAPK). Therefore, agents that can enhance MAPK activation like ROS may serve as good candidates for combination therapy with PTX. Recent studies have shown that carbon nanostructures increase the level of ROS and induce the activation of MAPK/ERK pathway in mammalian cells, so they can be used as a potential cotherapeutics with PTX [152,153]. In another separate study by Zhang et al., synergism between CNTs and PTX was again demonstrated by the ability of SWCNTs to sensitize human ovarian cancer OVCAR3 cells to PTX and resulted in higher cell death [154]. CNTs may thus serve 2 distinct functions in the delivery of PTX, i.e. as molecular carriers and as chemosensitizers.
2001
In one of the rst reports of delivering taxanes with CNTs, Chen et al. described a DDS comprising SWCNTs covalently functionalized with biotin and complexed to a 2nd generation taxoid (SB-T-1214), using a linker that can be easily cleaved by intracellular thiols [155]. In general, covalent chemical modication with linkers is known to decrease the potency of taxoid substantially. However in this study, after cellular internalization of the complex, the taxoid could be cleaved and became active again. Biotin functionalization conferred SWCNTs the ability to efciently recognize and target tumor cells with overexpressed surface biotin receptors. The efcacy of the construct was studied in vitro using leukemia cell line L1210FR (which overexpresses biotin receptors on surface). To further evaluate the cytotoxicity and the specicity of tumor-targeting via biotin, two other cell lines without overexpressed biotin receptors, namely W138 human lung broblasts and murine leukemia L1210 cells were used. The results conrmed the specicity of tumor targeting, as indicated by a signicant lower IC50 value of the biotinated-CNTtaxoid conjugate in L1210FR cell line in comparison to L1210 and W138 after 72 h of incubation. The drug, along with the CNT carriers, entered cells via receptor-mediated endocytosis and was then released, forming stable microtubuletaxoid complexes, halting mitosis and leading to apoptosis. Cellular uptake of this biotin SWCNTtaxoid conjugate was shown to be energy-dependent as it was hindered by metabolic inhibitors like NaN3. These results conrm that receptor-mediated endocytosis was by far the predominant mechanism accounting for internalization of this construct, with passive CNTs diffusion as a minor contributing pathway. In another attempt of covalent functionalization with cleavable bonds, a water soluble SWCNTPTX conjugate was designed by Liu et al. by attaching PTX to branched PEG PL chains adsorbed on SWCNTs via an ester bond [32]. The researchers reached a binding level of around 150 PTX molecules every 100 nm of CNTs. The SWCNTPEGPTX conjugate exhibited excellent stability without agglomeration in various biological media including serum. While the ester bond triggered chemical changes in PTX leading to its inactivation, in vitro cytotoxicity of the SWCNTPEGPTX conjugate was observed to be comparable to Taxol against 4T1 murine breast cancer cells, indicating that active PTX was able to be released following ester cleavage in cells without any loss of pharmacological activity. Sobhani et al. reported the use of hyperbranched polycitric acid (PCA) to functionalize MWCNTs in order to increase their solubility, followed by esterication of PTX to the carboxyl groups of PCA adsorbed on the CNTs [156]. This resulted in a MWCNTPCAPTX complex, which could be internalized by cells via endocytosis and released PTX once the ester bond was hydrolyzed in cytoplasm. Interestingly, in vitro drug release study showed that PTX could be released from the conjugates faster in acidic pH, making the system particularly suitable for specic delivery of drugs to acidic tumor tissues. However, from the release result, it can also be deduced that the cleavage of the ester bond can occur spontaneously even in the absence of esterases, which might be problematic when such complex is applied and delivered in vivo. Nonetheless, in vitro cytotoxicity tests conducted in A549 lung and SKOV3 ovary cancer cell lines indicated improved potency of PTX compared to carrier-free PTX. Contrary to the non-covalent approaches described above, Lay et al. have reported physical adsorption of PTX onto PEGylated SWCNTs and MWCNTs [147]. The advantage of physical incorporation over covalent conjugation via linkers is the lack of chemical changes to the structure of PTX. Lay et al. loaded physically PTX onto the sidewalls of CNTs by immersing PEGylated CNTs in a saturated solution of PTX in methanol. PTX loaded PEG-CNTs could be dispersed in aqueous solution well and without aggregation. The high loading capacity of 2636% w/w achieved may be attributed to strong hydrophobic interactions between PTX and CNTs in methanol. Using a dialysis method, the release proles of PTXPEG-CNTs and PTX alone were determined at pH 5 and 7.4. At pH 7.4, PTX could be released from PTXPEG-CNTs several times faster than free PTX that lacks aqueous solubility. Loading of PTX onto the
surface of CNTs diminished the tendency for PTX to aggregate, leading to higher free surface area that consequently increased the rate of dissolution and release of PTX. It should be noted that, at this physiological pH, the release rate of PTX from PEG-SWCNTs is higher than that from PEG-MWCNTs, presumably due to the higher surface area of SWCNTs and weaker hydrophobic interaction between SWCNTs and PTX caused by the smaller diameter of SWCNTs. The situation is however changed at pH 5 and the release levels, as compared to the same constructs at pH 7.4, were slower for SWCNTs but faster for MWCNTs. The presence of PTXPEG-SWCNTs aggregates, due to salt-out effects in acidic solutions that reduces the solubility of PEG, was observed, leading to lower surface area and slower rate of PTX release. This aggregation phenomenon was shown to be less signicant for PEG-MWCNTs than PEGSWCNTs. In terms of biological studies, PEG-CNTs used as a control exhibited low toxicity and slight cell proliferation effects on both tested cell lines MCF-7 and HeLa. PTXPEG-CNT, however, showed a high percentage of cell killing and lower IC50 than free PTX. Lee et al. described the use of synthetic polyamholyte poly [2(dimethylamino) ethyl methacrylate]-co-(methacrylic acid) (PDM), a water-soluble copolymer, and SWCNTs as a supramolecular complex [148]. This complex was then employed as a PTX carrier against human epithelial colorectal adenocarcinoma Caco-2 cells. The average amount of PTX in CNTPDMPTX was estimated to be around 126 g per mg of CNTs. This complex was able to increase the antitumor potency of PTX and possibly permeate tight junction barriers, which are of signicant physiological importance because they maintain electrolytic gradient and membrane polarity, as well as prevent macromolecules and xenobiotics such as anticancer drugs from entering cells. Following release after internalization of the complex inside cells, PTX could act on microtubules, weakening tumor cells in more effectively than PTX alone, and causing tight junction damage in Caco-2 cells. The carrier itself did not affect the resilience or formation of tight junctions in Caco-2 cells. The authors compared Caco-2 cells treated with free PTX and the complex SWCNTPDMPTX at an equivalent PTX concentration of 25 g/mL. The viability of cells was monitored for 120 h by MTT assay. To achieve 50% of cell death, 99.23 h of incubation with pure PTX were necessary. However, when treated with the CNT complex, the time needed was decreased by 30.01 h. The use of CNT-based drug carrier might also prevent PTX from being pumped out from the cells by P-gp. In a recent report by Moore et al., hydroxy-functionalized MWCNTs were coated with a biocompatible, amphiphilic block-co-polymer composed of poly(lactide) (PLA) and PEG and subsequently non-covalently loaded with PTX [157]. In this arrangement, the hydrophobic PLA encapsulates hydrophobic PTX while the hydrophilic PEG reduces CNT aggregation in aqueous solution. Uptake of PLAPEG-coated MWCNTs additionally functionalized with Alexa Fluor 647 was successfully observed in two different target lines: U87 human glioblastoma cells and human umbilical vein endothelial cells (HUVEC). In terms of the carrier's toxicity, in comparison with pristine CNT, coated CNTs showed no toxicity even at 10 fold higher concentration than pristine CNTs with Presto Blue cell viability assay in the two cell lines. Expression of both inammatory proteins IL-6 and ICAM-1 in rat epithelial cells, a different cell line from those tested for uptake and toxicity, after exposure to pristine CNTs was signicantly higher than those treated with coated CNTs. These results indicate that PLAPEG coating was able to signicantly decreased both toxicity and inammatory potential of MWCNTs. Regarding the drug loading potential of such coated CNTs, PLAPEGcoated MWCNTs were able to load 1.65% w/w of PTX and release PTX in a controlled manner for at least 1 week. Analysis of efcacy was conducted after incubating the PTX-loaded CNT construct or free PTX for 6 and 24 h in U87. While the results showed that with a shorter incubation time of 6 h, MWCNTPLAPEGPTX was able to achieve the same level of cell kill as free PTX at only half of equivalent PTX concentration (CNTPTX contained 70 nM PTX versus 140 nM of free PTX), longer period of incubation time with free PTX was shown to be more
2002
cytotoxic than MWCNTPLAPEGPTX at equivalent PTX dose. This inconsistency in cytotoxicity depending on incubation time was not thoroughly expounded by the authors, making one unsure if the construct was truly more efcacious in killing cancer cells as compared to free PTX. One way to achieve controlled and sustained release of drug is with the use of induction heating of template-synthesized CNTs. Wu et al. described an interesting new approach, which controls the release of two water-soluble drugs PTX and C6-ceramide from the interior of CNTs by inductive heating with an external alternating current or magnetic eld pulses [158]. The drugs were loaded by depositing the drug solutions in the upper part of vertical CNTs arranged on a membrane with a vacuum suction at the bottom. CNTs allowed the encapsulation of C6-ceramide and PTX at the same time. The drug concentration loaded into the CNTs was 100 times lower (PTX: 0.03 g/mL and C6-ceramide: 0.1 g/mL) than the concentration required for a comparable effect with exogenously applied treatment (PTX: 3 g/mL and C6-ceramide: 10 g/mL). Even at such high doses, PTX alone or C6-ceramide alone had limited effects on killing the three MDR pancreatic cancer cell lines L3.6, PANC-1, and MIA PaCa-2, achieving viability of as high as 7579%. However, combining these two agents at a relatively low dose, as that loaded into the CNTs, potently produced cell death with only 1727% viability, indicating the existence of a synergistic relationship between the 2 agents. Uptake of CNTC6-ceramidePTX construct by L3.6 was veried by labeling the CNTs with Texas red. The construct accumulated mainly within the perinuclear space. Also, alternating current magnetic eld resulted in controlled release of PTX and C6-ceramide from the CNTs, and this therapeutic drug delivery resulted in over 70% cell death in L3.6, as compared to almost 100% viability when the alternating current magnetic eld was turned off. The low electromagnetic eld required for release is advantageous, as it has no detectable adverse effect on cells. This delivery system enables the delivery of PTX and C6-ceramide at a much lower concentration with yet comparable killing rate and on-command release control. In a recent study, Das et al. presented the possibility of loading various drugs, including PTX, on a series of MWCNTs with different surface functionalizations, such as antifouling polymer (PEG), tumor recognition modules (FA, HA & ES), and uorophores (Rh B isothiocyanate/ Alexa Fluor) [84]. The authors then went on to explain how the surface functional molecules associated with each bio-functionalized MWCNTs inuenced their therapeutic potential. Release of PTX was measured at 2 different pH (7.4 and 5.5), and it was revealed that the release of PTX was slightly enhanced at pH 7.4 as compared to 5.5. Having said that, the rate of PTX released after 48 h of incubation at pH 7.4 was only 3032%. The slow release of PTX may be attributed to limited solubility of PTX and strong hydrophobic interaction between PTX and CNTs. Cytotoxic potential, cellular uptake and apoptosis-induction of the various MWCNTPTX complexes were evaluated in three different cancer cell lines, A549, HeLa and MCF-7. It was observed that the antiproliferative and proapoptotic activity of MWCNTPTX complexes were critically associated with the constructs' surface chemistry and their intracellular migration pattern. As microtubules are mainly present in the cytoplasm, it was found that PTX acted more efciently when its carriers were localized in cytoplasm such as with the use of FA-MWCNTs or HAMWCNTs, as compared to ES-MWCNTs where nucleus represents the construct's targeted destination. Besides PTX, other taxanes like DTX has also been delivered successfully with CNTs. DTX is a semisynthetic analog of PTX, which has achieved higher patient response rates and fewer side effects compared to PTX in the treatment of certain cancers, such as breast, ovarian and lung cancers. In the study by Wang et al., SWCNTs were rst conjugated with DTX via accumulation by nano-precipitation, followed by noncovalent functionalizing the SWCNTs with surfactant PVP K30 to confer water dispersibility [49]. Then the complex was linked with NGR (AsnGly-Arg) peptide, which targets CD13 tumor vascular antigen, to obtain a water-soluble and tumor-targeting SWCNTNGRDTX DDS. It was
hypothesized that DTX could be released from the DDS in a sustained fashion, and that the anticancer effect of DTX could be further enhanced through NIR mediated tumor photothermal destruction. Cellular uptakes were determined for SWCNTDTX and SWCNTNGRDTX combined with FITC in PC-3 cells. The results showed that the uptake was time-dependent and occurred faster for the SWCNTNGRDTX complex. The CNT complexes were all located in cytoplasm but not nuclei under the applied conditions. Wang et al. then analyzed the antitumor efcacy of this complex in PC-3 cells in vitro and demonstrated superior activity of the NGR-functionalized DTXCNTs construct in killing PC-3 cells in a dose- and time-dependent manner to free DTX and the construct without NGR peptide functionalization. Remarkably, the anticancer effect of SWCNTNGRDTX could be further augmented with NIR irradiation at 808 nm in an exposure time-dependent manner. 2.5. Other anticancer drugs Endocrine therapy, also known as hormonal therapy, has a special role in the treatment of breast cancers. Effective hormonal therapy hinges on the presence of ER or progesterone receptors in tumor cells. While not all breast cancer cells have these receptors, they are present in about 7580% of all breast cancers [159]. ERs are located both inside cells and on their surfaces. Upon binding estrogen, these receptors are activated, stimulating cell division as a result. Drugs that can competitively bind to and block ERs can therefore be used to sever this stimulation and impede the growth of breast cancer cells. Tamoxifen is a selective ER modulator that displays anti-tumor activity against both ER positive and, to a certain degree, ER negative breast cancers. Tamoxifen binds to ERs after endocytosis, thereby blocking estrogen annealing, modulating gene expression and inhibiting cell division [160]. Unfortunately, the use of tamoxifen is often associated with osteoporosis, tumor resistance and increased levels of uterine and endometrial cancer [159,161]. There is hence a need to look for a carrier for this drug. Already, there are many reports on the use of different carriers for tamoxifen, ranging from NP, nanoemulsions, dendrimers, polymers, QDs, micelles, molecular conjugates and liposomes [162,163]. However, only a few studies directly involve the application of CNTs to carry tamoxifen. While there have been reports of interactions between CNTs and tamoxifen, the details of these interactions have yet to be fully understood. Shahanipour et al. analyzed theoretically the interaction between tamoxifen and the open-ends of SWCNTs with the Gaussian 98 program and the effect of water on this interaction. In this work, they modeled structures of tamoxifen coupled with SWCNTs by selected atoms and concluded that this type of interaction between tamoxifen and SWCNTs has the theoretical basis for practical use to carry tamoxifen [164]. Subsequently, Nelson et al. described the synthesis and characterization of a tamoxifen-tethered SWCNTs conjugate, in which alcohol-functionalized tamoxifen derivative was covalently attached to carboxylic acidfunctionalized SWCNTs via esterication linked by octa(ethyleneglycol) [159]. Successful conjugation was indicated using many analytical techniques (FTIR, UVvis, Raman analysis, nuclear magnetic resonance). While it was suggested that this SWCNTtamoxifen conjugate could be useful in PTT and in multimodal endocrine therapy strategies for breast cancer treatment, no actual in vitro and in vivo biological studies were conducted. Further analyses are therefore required to validate the functionality of this DDS. Thalidomide is an antiangiogenic drug that inhibits the formation of blood vessels. Antiangiogenic strategy is used for cancer treatment and prevention of cancer recurrence and metastasis. In a study by Cheng et al., a multimodal PEGylated oxidized SWCNT-based DDS consisting of thalidomide, a targeting peptide ligand for v3 integrin receptors on activated endothelial and tumor cells (cyclic RGD) and a uorescent marker (Rh) was created [165]. The loading of thalidomide and cyclic RGD was around 0.334 mg and 0.125 mg per mg of CNTs, respectively. In vitro cellular uptake study of the CNT carrier without thalidomide
2003
after 4 h of incubation was conducted in 2 different mammalian cells, namely human glioblastoma U87MG cells (integrin v3 positive) and human breast cancer cells MCF-7 (integrin v3 negative). It was however noted that the uorescent tag employed in the in vitro uptake study was FITC and not Rh, which is the eventual uorescent tag used subsequently in the in vivo experiments. The authors did not provide the reason for this switch, and any potential difference in the uptake behaviors of the two different uorescently tagged CNT carriers was not accounted. Nonetheless, comparing to the same construct without cyclic RGD, which tended to accumulate in v3 negative MCF-7, the targeting efciency of FITCSWCNTRGD was demonstrated by its higher extent of internalization in v3 positive U87MG cells. Moreover, even without thalidomide, the drug-free targeted carrier was found to exhibit a certain degree of growth inhibition on U87MG cells as observed through morphological changes. Subsequently, the authors also investigated the biodistribution of the targeted CNT carrier and the efcacy of the CNTthalidomide complex in vivo in zebrash embryo models. They will be detailed in a later section of the review. Catechin (CT), a polyphenolic avonoid in green tea extract, is well known for its antioxidant and cancer chemopreventive effect against a broad spectrum of invasive solid cancers [166]. In a study performed by Cirillo et al., a new hybrid system developed on the basis of noncovalent inclusion of MWCNTs to the covalent complex of gelatinCT (GelCT) was analyzed. The aim was to evaluate a new DDS that would increase the antitumor activity of polyphenolic compounds bound to water-soluble protein macromolecules such as gelatin. Recent publications indicated the possibility of obtaining covalent bond conjugates between gelatin and antioxidants with increased biological activity [167,168]. Furthermore, non-covalent functionalization of CNTs with polymers was proposed as an efcient strategy to increase the water dispersibility and thus biocompatibility of CNTs. Therefore, this DDS was aimed to achieve two advantages, namely enhancement of the anticancer activity of CT by conjugation with gelatin and increase in CNT dispersibility with gelatin functionalization. As expected, the GelCT complex homogenously and stably dispersed CNTs over 3 months. It was observed in SEM that CNTs were almost completely coated by the polymeric matrix. Cytotoxicity tests performed on HeLa cells showed that Gelatin, GelCT and CNTs alone did not affect cell viability. Conjugation of CT with gelatin contradictorily decreased the activity of CT because the polymer prevented efcient entry of CT into cells. However, free CT killed 20% and 47% of cells, CNT-CT killed 38% and 48% of cells and CNT-GelCT killed 72% and 80% of cells at 150 g/mL and 300 g/mL, respectively. The signicant increase in the therapeutic activity of CT in the presence of CNTs was presumably due to improved cellular penetration and the ability for the macromolecular system to stabilize the avonoid, thus enhancing its biological properties. Conjugating CT to gelatin further enhanced the construct's antitumor efcacy due to the formation of covalent GelCT complexes with increased biological activities. Overall, this study reveals the great potential for such hybrid DDS to augment the activity of CT. Hexamethylmelamine (HMM), also known as altretamine, is an antineoplastic agent used for the treatment of refractory ovarian cancer. While its precise mechanism of action is still unclear, HMM is postulated to exert its anticancer effect through producing formaldehyde, which is a weak DNA alkylating agent. Previously, our group has attempted to encapsulate HMM into SWCNTs or double-walled CNTs (DWCNTs) and to seal the opened ends of CNTs with C60 [121]. C60 protected the tubes from premature drug leakage and the prepared nanotubes acted as closed nano-bottles for HMM. TEM allowed the identication of the ends of CNTs incorporated with C60. Notably, there were no C60 in the middle part of CNTs previously loaded with HMM, which was in contrast to HMM-free CNTs, where C60 were distributed all along the CNTs. The above observations were conrmed using Raman spectroscopy (in particular radial breathing mode) and XPS. To analyze the release of HMM from C60-capped CNTs, an in vitro release study was conducted with dichloromethane as solvent because of its strong ability to dissolve
both C60 and HMM. Infrared spectrum conrmed efcient washout of HMM from the insides of CNTs. While this paper presented the interesting possibility of packaging drugs inside CNTs in the form of nanobottle, no cytotoxicity tests on cell cultures (that are necessary to conrm the therapeutic effectiveness of the system) were conducted. Moreover, the use of dichloromethane to remove the C60 caps, although used as a proof-of-concept, might not translate well to biological systems that require aqueous environment. Chlorin e6 (Ce6) is a heterocyclic aromatic molecule with photosensitizing properties that can be used in photodynamic therapy (PDT), a process involving activation of molecular oxygen under light irradiation in the presence of photosensitizers that have been selectively accumulated in target tumor tissues. Xiao et al. reported a complex between SWCNTs and Ce6 via non-covalent stacking, van der Waals and hydrophobic interactions [169]. To enhance the solubility and biocompatibility of the complex, CHI was employed to wrap around Ce6SWCNTs. High cellular uptake, low inherent toxicity and efcient PDT efcacy of CHICe6SWCNTs were observed against HeLa cells compared to cells treated with only free Ce6. In another proof of concept study, controllable release and activation of Ce6 were demonstrated by Zhu et al. by wrapping oxidized SWCNTs with single stranded DNA aptamers covalently linked to Ce6 [170]. In the absence of the aptamer's target (in this case thrombin), generation of singlet oxygen was hampered because the photosensitizing and uorescent properties of Ce6 were effectively quenched by its close proximity to SWCNTs. Binding between aptamers and thrombin signicantly weaken the interaction between the aptamers and SWCNTS, leading to dissociation of Ce6aptamerthrombin complex from SWCNTs, restoring the photodynamic activity of Ce6. Specically, introduction of thrombin enhanced the generation of singlet oxygen, as tested using singlet oxygen sensor green, by 13 fold following irradiation of visible light at 404 nm. The binding event was specic to thrombin as incubation with other proteins like BSA, protein A, protein L, NeutrAvidin and immunoglobulin G failed to trigger efcient singlet oxygen generation. Additionally, preliminary in vitro data showed that the phototoxicity of the CNT complex on human Burkitt lymphoma Ramos cells could be mediated through thrombin. Notably, besides regulating the activity of Ce6, the use of aptamers could also confer this DDS the ability to target specic tumor cells. The current system developed, however, relies on exogenous activation by thrombin. Given the promising controlling and potential targeting capabilities of aptamers, more efforts could be directed in devising similar DDSs that can recognize specic endogenous tumor markers. As a precursor of a photosensitizer protoporphyrin IX, 5aminolevulinic acid was electrostatically adsorbed onto the surface of PAMAM dendrimer modied MWCNTs and successfully delivered into the cytoplasm of human gastric cancer cells MGC-803 [171]. Protoporphyrin IX was formed after endocytosis of the CNT complex, resulting in intense homogenous red uorescent signals in the cell cytoplasm. The agent retained its photosensitizing ability after laser irradiation at 632 nm in MGC-803 cells. However, since no direct comparison was made to free 5-aminolevulinic acid, it is unable to assert conclusively that the use of CNT has enhanced the permeation and photodynamic activity of 5-aminolevulinic acid. A novel Bodipy-based PDT sensitizer containing a distyrl-Bodipsy red light absorbing chromophore with intersystem crossing promoter heavy atoms iodine on the Bodipy core, a pendent PEG2000 as the solubilizing group and 2 pyrene groups on the distyrl branches was synthesized by Erbas et al. and non-covalently functionalized onto SWCNTs via strong pyreneCNT stacking [172]. The Bodipy-based derivative retained its ability to generate singlet oxygen after SWCNTs functionalization upon red light irradiation, as determined by the use of selective singlet oxygen trap, diphenylisobenzofuran, albeit with slightly lower activity compared to the free compound. The delivery potential of this system was however not explored in this study as no in vitro cell work was conducted.
2004
Capitalizing on the ability for CNTs to be used for PTT by absorbing NIR, a dual-function CNT-based DDS capable of engaging both PDT and PTT was envisioned. In an attempt to reap the potential benets of combining PDT and PTT, and to achieve specic tumor targeting, Shi et al. has recently developed a multimodal CNT-based DDS consisting of amine-functionalized SWCNTs covalently linked to HA with physically adsorbed hematoporphyrin monomethyl ether (HMME), a new generation prophyrin-related photosensitizer [173]. The CNT system could respond to 808 and 532 nm to trigger photothermal and photodynamic killing respectively. At a HMME loading of around 230% w/w, consecutive irradiation of lasers at 808 and 532 nm led to the most decrease in B16-F10 cell viability, as determined by sulforhodamine B cytotoxicity assay, compared to just irradiation of 808 nm or 532 nm alone. The improved efcacy was postulated to be a result of transient increase in cell membrane permeability in response to hyperthermia induced by PTT, which had consequently increased the cellular uptake of HMME and promoted more efcient photodynamic killing. The targeting function of HA was however not assessed in this study, as a non-targeting control devoid of HA was not created and the experiments were not repeated on other cancerous or normal cell types that do not overexpress HA. Furthermore, the uptake of this CNT complex was only compared to that of free FTIC, which was irrelevant in assessing the ability of CNTs to improve the cellular uptake of HMME. A more valid comparison should be made against appropriately tagged free HMME instead. 3. Delivery of non-anticancer drugs with carbon nanotubes While the delivery of cancer chemotherapeutic agents with CNTs has been more widely attempted and researched, CNTs have also demonstrated promising potentials to be used as either carriers or additives to deliver many non-anticancer drugs, such as antimicrobial, antiinammatory, antihypertensive and antioxidant agents. These CNTbased DDSs, despite being less frequently reported, have been shown to successfully enhance the therapeutic efcacy and modulate the release prole of various non-anticancer drugs. 3.1. Antimicrobials Antimicrobial represents one of the most popular classes of nonanticancer agents to be studied and delivered with CNTs. Sharing very similar concepts with anticancer drugs, antimicrobials are substances that either kill or inhibit the growth of microorganisms, like bacteria, fungi, viruses or even parasites, instead of cancer cells. Amphotericin B (AMB) is one of the rst antimicrobials to be delivered with CNTs. As a polyene macrolide, AMB is considered as one of the rst line agents to combat opportunistic systemic fungal infections in immunocompromized patients. Despite its broad activity spectrum, high effectiveness and relatively low cost, the use of AMB is however limited by numerous undesirable side effects, ranging from infusion reaction to nephrotoxicity. One of the factors contributing to the toxicity of AMB lies in its natural tendency to aggregate due to its low water solubility. CNTs chemically derivatized with AMB were hence attempted, with the aim to improve the solubility of AMB and decrease its aggregation potential. Indeed, double orthogonal functionalization of oxidized MWCNTs with AMB and FITC was performed [34]. At equivalent AMB concentration of 10 g/mL, this CNT conjugate was found to be signicantly less cytotoxic to Human Jurkat lymphoma T cells as compared to pristine AMB. This CNTAMB construct was able to penetrate cells rapidly within 1 h of incubation by spontaneous piercing mechanism without causing cell death. More importantly, modied versions of the construct without FITC on both SWCNTs and MWCNTs, with an AMB loading of around 33%, was found to be more potent than free AMB against Candida and Cryptococcus fungi species, possibly due to enhanced drug solubility and the presence of multiple copies of AMB per CNT molecule (i.e. the multivalence effect) leading to improved binding afnity between
the drug and its target. In another separate paper by the same group, a similar MWCNTAMB construct and a newly designed SWCNTAMB conjugate with PEG linker were prepared with AMB loading of 25% and 10% w/w, respectively [33]. These conjugates were tested for their antifungal activities against a collection of reference and clinical fungal strains, in comparison to pristine AMB and a conventional colloidal dispersion AMB deoxycholate formulation. Both CNT conjugates displayed broad spectrum of antifungal activity that was considerable more potent than AMB alone. The MWCNT-based AMB conjugate, in particular, was observed to be active in several AMB-resistant fungi, and demonstrated a nonlytic mechanism of action, where the conjugate only permeabilized fungal membrane after extended period of incubation and induced membrane depolarization at slow kinetics. Other than serving as an antifungal agent, AMB can also be used to cure visceral leishmaniasis, a fatal intracellular infection of macrophages caused by protozoan parasites of the Leishmania genus. Chemical linkage of AMB onto ethylenediamine-functionalized MWCNTs was attempted [174]. Unfortunately, the exact chemistry underlying the conjugation reaction was not clearly reported, due to discrepancy between the text and the chemical scheme provided. Loading of AMB was estimated to be around 72.4%. Compared to free AMB, AMBloaded MWCNTs were tested to be around 14 times more potent against intramacrophage amastigotes in vitro. In another study performed by Pruthi et al., instead of chemical functionalization, AMB was physically adsorbed on mannosylated MWCNTs to achieve specic delivery of the drug to macrophages for the potential treatment of leishmaniasis [175]. With around 0.66 mM of mannose present in every g of MWCNTs, the mannosylated MWCNTs possessed high afnity to bind lectins (Concanavalin A) and were able to be uptaken by J774 macrophages in sufcient amount, as claimed by the author. The result of the uptake study is however insufcient to support macrophage-targeting because a control experiment with non-mannosylated MWCNTs was not performed. Notwithstanding the poor resolution of the uorescent microscope images provided, the use of Rh B-loaded mannosylated MWCNTs for intracellular uorescence imaging of macrophages is also inconclusive as Rh B could be detached from the MWCNTs before even entering macrophages, causing the uorescence observed to be due to free Rh B instead of MWCNTs-loaded with Rh B. Nevertheless, AMB was adsorbed onto the mannosylated MWCNTs with good entrapment efciency of around 75%, and demonstrated a sustained in vitro release prole. Apart from AMB, dapsone is another example of antimicrobial that has been successfully delivered with CNTs. Possessing both antimicrobial and anti-inammatory properties, dapsone is used clinically for the treatment of malaria, leprosy, pneumocystis pneumonia and toxoplasmosis associated with Acquired Immunodeciency Syndrome and certain dermatitis conditions [176]. With rising resistance of intracellular bacterial infection, it is therefore of utmost interest to devise a strategy to improve the cellular uptake of dapsone and eventually its therapeutic efcacy. Dapsone was chemically functionalized onto the surface of oxidized MWCNTs to form Dap-MWCNTs [177]. Chemical conjugation of dapsone was conrmed and the loading of dapsone was estimated to be around 15% w/w. Dapsone functionalization also conferred the MWCNTs better aqueous dispersibility, a pivotal attribute for biological applications. When tested on rat peritoneal macrophages, Dap-MWCNTs were rapidly ingested with predominant endosomal localization and without any signicant cytotoxicity, much like the unconjugated oxidized MWCNTs. Higher concentration of CNTs, however, could induce apoptosis, with oxidized MWCNTs causing more extensive apoptosis than Dap-MWCNTs due to elevated oxidative stress. It is noted also that Dap-MWCNTs demonstrated delayed apoptosis of rat peritoneal macrophages after 3 days with lower degree of oxidative stress. This phenomenon could be explained by the inherent antiapoptotic activity of dapsone and the protection of reactive carboxylic functional groups on the CNTs by dapsone functionalization. While it is clearly demonstrated that DapMWCNTs were able to be internalized by rat peritoneal macrophages,
2005
the efcacy of such conjugates against intracellular bacterial infection were not examined in this study. Moreover, the delayed apoptosis manifested by Dap-MWCNTs may be counterintuitive in the treatment of infection since apoptosis of macrophages is essential for timely clearance of intracellular pathogens. Pazuoxacin mesylate, an antibiotic belonging to the class of uoroquinolones, was adsorbed onto MWCNTs functionalized with ethylenediamine [178]. In vitro release suggested that the adsorption of pazuoxacin was reversible and its release prole consisted of an initial rapid burst release followed by a period of sustained release. Interestingly, it was revealed that the total amount of pazuoxacin released from the amino-functionalized CNTs was almost a fold higher at pH 5.7 than pH 7.0, possibly due to enhanced hydrophilicity of protonated pazuoxacin in acidic condition that consequently diminished their hydrophobic interaction with the CNT carriers. The sensitivity of this DDS to pH could also be advantageous in the treatment of infections, where the intracellular environment of infected cells is likely to be more acidic. Unfortunately, the efcacy of this DDS was not accessed in the study. Hence there is no indication if such delivery system is superior to free pazuoxacin. An aminoglycoside antibiotic, gentamicin, was incorporated into bullfrog collagen hydrogel doped with 1% w/w CNTs [179]. In this context, CNTs served as an additive to enhance the physical stability of the hybrid hydrogel and retard the release of gentamicin. The release modulation effect was attributed to the formation of irregular CNT network in the hydrogel that impeded solvent diffusion, and also the presence of carboxylic functional groups on the CNTs that could interact with the amine groups of gentamicin. However, there was no mention on the extent of gentamicin loading of both the CNT/collagen hybrid gel and the control collagen-only gel. The presence of CNTs might improve drug loading by simple chemical attraction between CNTs and gentamicin, potentially resulting in differential drug loading in both control and CNTladen hydrogels, thereby affecting the rate of drug release. Chloroquine is an anti-malarial drug that also possesses lysomotropic effect. Due to their ability to cause swelling and rupture of endocytic vesicles, lyosomotropic compounds have been proposed to be used as an additive to enhance gene transfection by enabling timely escape and minimizing degradation of delivered genetic material in lysosomes. In order to further enhance gene delivery efciency with CNTs, chloroquine was non-covalently loaded onto DWCNTs coated with cationic polymer PEI and plasmid encoding for luciferase [180]. Being a hydrophobic weak base with quinolone aromatic ring, chloroquine is able to interact with both the exterior and interior of CNTs via hydrophobic stacking, and be released readily from the CNTs in acidic condition where chloroquine is protonated. With respect to the loadings of chloroquine, they were not precisely measured in all samples, where different polymers were used to coat the chloroquine-loaded CNTs. The method of chloroquine quantication involving gel electrophoresis was also unorthodox, especially since chloroquine is inherently uorescence. Loading of chloroquine was heavily affected by subsequent coating with cationic polymer and plasmid, as seen by the drastic drop in chloroquine content with additional experimental maneuvers. While it was noted that introduction of chloroquine was able to improve transfection efciency by 5 fold compared to a non-chloroquine loaded control, the efciency was only at 10% of conventional transfecting agent, i.e. lipofectamine, in HeLa cells. Moreover, the construct was also found to be more cytotoxic to cells than the non-chloroquine loaded control and free chloroquine, which may potentially limit its use as a biocompatible transfecting agent. Having said that, the construct might prove to be advantageous for the treatment of intracellular malaria infection, as the higher cytotoxicity observed could be a result of improved intracellular delivery of chloroquine. 3.2. Anti-inammatories Anti-inammatory agents can be broadly classied into two major classes, the steroids, specically glucocorticoids, and the non-steroidal
anti-inammatory drugs (NSAIDs). Both of these anti-inammatory agents have been delivered with CNTs acting as either the main drug carrier or as adjuncts to assist or modify their release from another parent delivery systems. Using MWCNTs as nanoreservoirs, oxidized MWCNTs loaded with DEX phosphate, a steroidal anti-inammatory drug, were sealed and incorporated in a lm of polypyrrole via electropolymerization [35]. The use of CNTs and polypyrrole complemented each other by mitigating each other's weaknesses. While polypyrrole, being a conducting polymer, is able to release drug in response to electrical stimulation, it is limited with issues such as low drug loading and non-sustainable drug release per stimulation. On the other hand, while the interior of CNTs with open ends can be lled with drugs, drug leakage from CNTs could lead to uncontrollable drug release. By combining the two components, polypyrrole lm could be used to seal the open ends of drugladen CNTs to abate leakage. In exchange, drug-loaded CNTs could serve to raise the system's overall drug loading capacity and to modify the release prole of encapsulated drug. Indeed, DEX was successfully loaded within the inner cavity of oxidized MWCNTs and the polypyrrole coating was able to store the encapsulated DEX effectively, without any signicant leakage in the absence of electrical stimulation. Drug loading was increased by almost a fold with the incorporation of CNT nanoreservoirs. Upon the application of cyclical negative bias, DEX was released out of the polypyrrole lm via a de-doping process where positive charges on the polymer backbone were neutralized to promote the dissociation of negatively charged DEX phosphate. The CNT-containing lm was able to release DEX in a sustained and linear manner due to the extra drug stored in the nanoreservoirs. More importantly, DEX retained its pharmacological activity after loading and stimulated release, as demonstrated by measuring the amount of nitrogen oxide, an inammatory product, secreted by lipopolysaccharideactivated microglial cells. Such delivery system is useful in the development of implantable microelectrodes as neuronal prosthesis that can deliver anti-inammatory agents to combat inammationinduced neuronal loss and scar formation due to chronic application of microelectrodes. DEX phosphate was also incorporated into an electro-responsive CHI/SWCNT hybrid lm, achieving a near to complete release of DEX with the application of negative potential due to electrostatic repulsion between SWCNTs and negatively charged DEX [181]. Adding SWCNTs into CHI slowed down the release of DEX due to the presence of attractive interaction between DEX and SWCNTs. Contrary to the result obtained with negative potential, application of positive potential further decreased the rate and extent of DEX release from the hybrid lm. While controllable release of DEX is achievable with this delivery strategy, the effects of different CNT concentrations on the release properties of DEX were not investigated. Transdermal delivery of NSAIDs is particularly useful in alleviating pain associated with arthritic conditions and musculoskeletal damage. While transdermal drug delivery causes less systemic toxicity compared to conventional routes such as oral administration, release of drugs from transdermal devices is unfortunately often low and lacks precise control. Capitalizing on the excellent electrical conductivity of CNTs, CNTs have been used as an additive in electro-responsive gel systems to modulate and enhance the release of several NSAIDs at a relatively low voltage that is non-irritating to skin. Diclofenac sodium, for instance, has been incorporated into spherical gelatin/MWCNT hybrid microgels to form an electro-responsive DDS [182]. CNTs offer the gelatin microgels greater thermostability and electrical conductivity. Interestingly, incorporation of CNTs of as high as 35% w/w did not affect drug loading, and resulted in higher rate and total amount of drug released. The authors suggested higher gel swelling arising from larger capillary networks formed at higher CNT concentration to be a reason explaining the phenomenon. The release of diclofenac from the hybrid microgel was further increased by 20% with voltage application due to electrically induced shrinkage. In another study, the same drug diclofenac sodium
2006
was loaded into carboxymethyl guar gum and oxidized MWCNTs hybrid polymer hydrogel at different CNT concentration (0.5, 1.0 and 3.0% w/w) [183]. It was depicted that 1.0% CNT w/w hybrid hydrogel possessed the highest drug loading and slowest rate of drug release as a result of strong drugCNT association and high matrix viscosity. Higher CNT concentration of 3% w/w, however, gave poorer gel performance, as CNTs were more likely to aggregate and reduce available free surface area. There seem to be a discord between the two studies relating to the concentration of CNTs incorporated and their eventual effects on the mechanical properties and the release prole of diclofenac of CNT-hybrid gel DDSs. Ketoprofen is another example of NSAIDs that has been incorporated into an electro-sensitive transdermal DDS impregnated with CNTs. Specically, a semi-interpenetrating polymer network composed of poly-ethylene oxide (PEO) and pentaerythritol triacrylate interspersed MWCNTs (10% with respect to PEO) and ketoprofen were electrospun into bers [184]. As expected, the electrical conductivity of the bers was increased with the addition of MWCNTs. In the absence of voltage application, in vitro skin permeation of ketoprofen was slightly lowered with the introduction of MWCNTs blocking available space for diffusion. Under electrical stimulation, however, CNTs accelerated the effect of electric voltage, leading to higher drug release. The CNT-laden ber also did not affect the viability of mouse L929 broblasts, suggesting the biocompatibility of such transdermal DDS. Other than transdermal delivery, prolonged and sustained release of NSAIDs can also be achieved with the use of membrane technology, where a drug-core is surrounded by layers of membranes that control drug release via diffusion. Oxidized MWCNTs containing semipermeable cellulose acetate polymer membrane was used to coat an indomethacin osmotic pump tablet system, and the effect of CNTs on the release characteristics of indomethacin was investigated [185]. At 0.01% w/w of CNTs against cellulose acetate, higher amount of indomethacin was released in a zero-order release pattern, as a result of increased membrane porosity and higher extent of water uptake attributed to the presence of oxidized CNTs with hydrophilic groups. Higher concentration of CNTs however failed to form homogenous and porous membranes because of aggregation and reduction in the effective surface area of CNTs. From the result of this study, it is once again suggested that there exists an optimum concentration of CNTs to be used as an additive to modulate drug release. Excessive amount of CNTs could be counter-productive, especially if they aggregate and form bundles with reduced surface area. 3.3. Antihypertensives Treatment of hypertension typically involves oral administration of a cocktail of different antihypertensives with varying mechanisms of action. Examples of the different classes of antihypertensives include diuretics, angiotensin converting enzyme inhibitors, angiotensinogen receptor blockers, blockers and calcium channel blockers. In addition to lowering blood pressure, some of these antihypertensives are also useful in the treatment and management of other cardiovascular diseases, such as angina, ischemic heart disease and heart failure. While these agents are typically administered orally, increasing attention has been devoted to the development of novel DDSs that can prolong the drug's half-life and enable facile administration with reduced frequency to improve patient compliance. One way to achieve such aim would be through the development of transdermal DDSs, where drugs can be released from a reservoir in a sustained and controlled manner topically. A transdermal composite membrane consisting of PVA and carboxylated MWCNTs was prepared and loaded with diltiazem hydrochloride, a water soluble non-dihydropyridine calcium channel blocker [186]. Incorporation of CNTs increased the tensile modulus, ultimate tensile strength, percent elongation and viscosity of the composite membrane, resulting in low bursting tendency and sustained release of diltiazem when tested
with a Franz ow diffusion cell. In addition, CNTs were also able to adsorb diltiazem and retard its release. Further increase of CNT concentration past 1% w/w, however, led to poorer membranes' mechanical properties and performance due to the formation of CNT aggregates. Another way of achieving controlled and sustained release of highly water-soluble drugs is through microencapsulation. Metoprolol tartrate, a water soluble antihypertensive belonging to the class of blocker, was chosen to be the model drug to formulate a sustained release polymeric microparticulate formulation with ethyl cellulose (EC) microsphere impregnated with MWCNTs via solvent evaporation technique with acetone [187]. The ability for metoprolol to be homogenously adsorbed onto CNTs minimized leaching of the drug from EC, leading to elevated drug loading and also sustained drug release prole up to 24 h in CNT-laden EC microspheres compared to plain EC microspheres. In both cases of diltiazem and metoprolol delivery aided by CNTs, however, efcacies of the DDS were not demonstrated and no comparisons with existing oral therapies were made. In addition, there was also a lack of toxicity studies conducted on the two DDSs developed. Long-term safety evaluation is critical, especially for antihypertensives where chronic drug intakes are required. Candesartan cilexetil and diltiazem hydrochloride, belonging to the drug class of angiotensinogen receptor blockers and nondihydropyridine calcium channel blockers respectively, were noncovalently loaded successfully onto MWCNTs by nanoprecipitation [188]. The differences in hydrophobicity of the two drugs led to different release proles, where hydrophobic candesartan cilexetil demonstrated slow release of only 20% while highly water soluble diltiazem hydrochloride was released rapidly to almost 90% within 15 h. Unlike the previous antihypertensives, carvedilol is a water insoluble blocker, and its drug loading mechanism was investigated on MWCNTs [189]. Three different methods of non-covalent attachment, namely the fusion method, the incipient wetness impregnation method and the solvent method, were explored, with each method yielding different drug loading, distribution and dissolution results. Carvedilol was distributed both on the surface and inside of CNTs using the fusion method, with higher drug content at the nozzles. The physical states of carvedilol also changed with increasing drug loading concentration, transitioning from a mixture of molecular and amorphous forms to crystalline layers on the exterior of CNTs when the maximum drug loading for carvedilol was exceeded. In the incipient wetness impregnation method, molecular monolayers of carvedilol were formed both inside and outside individual MWCNTs, although fewer drugs were distributed in the nanotube center due to rapid solvent evaporation. Finally, the solvent method permitted sufcient time to ll the interior of CNTs via capillary forces, thus resulting in more uniform intratubular distribution of carvedilol. Again, no efcacy or toxicity studies were conducted on these systems.
3.4. Antioxidants With the ability to scavenge free radicals, antioxidants are a class of compounds that can reduce cellular oxidative stresses related to aging and a variety of human disease states, such as Alzheimer's disease, Parkinson's disease, cardiovascular diseases and cancers. Co-delivery of antioxidants with CNTs is particular relevant in mitigating some of the nanotoxicological concerns associated with SWCNTs, specically antioxidant depletion and ROS generation [190,191]. Tocopheryl PEG succinate (TPGS) is a synthetic water-soluble precursor of the natural antioxidant -tocopherol (Vitamin E), commonly used as surfactant to enhance the solubility of drugs in pharmaceutical industries. In a study by Yan et al., TPGS was found to be capable of dispersing both MWCNTs and SWCNTs better than the conventional nonionic surfactant triton X-100. TPGS formed amorphous coating on the CNT surface after drying, indicating the possibility for such system to be a potential delivery system for Vitamin E [192].
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Quercetin and rutin, belonging to the avonoid class of antioxidants, were co-precipitated with SWCNTs with ultrasonic eld and the antioxidant ability of the biohybrid conjugates were evaluated with chemiluminescence assay with luminol and hydrogen peroxide [193]. While the SWCNT biohybrids produced greater antioxidant ability than their respective avonoid counterparts, the method of determining avonoid loading was not mentioned in the article. Intriguingly, plain SWCNTs without any avonoid attached also demonstrated, albeit lower, antioxidant property, an observation that is contradictory to previous reports of oxidative damage induced by SWCNTs [190,191]. With the ability to scavenge free radicals, gallic acid is a polyphenolic antioxidant that has been used in food and cosmetics to prevent rancidity of fats and oils. Covalent attachment of gallic acid onto pristine MWCNTs by free radical grafting with hydrogen peroxide and ascorbic acid as biocompatible redox initiators pairs was conducted to form an antioxidantCNT bioconjugate [194]. With a gallic acid loading of around 2 mg per g of CNTs, the conjugate retained the antioxidant activity of gallic acid and demonstrated signicant higher ability to scavenge 2,2-diphenyl-1-picrylhydrazyl, hydroxyl and peroxyl radicals than non-gallic acid loaded CNT control. The biocompatibility of the antioxidantCNT conjugate was also assessed with hen's egg testchorioallantoic membrane (HET-CAM). The conjugate was found to be non-irritant and non-inammatory as no signicant hemorrhage, intravasal coagulation or lysis of blood vessels were observed on the membrane after applying the construct for 5 min and washing with normal saline. Interestingly, in addition to its ability to act as an antioxidant, the CNTgallic acid conjugate also possessed good activity at inhibiting cholinesterase, which may be benecial for the treatment of Alzheimer's, where ROS damage is already contributing to the pathogenesis of the disease. Despite the successful demonstration of the conjugate's chemical functionality, no actual biological in vitro efcacy studies on the conjugate have been conducted. Furthermore, the length of the MWCNTs employed in this study, as they are not oxidized, was in the range of 1030 m, which might be too long for efcient cell entry.
perhaps antibiotics or anti-inammatory agents, for specic intestine or colon delivery. 4. Progress of in vivo research on CNT-based drug delivery systems Even though many of the previously discussed CNT-based DDSs have shown encouraging results in various in vitro cell based assays, it is still necessary and important to conduct proper in vivo efcacy and toxicity studies to better evaluate the clinical roles of these DDSs in treating cancer and non-cancer related conditions. 4.1. Anticancer drugs 4.1.1. Topoisomerase inhibitors The covalent complex formed between HCPT and MWCNTs with diaminotriethylene glycol linker developed by Wu et al. was tested in vivo in hepatic H22-bearing mice to assess its biodistribution, efcacy and toxicity [48]. To conduct the biodistribution study, the CNT construct was rst labeled with radioactive nuclide technetium-99m and injected into the tail vein of mice. Single photon emission computed tomography imaging showed that the complex was rapidly distributed throughout most tissues via blood circulation, with stronger signals coming from liver, spleen, lung and heart and weaker signals generated from intestine, skin and muscle. Comparable biodistribution pattern were seen with ex vivo gamma scintillation counting of selected organs. Similar biodistribution results were also observed in healthy non-tumor bearing mice. Interestingly, no signicant changes in radioactive intensity were observed over time, indicating slow excretion and possibly prolonged circulation time for the complex. In fact, the half-life of the CNTHCPT hybrid was estimated to be around 3.6 h, which was significantly longer than that reported for HCPT of only 30 min. Even though the biodistribution of the hybrid was not specic due to the absence of targeting moieties, the CNT construct was found to be enriched in tumors over the entire experimental duration of 22 h. In terms of efcacy, the CNTHCPT hybrid exhibited the best tumor growth inhibition in a dose-dependent manner that was almost twice of free HCPT injection at equivalent dosages, and induced more necrosis in resected tumors. As for the construct's toxicity prole, no signicant weight loss and pathological lesion were observed in lung, kidney, liver and spleen, suggesting at least the absence of overt short term toxicity over 15 days. However, long-term toxicity would have to be evaluated to better assess the clinical potential and safety prole of this CNT conjugate. Having developed a DDS consisting of branched PEG-functionalized SWCNTs non-covalently loaded with DOX, Liu et al. went on to investigate the in vivo biodistribution and efcacy of the DDS in severe combined immunodecient (SCID) mice with Raji lymphoma xenografts with weekly intravenous administration [57]. Compared to free DOX, the circulation half-life of DOX delivered by SWCNTDOX was lengthen by almost 10 fold, with higher total area under curve and doubling of uptake by tumors. The improved circulation half-life demonstrated by the construct could be attributed to the inability of the large size SWCNTDOX construct to be ltered through kidney glomeruli, and the presence of branched PEG which slowed down recognition by opsonins and diminished RES clearance by macrophages. Increase in circulation time then facilitated tumor uptake by enabling repeated passing of the CNT construct through tumor vasculature via the EPR effect. In terms of tumor suppression, at 5 mg/kg of equivalent DOX concentration, the CNT construct inhibited tumor growth to a greater extent compared to free DOX, but failed to reach partial tumor regression that was otherwise achieved by Doxil, a liposomal formulation of DOX. On the other hand, at the same concentration of 5 mg/kg DOX, mice treated with DOX and Doxil experienced signicant weight loss and higher mortality, which was drastically different from the SWCNTDOX treated group where no sign of toxicity and mortality was observed. Due to the lack of toxicity observed, higher doses of DOX were tried. Further
3.5. Other non-anticancer drugs As a naturally occurring neutrotransmitter, acetylcholine plays an imperative role in the process of signal transmission between neurons, both in the central and peripheral nervous system. Deciency of acetylcholine could potentially lead to the development of Alzheimer's disease, a neurodegenerative disease affecting memory and cognition. Since it is difcult for hydrophilic acetylcholine to cross the hydrophobic BBB, there is therefore a need for novel strategies to deliver acetylcholine across BBB and into brain effectively. SWCNTs non-covalently loaded with acetylcholine (around 200 mg/g of CNT) were able to penetrate BBB and effectively reverse learning and memory loss in kainic acidinduced Alzheimer's mice model [195]. Theophylline is a xanthine derivative commonly used for the treatment of respiratory diseases. Using it as a model drug, theophylline was incorporated into hybrid microsphere comprising alginate and CNTs [196]. Incorporation of CNTs conferred greater mechanical resistance and stability to the microspheres. In addition, introduction of CNTs also enhanced drug loading, diminished drug leakage and slowed down the rate of theophylline release from the hybrid microspheres. Lastly, the CNT/alginate microspheres also lacked signicant cytotoxicity on mouse broblasts L929, achieving similar cell viability as neat alginate microspheres. While it was suggested that such delivery system was able to deliver drugs specically to intestines and colon, as the microspheres are stable in acidic pH and only swell and release drug cargo in neutral pH, there is no mention of the relevance of such targeted delivery with respect to theophylline and any reasons for the choice of theophylline as a model drug in this study. Nonetheless, the results of this study could serve as a reference for future exploration of other drugs,
2008
improvement in tumor growth inhibition was observed for SWCNT DOX at 10 mg/kg, albeit still less efcacious than Doxil, without any signicant toxic effects. Overall, the construct demonstrated comparable antitumor activity with greater selectivity and safer toxicological prole as compared to free DOX. More studies, however, are still needed to compare the efcacy and toxicity of SWCNTDOX and Doxil at various doses. Long term monitoring of toxic effects, especially cardiotoxicity, should also be conducted to assess the safety prole of this DDS. SWCNTs non-covalently modied with Cremophor EL (polyoxyl 35 castor oil) and DOX were investigated for their in vivo biodistribution and efcacy in mice inoculated with S180 mouse sarcoma cells [72]. Longer drug retention and higher drug accumulation (27.6 fold higher) in tumors, as well as correspondingly lower DOX accumulation in other solid tissues like liver, spleen, lung and heart, were observed after intratumoral administration of the CNT construct as compared to free DOX. The elevated intratumoral DOX level subsequently translated to higher tumor growth suppression of 1.5 fold of free DOX, without causing overt toxicity to other organs based on histological observations. The mechanism accounting for the tumor specicity of such construct without any targeting group was not expounded, but can be postulated to be due to the EPR effect [40]. However, the intention or advantage of using Cremophor EL as a dispersing agent for CNTs was not clearly stated by the authors. This is especially relevant because clinical usage of Cremophor EL-containing drug formulation has been associated with severe anaphylactic hypersensitivity reactions, and had since fallen out of favor as a pharmaceutical excipient [197]. With targeting purposes in mind, a HA-tethered MWCNTs supramolecularly loaded with DOX was tested intravenously for its in vivo biodistribution in Ehlrich ascites tumor (EAT) bearing mice model, and its efcacy and toxicity in Sprague Dawley (SD) rats with chemically induced mammary tumors [73]. As EAT bearing mice is a well-established model for HR CD44 overexpressing tumors, the biodistribution and the targeting ability of HA-MWCNTs with additional 99m Tc radiolabel was assessed in this mice model. The result indicated that HA-MWCNTs could accumulate in tumors with higher efciency than free DOX and MWCNTs without HA functionalization. In terms of in vivo efcacy and toxicity, the HA-targeted construct was found to be the most effective in inhibiting tumor growth followed by a nontargeted construct and lastly free DOX. No overt toxicity to heart, kidney and liver were detected for the HA-tethered MWCNTDOX construct, which was conversely apparent in free DOX. Overall, delivery of DOX with MWCNTs functionalized with HA was able to selectively target tumors and raise the potency of DOX without causing signicant toxicity. Having said that, the targeting ability of the construct could actually be further substantiated by evaluating its pharmacokinetic and pharmacodynamic proles in other in vivo tumor models, especially those without HR overexpression. Using FA instead as the targeting moiety, FA-functionalized CHIcoated SWNCTs with supramolecularly attached DOX demonstrated greater degree of tumor inhibition than free DOX at equivalent of 5 mg DOX/kg in nude BALB/c mice inoculated with SMMC-7721 after intravenous administration [79]. Analysis of mice body weight, blood count, serum biochemical markers and histology of liver and kidney revealed lack of in vivo toxicity for this DDS. This observation was in stark contrast to that of free DOX, where mice experienced signicant weight loss after just 1 injection, increased platelet and aspartate aminotransferase level and gross histological changes to liver and kidney. Cardiotoxicity, which is one of the most notorious adverse effects of DOX, was regrettably not assessed in this study. Targeted delivery of DOX to brain glioma with PEGylated oxidized MWCNTs modied with angiopep-2 was investigated in vivo using BALB/c mice xenografted with C6 glioma cells [80]. Mimicking the results of the in vitro uptake of this construct in BCEC and C6, angiopep2 modication was able to equip MWCNTs the ability to target both brain and glioma. Higher uorescence intensity of DOX observed in the brains of mice injected intravenously with angiopep-2-modied
PEGylated CNTs loaded with DOX was observed as compared to free DOX and a non-targeted construct without angiopep-2, possibly due to enhanced receptor-mediated endocytosis arising from interaction between angiopep-2 and LRP. Improvement of DOX delivery and accumulation in brain and glioma consequently led to prolonged mean survival time of xenografted mice from around 33 days in free DOX to 43 days. While the increase in mean survival time was considered statistically signicant, the question of whether it can be considered clinically signicant remained to be answered. Having said that, the construct, in comparison with DOX, exhibited reduced renal and pulmonary accumulation and decreased myocardial ber rupture. In vivo results of CD68 immunohistochemical staining and hematology analysis also indicated the lack of toxicity for this construct and its potential biocompatibility. In vivo efcacy study on SWCNTs attached with pyreneDOX covalent complex was conducted in C57/Bl/6 mice subcutaneously inoculated with B16-F10 melanoma cells [99]. It was revealed that this CNTbased DDS was able to attain similar reduction in average tumor volume without causing severe weight loss like in the case of free DOX. Other toxicity parameters such as organ tissue histology and blood tests were however not assessed in the study. In another approach of covalent conjugation, PL-branched PEG functionalized SWCNTs esteried with pirarubicin exhibited superior efcacy in vivo in rat bladder cancer model with reduced systemic toxicities to free pirarubicin [104]. Unfortunately, the method of quantifying in vivo efcacy employed in this study of measuring the extent of tumor tissue apoptosis induced by the various test agents 2 days after intravesical administration may not be an accurate reection of the ability of the test agents or DDS to effectively suppress tumor growth, especially over a longer time period. 4.1.2. Platinum-based drugs In vivo studies are inevitable and indispensable in the process of drug discovery in terms of assessing safety and efcacy of bioactive molecules. While intense efforts have been dedicated in developing CNTbased DDSs for Pt anticancer drugs, translation of in vitro ndings into in vivo settings is still at its infancy stage and only a few in vivo rodent studies have been reported. One of the earliest in vivo assessments of Ptbased CNT DDSs was a continuation to the in vitro investigation of SWCNTCDDPEGF reported by Bhirde et al. [116]. After demonstrating that SWCNTCDDPEGF selectively targeted EGFR and displayed superior in vitro efcacy to free CDDP, the authors proceeded to investigate in vivo tumor targeting of SWCNTCDDPEGF in head and neck squamous carcinoma cells (HN12) xenograft mouse model. By noninvasive two-photon video imaging, SWCNTQDEGF could be observed moving along with blood ow, diffusing out from vasculatures within around 20 min after systemic injection and rapidly accumulating within the tumor mass in mice. On the contrary, control constructs of SWCNTQD without EGF were rapidly cleared from blood stream and absent in tumor mass. Tumors from SWCNTQDEGF and SWCNTQD treated mice were analyzed by confocal microscopy and only the bioconjugates containing EGF targeting moiety showed extensive intratumoral accumulation. In addition, tumors from SWCNTCDDP EGF treated mice displayed SWCNT signature G-band with Raman characterization and was conrmed by TEM to contain nanotube-like structures. Such observations were not found in SWCNTCDDP treated mice. Importantly, mice treated with targeted SWCNTCDDPEGF experienced stalled tumor growth at around 500 mg in volume, whereas unguided SWCNTCDDP treated mice demonstrated progressive tumor growth to a volume of 2000 mg at 10 days post rst injection. Short-term biodistribution study revealed that SWCNTCDDPEGF accumulated more at tumor microenvironment at 45 min post-injection compared to non-targeted control, with smaller amount of nanotubes present in spleen, lung, liver, kidney, and heart. Nevertheless, comparison of SWCNTCDDPEGF with clinically relevant nano-formulated CDDP for example Lipoplatin [198] was not substantiated in this study. A comprehensive toxicity evaluation was also lacking.
2009
Hence, in a follow up study, the same group wrapped SWCNTs with PEG5000 by reacting amine-terminated PEG with EDC-activated SWCNTCOOH [199], as PEG is able to improve the water solubility of CNTs and reduce their cytotoxicity after being coated onto nanomaterials for drug delivery [26]. Numerous reports had elucidated the benet of PEG chain decoration on CNTs from a biomedical point of view, for example PEGfunctionalized SWCNTs being able to exhibit longer circulation time, less toxicity and more effective clearance in mice [60]. In addition, PEGylation of SWCNTs had also led to increased CNT dispersibility and hydrodynamic size and decreased immunogenicity [200]. Consistently in this study, PEG-functionalized SWCNTs (SWCNT-PEG) displayed better dispersibility and stability in aqueous solution. After showing that SWCNTs and SWCNT-PEG did not induce early apoptosis at concentration of 0.5 mg/mL after 24 h incubation in vitro, SWCNTs and SWCNTPEG were injected intravenously in mice. While both constructs did not cause toxicological changes in liver, kidney, and spleen as demonstrated by histopathological staining, lung tissues from both constructsinjected mice were observed to contain dark particulate materials likely to be CNTs. In spite of the ndings that the presence of black granular concretions and inltration of mononuclear reactive cells with SWCNT-PEG treatment were less than SWCNT alone, the results still signied the presence of mild tissue reactions and inammations even with PEG functionalization. Intriguingly, Raman analysis of solid excreted feces from mice treated with SWCNT-PEG displayed a slow but persistent decrease of SWCNT Raman G band, suggesting that biliary clearance of SWCNTs was occurring in the mice. On the other hand, Raman G-band of feces from SWCNT-treated mice diminished quickly to zero on day 3, suggesting that the SWCNTs was either excreted rapidly within 1 or 2 days, or had became lodged in various vital organs and were not excreted at all. Nevertheless, the inference made from the in vivo efcacy study in HN12 xenograft mice model is only that EGF targeting is important for tumor regression as PEG-functionalized SWCNTCDDPEGF was more effective than PEG functionalized SWCNTCDDP. Again, long-term toxicity and additional comparison with established CDDP treatment regimen are warranted. 4.1.3. Antimetabolites Delivery of GEM with magnetic CNTs has been attempted by Yang et al. by physically adsorbing GEM onto hydrophilic magnetite NPdecorated MWCNTs (MN-MWCNTs) [31]. The aim of this study was to develop an efcient lymphatic targeted DDS to counter lymphatic metastasis. Using SD rats, the authors reported preferential uptake of MN-MWCNTs into lymph vessels and efcient delivery of GEM specically to lymph nodes under the guidance of magnetic eld, aided also by the EPR effect inherent to most nanocarriers. Subcutaneous injection of the drug-free MN-MWCNTs carrier instigated no adverse local toxicity and showed rapid accumulation to left popliteal lymph nodes, without any presence in major internal organs such as liver, spleen, kidney, heart and lung. Targeted delivery of GEM into lymphatic system was conducted by determining the concentration of GEM in left popliteal lymph nodes and blood at different time points. GEM concentration in lymph nodes was low in the free GEM control group, indicating that GEM was not preferentially distributed to lymphatic system without any drug carrier. On the other hand, high level of GEM was detected in lymph nodes with the application of CNTs carrier and external magnetic eld. While the results of this study indicate that MN-MWCNTs is a very promising nanoplatform for future lymphatic cancer therapeutics, the antitumor efcacy of this system is alas not assessed. In a subsequent paper by the same group, a continuation of the experiments described above was presented with specic emphasis on the construct's anticancer activity [140]. With similar methods described in their previous paper, the authors physically loaded GEM onto magnetic MWCNTs. Using metastatic nude BALB/c mice subcutaneously inoculated with metastatic BxPC-3 cells, the authors reported superior activity of the magnetic MWCNTGEM complex in decreasing metastatic lymph node volume regardless of the use of magnets. In
contrast, free GEM was totally ineffective. MN-MWCNTs with GEM were also compared with magnetic activated carbon particle (mAC) GEM. While the authors claimed that the therapeutic effectiveness of MN-MWCNTGEM was better than that of mACGEM, the difference appeared to be rather insignicant. The toxicity of both MN-MWCNT and mAC loaded with GEM, regardless of application of external magnetic eld, were observed to be similar to that of free GEM. Overall, while the CNTGEM construct was effective in inhibiting tumor growth compared to free GEM, the effect or advantage of magnetic targeting was unfortunately not clearly demonstrated in this study. In vivo biodistribution and PK studies were conducted by Singh et al. for the MWCNTGEM complex with additional FA targeting functionalization on albino rats [141]. At equivalent intravenous GEM concentration, delivering GEM with FA-CNTs resulted in substantially lower amount of GEM being accumulated in major organs like liver, lung, kidney and spleen as compared to free GEM, suggesting preferential release of GEM in systemic circulation rather than solid organs. This observation is slightly in disagreement with the in vitro release data, which suggested higher rate of drug release at acidic lysosomal pH that could only occur intracellularly. Nevertheless, it was observed that the construct was able to prolong the circulation half-life and improve the bioavailability of GEM as compared to free GEM. The role of FAconjugation on the biodistribution and PK prole of GEM was not thoroughly explored as only healthy non-cancerous rats were used for the study. Also, it is noted that no in vivo efcacy or toxicity experiments were conducted in this study. 4.1.4. Antimicrotubules While only comparable efcacy with free PTX (Taxol) was demonstrated in vitro, the water soluble conjugate of SWCNTs non-covalently functionalized with PL branched PEG chains terminally conjugated with PTX via cleavable bonds (as designed by Liu et al.) was subjected to further in vivo efcacy and toxicity testings [32]. Contrary to the in vitro results, in vivo efcacy study revealed increased tumor shrinkages in mice treated with PTXPEG-CNT compared to Taxol and PTXPEG. In female BALB/c mice subcutaneously injected with 4T1, PEGylated CNTs extended the circulation time of PTX and improved its cellular uptake. Higher ratios of tumor-to-normal organ PTX uptake and low toxicity were observed due to rapid clearance of PTXPEG-CNT from the mice by biliary excretion. PEGylated SWCNTs were found to be non-toxic to mice monitored over many months. These advantages make this system a promising carrier for PTX. However, in order to fully predict the clinical role of such delivery system, comparison should be made to the more clinically used microalbumin formulation of PTX (Abraxane) instead of Taxol. PLAPEG-coated MWCNTs loaded with PTX developed by Moore et al. were investigated for their biodistribution pattern, toxicity and potential to trigger inammatory response in vivo using mouse model [157]. It is noted, however, that all these in vivo studies were performed on only the carrier (i.e. PLAPEG-coated MWCNTs) without PTX, and the results were compared to that of pristine CNTs, mainly to elucidate the biological functions and effects of PLAPEG coating. Biodistribution study conducted in BALB/c mice intravenously injected with uorescently-labeled PLA PEG-coated CNTs showed that the carrier was cleared by both renal and RES pathways. Compared to pristine CNTs that accumulated as aggregates in lungs, liver and spleen, coated CNTs could also be found in those organs but without aggregation, as observed under histological staining. The ability for PLAPEG-coated CNTs to induce inammatory response was analyzed following pulmonary administration of both coated and uncoated CNTs in C57BL/6 mice. A lack of inammatory response with MWCNT PLAPEG compared to non-coated CNTs was observed. The polymer coating also increased the CNTs' maximum tolerated dose in BALB/c mice by almost two times. Overall, while the carrier was proven to be relatively non-toxic, which indicates the biocompatibility of such polymer-coated CNTs, the in vivo antitumor efcacy and the ability for PLAPEG coated MWCNTs to deliver PTX are still not demonstrated.
2010
A multimodality SWCNT-based DDS comprising of physically adsorbed DTX, targeting NGR peptide and NIR-induced phototherapy was developed by Wang et al. DTX's PK and biodistribution were studied in healthy mice, while its efcacy and toxicity proles were investigated in murine S180 cancer model in BALB/c mice [49]. Comparing between DTX, SWCNTDTX and SWCNTNGRDTX, the use of CNTs clearly increased the blood circulation time of DTX, with SWCNT NGRDTX showing preferential accumulation in tumors. Intravenous administration of SWCNTNGRDTX demonstrated higher efcacy than free DTX and SWCNTDXT in suppressing tumor growth. Again, similar to the in vitro results, NIR irradiation was able to further improve the therapeutic efcacy of this DDS. In terms of toxicity, no signicant toxicities to the various harvested organs were detected by histological observations. This SWCNTNGRDTX DDS may be promising for high treatment efcacy with minimal side effects in future cancer therapy. 4.1.5. Other anticancer drugs The triple-functionalized SWCNT-based DDS consisting of thalidomide, cyclic RGD and uorescent Rh tag designed by Cheng at al. was subjected to a variety of in vivo biological studies in zebrash embryo models to investigate the angiogenic potential of this nanoconjugate [165]. Biodistribution conducted in wild-type zebrash embryos by microinjection showed that the carrier RhSWCNTRGD (without thalidomide) was mainly distributed to blastoderm, a region where most important cellular development events take place, but not yolk. While this result is relevant in evaluating the in vivo biological effects of the CNT construct in embryos, it reveals no information regarding the biodistribution of the construct in mature animals and its pattern of organ accumulation. Transgenic zebrash embryos with endothelial cells capable of producing green uorescent proteins were used to study the antiangiogenic ability of this construct. RhSWCNTRGD was shown to co-localized with articially induced wound site in transgenic zebrash embryos, presumably due to the ability for RGD to recognize integrin overexpressed on activated endothelial cells during wound healing. However, this experiment was not repeated on CNTs without RGD, hence it was not certain if RGD indeed afforded the targeting ability. After demonstrating that covalent attachment of thalidomide on CNTs did not eliminate the ability for thalidomide to inhibit angiogenesis of normal blood vessels, xenograft angiogenesis assay was conducted to evaluate the anticancer properties of this construct. Compared to control embryos injected with only Matrigel, HT1080, being a proangiogenic mammalian tumor cells, demonstrated increased ectopic subintestinal vessel angiogenesis. Co-administration with the nanoconjugate was able to inhibit this ectopic angiogenesis. No valid comparison, however, was made to free thalidomide and RGD-free construct. Thus, it is not possible to ascertain if the antiangiogenic potential of thalidomide was modulated by CNTs and/or RGD targeting peptide. Recently, a multimodal HA-derivatized SWCNT-based delivery system for a photosensitizer, HMME, amendable for both PDT and PTT was developed by Shi et al. and tested in vivo against female C57 mice subcutaneously xenografted with B16-F10 cells [173]. Similar to the in vitro ndings, the combination of both radiation at 532 and 808 nm resulted in the most drastic decrease in tumor volume by inducing both PDT and PTT. The enhanced efcacy was attributed to a few reasons, namely the ability for CNTs to carry more HMME to tumor sites via the EPR effect, the targeting ability of HA against HR-expressing B16-F10 xenografts and potential synergism between PTT and PDT. In addition, no signicant toxicities, such as weight loss or histological changes to vital organs, were detected. 4.2. Non-anticancer drugs The antileshmanial activity of amine-functionalized MWCNTs covalently conjugated to AMB was assessed in vivo after demonstrating superior activity to free AMB in vitro against intramascrophage amastigotes [174]. The construct was more effective in inhibiting
amastigote replication in Syrian golden hamster than free AMB, and demonstrated lack of renal and hepatotoxicity in healthy BALB/c mice. Despite having demonstrated both in vitro and in vivo efcacy of the MWCNT AMB conjugate, the authors had failed to address several critical issues. For instance, the authors did not provide any explanation for the negligible in vivo efcacy of amine-functionalized CNTs without any AMB, where the same construct was found to be potent in inhibiting the growth of amstigotes in vitro with a potency that was only a fold lower than that of AMB conjugate. Subsequent study by the same group revealed that the same conjugate was orally active against experimental visceral leishmaniasis in hamsters [201]. At a dose of 15 mg/kg for 5 days, oral administration of the CNT conjugate was as effective as intraperitoneal administration of liposomal AMB formulation and the CNT conjugate at 5 mg/kg. More impressively, the CNT conjugate was found to possess greater potency than the only available oral antileishmanial agent, miltefosine, at equivalent dose. Further study on safety and PK prole, however, are still needed to establish the role of this MWCNTAMB conjugate in treating leishmaniasis. In another study where AMB was physically adsorbed on mannosylated MWCNTs for targeted delivery to macrophages, intravenous administration of the construct in albino rats experienced preferential disposition in macrophage-rich organs, like liver and spleen, and lower nephrotoxicity compared to free AMB [175]. The antileishmanial efcacy of the construct, however, was not investigated in the study. SWCNTs non-covalently loaded with acetylcholine were able to accumulate in the lysosomes of neurons after BBB penetration following gastrogavage and demonstrated superior effectiveness in Alzheimer's mice model to free acetylcholine [195]. While administration of the CNT construct at doses under 300500 mg/kg caused insignicant changes in mice body weight, blood count, liver and renal functions, in vitro toxicological studies indicated the occurrence of overt mitochondrial and lysosomal damages at doses of 400500 mg/kg on the cellular level. Moreover, since the in vivo toxicological study was only conducted over a relatively short time period, the toxicity nding of this article may hence not be able to accurately reect actual clinical management of Alzheimer's disease where chronic daily therapy is needed. There lies a possibility for any clinically signicant adverse effects of the CNT-based DDS to manifest only after prolonged and repeated application. Furthermore, the excretion prole of this CNT carrier was also not assessed, leaving one to ponder if they can be cleared from brain after delivering acetylcholine. A detailed PK study focusing on the clearance of CNTs and a long term in vivo toxicological study are therefore in order, especially since the toxicities of non-functionalized SWCNTs still remain controversial, for one to accurately evaluate the clinical applications of this CNT-based DDS. 5. Concerns regarding CNT-based drug delivery systems As CNT interaction with living systems has not been fully elucidated, imbalances between wide-spread commercial applications and proper regulation might pose serious threats to environmental issues and human health. Combining the immense interest of developing CNTs as potential DDSs for biomedical applications, it is therefore imperative to investigate and scrutinize their effects on organisms' viability. Over the past decades, numerous reports have been published on the interactions between CNTs with cells in vitro in terms of uptake [42,202204], intra-cellular distribution [205207], potential expulsion [208,209] and even destruction/metabolism [210,211]. However, with current technologies, it is impossible to precisely control every parameter (i.e. length, diameter, surface defects, etc.) during CNTs production. Therefore, huge dissimilarities pertain among CNT batches employed in different research groups, and drawing conclusions from cytotoxicity ndings of diverse CNT materials becomes challenging. Although debates are on-going with concerns on CNTs' long-term biocompatibility, reports of CNTs' toxicity should be taken into consideration carefully when designing CNTs as drug carriers: this is because
2011
it has been demonstrated that the biodistribution, long-term fate and toxicity of CNTs are closely associated with their surface chemistries, sizes, doses, and administration routes [60,63,212215]. For example, it is generally recognized that pristine (i.e. non-functionalized) or functionalized CNTs intratracheally (IT) instilled into animals cause pulmonary toxicity including unusual inammation and brotic reactions stemming from aggregation of CNTs in airways [216220]. These results suggest that aerosol exposure of CNTs in workplace should be avoided to protect human health. Nevertheless, toxicities observed by intratracheal instillation of CNTs might not be relevant to some of the CNTs' biomedical applications, where highly functionalized, water-soluble CNTs are employed and administrated through intraperitoneal (IP) and intravenous (IV) injections rather than intratracheal instillation, i.e. lung airways are not exposed to CNTs. There are also widespread concerns for biomedical applications of CNTs as CNTs are particularly strong in structure and intensive and systematic studies on possible degradation by cells, especially macrophages, hepatocytes, and kidney cells, are lacking. Hence, the risk is that, once CNTs are administered into human body, long-lasting scavenging and inammatory activities towards this non-degradable or less bio-degradable nanomaterial might result in continuous oxidative stress at their deposited sites. This determines a greater tendency for tissue destruction or carcinogenesis. Intriguingly, despite discrepancies in ndings on the clearance mechanism of nanotubes, majority of the studies have suggested that functionalized CNTs, when intravenously injected into animals such as mice or rats, are prone to accumulate in the RES, for example liver and spleen, and then gradually excreted, possibly via both fecal and renal routes [6062,130,199,221,222]. Generally, it is recognized that larger and pristine CNTs, which are hardly suspendable in aqueous solution, would form bundles and aggregates that induce inammation and granuloma formation [58,223], whereas such toxicities are not observed with smaller and individualized CNTs [130,216,220,221,224]. In addition, highly-functionalized CNTs with well-known biocompatible moieties (such as PEG) have demonstrated reduced in vivo toxicity after being intravenously injected into animals as compared to their raw, un-functionalized counterparts [52,60,215]. Nevertheless, issues of CNTs' effects on reproductive functions and elevated immune responses have only been partially addressed [90,225227] and it is imperative to further investigate these two areas comprehensively before advancing CNTs into clinical stage as drug carrier (please refer to another article in the issue for more details on the toxicity of CNTs). 6. Conclusion & future perspectives The use of CNTs as novel DDSs has been widely investigated with encouraging results. Several characteristics of CNTs, such as high aspect ratio and surface area, ability to attach additional molecules for targeting or imaging purposes, elevated cellular internalization and preferential tumor accumulation, have made CNTs versatile and effective drug delivery carriers for many small molecule drugs, including drugs for both cancer and non-anticancer indications. Generally, there are 4 ways in which CNTs can be loaded with drugs and be used for drug delivery. Firstly, drugs can be chemically functionalized either permanently or via cleavable linkers onto the surface of CNTs. Secondly, drugs that possess conjugated aromatic ring systems are able to physically adsorb onto the surface of CNTs via non-covalent and hydrophobic interactions. Thirdly, some drugs can be encapsulated within the interior cavity of CNTs. Lastly, instead of being the main carrier, CNTs can also be incorporated into other parent DDS, for instance hydrogels or electron spun bers, as adjunct material to modulate the loading and release of co-encapsulated drugs. In order to further enhance the therapeutic efcacy and diminish the inherent cytotoxicity of CNTs, other molecules are used to functionalize CNTs. Polymers such as PEG and CHI are popularly used to impart water dispersibility to CNTs and offer additional sites for conjugation of other
molecules. Some CNTdrug constructs are tethered to targeting molecules, be it small chemical molecules, complex biological entities or even magnetic NP, to selectively target certain cells or tissues. Transmembrane uptake and intracellular distribution of CNTdrug complexes can also be systematically studied with additional imaging probe functionalization. In vitro studies conducted on the various CNTdrug constructs developed predominantly supported the use of CNTs as drug delivery carriers. Enhanced cellular uptake, improved potency and selective targeting were often demonstrated for CNTdrug complexes compared to their respective free drugs. In spite of their lower number, in vivo studies performed (mostly on mice) also lent considerable credence to the superiority of CNT-based DDSs. CNTdrug complexes generally exhibit preferential uptake and distribution into tumor or other target tissues, increased therapeutic efcacy and most importantly negligible toxicity. One thing to note, however, is that most studies only compared the effectiveness of CNTdrug constructs with their respective free drugs, with only a few attempted to make head-on comparison with their clinically available nanoparticulate formulation counterparts (such as Doxil, Lipoplatin, Abraxane and AmBisome). Despite the various successes and promising potentials that CNTbased DDSs have demonstrated in vitro and in vivo, there still exist a few concerns regarding their clinical applications. Most notably, the issues of CNT toxicity still remain contentious to this date. Long-term toxicity studies on CNT carriers are also lacking, as most toxicity studies conducted in vivo were relatively short course. Depending on the surface functionalization and the physical attributes of CNTs, different toxicity ndings can be obtained. Another issue limiting the clinical application of CNTs lies in the ability for CNT-based DDSs to liberate drugs adequately and controllably. Some CNTdrug complexes, for instance, were found to be less active than their respective free drugs due to uncontrollable and low rate of drug release. Lastly, as CNTs are inherent heterogeneous, it is thus challenging to ensure the uniformity and batch-to-batch consistency of fabricated CNTdrug complexes. These only represent some of the challenges that need to be addressed in order to advance CNTs into actual clinical settings for the delivery of small molecule drugs and to become commercially viable. Acknowledgment This research has been supported by the National University of Singapore, Department of Pharmacy ((AcRF) Tier 1-FRC grant R-148000-164-112) and by MOE of Singapore (grant MOE2009-T2-2-011, R-398-000-068-112). The authors G.P., H.K.H. and W.H.A. also acknowledge support from A*STAR SERC TSRP-Integrated Nano-Photo-Bio Interface grant (Project Number: 102 152 0016). References
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Carbon Nanotubes For Delivery of Small Molecule Drugs

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Advanced Drug Delivery Reviews 65 (2013) 19642015

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Carbon nanotubes for delivery of small molecule drugs

B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015

Covalent conjugation via ester linkage

Uptake & cytotoxicity in MKN-28

Oxidized MWCNTs functionalized with PVA

Cytotoxicity in MDA-MB231 & A-5RT3

Oxidized MWCNTs coated with Pluronic P123

Uptake & cytotoxicity in HeLa

MWCNTs with open tips

Acidic pH due to CHI disruption

EGF against EGFR

Uptake & cytotoxicity in A549

SWCNTs functionalized with PEG with cyclic RGD

Acidic pH CNT diameter

Cyclic RGD against integrin v3

Uptake & cytotoxicity in U87MG & MCF-7

MWCNTs dispersed with Pluronic F127

SWCNTs functionalized with branched PEG

Antibody against CEA

(continued on next page)

B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015

SWCNTs covalently linked to P-gp antibody labeled with FITC

Antibody against P-gp

Uptake & cytotoxicity in K562 sensitive & resistant cell lines

SWCNTs functionalized with FA-conjugated CHI

Acidic pH disruption of CHI

MWCNTs dispersed with PEG-PSS labeled with FITC

Uptake in HeLa. Cytotoxicity in MDA-MB435

MWCNTs functionalized with FA-hexamethylenediamine conjugate & iron NP.

FA against FR Iron oxide NP for magnetic targeting

Uptake & cytotoxicity in HeLa

Acidic pH Serum protein Shorter loading time

SWCNTs functionalized with branched PEG 2500-NH2 & FA

Acidic pH Serum protein

Uptake & cytotoxicity in HeLa

B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015

PAA grafted MWCNTs functionalized with FA & iron NP

Acidic pH Iron oxide NP for magnetic targeting

Uptake in U87, cytotoxicity in U87 & 3 T3

EDBE-conjugated MWCNTs covalently functionalized Physical adsorption with HA

Uptake & cytotoxicity in A549

(continued on next page)

Transferrin against Iron NP for magnetic targeting

Uptake in HeLa & HEK 293 Cytotoxicity in HeLa

B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015

CHI-coated SWCNTs covalently functionalized with FITC

CHI-coated SWNCTs chemically attached with FA

PEGylated oxidized MWCNTs modied with angiopep 2

Angiopep 2 peptide against LRP receptors

Uptake & cytotoxicity in C6 & BCED

B.S. Wong et al. / Advanced Drug Delivery Reviews 65 (2013) 19642015

Amine-MWCNTs conjugated covalently with DEX mesylate

DEX mesylate for nuclear targeting

Uptake & cytotoxicity in A549

SWCNTs non-covalently functionalized with FAterminated methoxy-PEG

Uptake & cytotoxicity in HeLa & 3 T3

MWCNTs linked with EDBE conjugated with FA, HA or -estradiol-17-hemisuccinate

FA against FR HA against HR ES against ER

Uptake & cytotoxicity in A549, HeLa & MCF-7

(continued on next page) 1971