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Drug-delivery systems with nanoparticles show a clear potential for cancer treatments. The idea of directing a therapeutic agent to the proximity of a damaged tissue was postulated by the Nobel Prize winner P. Ehrlich already in 1906. In the u.s. Alone the demand is expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007.
Drug-delivery systems with nanoparticles show a clear potential for cancer treatments. The idea of directing a therapeutic agent to the proximity of a damaged tissue was postulated by the Nobel Prize winner P. Ehrlich already in 1906. In the u.s. Alone the demand is expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007.
Drug-delivery systems with nanoparticles show a clear potential for cancer treatments. The idea of directing a therapeutic agent to the proximity of a damaged tissue was postulated by the Nobel Prize winner P. Ehrlich already in 1906. In the u.s. Alone the demand is expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007.
Despite the significant progress in the development of anticancer technology, there is still no common cure for patients with malignant diseases. In addition, the long- standing problem of chemotherapy is the lack of tumor-specific treatments. Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic agents have very little or no specificity, which leads to systemic toxicity, causing undesirable severe side effects such as hair loss, damages to liver, kidney, and bone marrow. Nowadays, drug-delivery systems with nanoparticles show a clear potential for cancer treatments in view of advantages such as, i) the ability of targeting specific locations in the body, ii) the ability of reducing the quantity of drug that needs to be delivered to attain a particular concentration level in the vicinity of the target, and iii) the ability of decreasing the concentration of the drug at non-target sites. 1
As a consequence, controlled drug delivery is one of the fastest-growing segments of the pharmaceutical market, and in the United States alone the demand is expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007. 2
1.1.-Preparation of nanoparticles for drug delivery. The idea of directing a therapeutic agent to the proximity of a damaged tissue was postulated by the Nobel Prize winner P. Ehrlich already in 1906 with the concept of magic bullets which were compounds that would have a specific attraction to disease-causing microorganisms. He envisioned that these magic bullets would seek out these organisms and destroy them, avoiding other organisms and having no side effects on healthy tissue. 3
In general, there are two possibilities of localizing a drug in the proximity of a target (i.e., tumor): passive and active. The former implies using characteristic properties of the tumor to locate in its proximity an encapsulated, bonded or adsorbed drug on nanoparticles. That is by using the characteristic enhanced permeation and retention of the vasculature of the tumor (EPR-effect) is possible to concentrate drug-loaded nanoparticles within the tumor tissue. This is because
1 J. Ritter, A.Ebner; K. Daniel, K. Stewart. Application of high gradient magnetic separation principles to magnetic drug targeting. J. Magn. Magn. Mater. 2004, 280, 184. 2 S. K. Sahoo, V. Labhasetwar. Nanotech approaches to delivery and imaging drug. Drug Discov. Today. 2003, 8,1112. 3 T. M. Fahmy, P. M. Fong, A. Goyal and W. M. Saltzman, Targeted for drug delivery, Mat. Today. 2005, 8, 18. 8 tumors develop a leaky vasculature as well as a poor lymphatic drainage. 4 The majority of solid tumors exhibit a vascular pore cut-off size between 380 and 780 nm. 5 The healthy capillary vessels are permeable showing a pore size cut-off depending on the location and kind of blood capillary. The tight-junction capillary (blood-brain barrier), including the central nervous system has exhibited a vascular pore cut-off size < 0.1 nm; continuous capillaries, including most tissues, such as muscle, lung, and skin (cut-off < 6 nm); fenestrated capillaries, including kidney, intestine, and some endocrine and exocrine glands (cut-off < 50-60 nm); and sinusoid capillaries, including liver, spleen, and bone marrow (cut-off 100-1000 nm). 6 Therefore it is possible to synthesize drug-loaded nanoparticles with a tailored size and with a shell that delays or avoids the action of the reticulo- endothelial system (RES) expecting to be naturally located within a specific organ. Other examples of passive targeting imply using specific affinity or physicochemical properties of the tumor tissues. For instance, Kukowska-Latallo et al. 7 used the high affinity folate receptor for the vitamin folic acid as a target for the delivery of folate-conjugated drugs to cancer tissue. Another example of passive targeting with nanoparticles uses pH-dependent drug release due to the tumor has a pH slightly lower than healthy tissues. Most solid tumors have pH values of less than 7.2 8
contrary to the normal blood pH of 7.4 0.05. 9 When these pH sensitive nanocarriers (i.e., polymers, liposomes) encounter acidic environments such as tumor tissues, they break apart and release the molecules they contain. 10,11
Another physical property of tumor tissue is its higher temperature (hyperthermia) compared to healthy tissues 12 and it is considered another utilization of passive
4 M. J. Vicent and R. Duncan, Polymer conjugates: nanosized medicines for treating cancer, Trends Biotech., 2006, 24, 39. 5 S. K. Hobbs, W.L. Monsky, F. Yuan, W.G. Roberts, L. Griffith and V.P. Torchilin, R. K. Jain, Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment, Proc. Natl. Acad. Sci. 1998, 95, 4607. 6 Y. Okuhata, Delivery of diagnostic agents for magnetic resonance imaging, Adv. Drug Delivery Rev., 1999, 37, 121. 7 J. F. Kukowska-Latallo, K. A. Candido, Z. Y. Cao ZY, S. S. Nigavekar, I. J. Majoros, T. P. Thomas, L. P. Balogh, M. K. Khan, J. R. Baker, Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer, Cancer Res., 2005, 65, 5317. 8 O. M. Koo, I. Rubinstein and H. Onyuksel, Role of nanotechnology in targeted drug delivery and imaging: a concise review, Nanomed.: Nanotech. Biology Med. 2005, 1, 193. 9 G. Gaucher, M. H. Dufresne, V. P. Sant, N. Kang, D. Maysinger and J. C. Leroux, Block copolymer micelles: preparation, characterization and application in drug delivery, J. Controll. Release. 2005, 109, 169. 10 T. Ishida, M. J. Kirchmeier, E. H. Moase, S. Zalipsky and T. M. Allen, Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells, Biochim. biophys. acta-biomem. 2001, 1515, 144. 11 D. E. L. de Menezes, L. M. Pilarski, A. R. Belch and T. M. Allen, Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo, Biochim. biophys. acta-biomem. 2000, 1466, 205. 12 V. P. Torchilin, Drug targeting, Europ. J. Pharmac. Sci., 2000, 11, S81. 9 targeting by using temperature-sensitive release nanoparticles. 13 The PathFinder technology is based on the identification of naturally occurring mechanisms for the localization of material (i.e., proteins) indifferent parts of the body and uses these principles for the controlled production of site-specific nanoparticulate carriers. 14
On the other hand, active targeting is based on inducing external properties to the nanoparticles to target them to specific tumor tissues and involves the use of light, magnetism, and the specific recognition mechanisms (i.e., interaction antigen- antibody, peptide-based targeting, DNA or RNA-based ligands, etc.) Magnetic drug delivery is not a new therapeutic tool; in 1978 Senyei et al. 15 used an in vitro analog of the human circulatory system to test the magnetic retention of magnetic microspheres which consisted of adriamycin hydrochloride and ultrafine magnetite encapsulated in an albumin matrix. From then to now, several scientific papers have been published describing different drug-loaded magnetic nanoparticles encapsulated in polymeric or inorganic matrices. 16,17,18 Currently, magnetic nanoparticles are commercially used to treat tumors by hyperthermia and thermoablation (Magforce Nanotechnologies AG, Germany) and by using the magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix , France, Alnis BioScience, USA). The TNT System (Triton Biosystems) consist of polymer-coated iron oxide nanoparticles tagged with an antibody, and an external magnetic field is used to kill diseased cells. Pre-clinical animal models test not show any side effects in healthy tissue. On the other hand, active targeting using the specific interaction antigen-antibody is widely studied 19,20 and currently exist commercially a drug conjugated with an antibody (Mylotarg) used to treat a form of bone marrow cancer (CD33 positive acute myeloid leukemia). Several papers have been published using dendrimers, dendritic polymers and inorganic matrices with a loaded drug and with an antibody functionalized surface.
13 A. Chilkoti, M. R. Dreher, D. E. Meyer and D. Raucher, Targeted drug delivery by thermally responsive polymers, Adv. Drug Delivery Rev. 2002, 54, 613. 14 R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise of nanomedicine, Chem. Eng. Prog. 2006, 102, 35. 15 R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise of nanomedicine, Chem. Eng. Prog. 2006, 102, 35. 16 [16] X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung and S. M. Nie, Magnetic nanoparticle design for medical diagnosis and therapy, J. Mater. Chem. 2004, 14, 2161. 17 A. K. Gupta and M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications, Biomater. 2005, 26, 3995. 18 Z. P. Xu, Q. H. Zeng, G. Q. Lu and A. B. Yu, Inorganic nanoparticles as carriers for efficient cellular delivery, Chem. Eng. Sci. 2006, 61, 1027. 19 E. R. Gillies and J. M. J. Frechet, Dendrimers and dendritic polymers in drug delivery, Drug Discov. Today, 2005, 10, 35. 20 X. Wu and I. Ojima, Tumor specific novel taxoid-monoclonal antibody conjugates, Current Medicnl. Chem. 2004, 11, 429. 10 We can separate the drug-loaded nanoparticles in two groups according to their structure, core-shell and matrix-like nanoparticles. Nanoparticles are defined as solid, submicron-sized drug carriers that may or may not be biodegradable. The term nanoparticle is a collective name for both nanospheres and nanocapsules. Nanospheres have a matrix type of structure. Drugs may be absorbed at the sphere surface or encapsulated within the particle. Nanocapsules are vesicular systems in which the drug is confined to a cavity consisting of an inner liquid core surrounded by a inorganic or polymeric shell. In this case the active substances are usually dissolved in the inner core but may also be adsorbed to the capsule surface. Nanoparticles are receiving considerable attention for the delivery of therapeutic drugs.
1.1.1.-Core-shell based nanoparticles Core-shell nanoparticles have recently attracted a huge scientific effort due to the possibility of combining different properties in individual particles, based on different compositions of the core and the shell. In addition, many interesting technological applications can be foreseen for this kind of materials, including analytical chemistry (chromatography), separation technology (ion exchange), catalysis, biochemistry and medicine, etc. 21 These types of particles can be defined by their different core and shell composition. The core often shows a useful physical property, e.g. semiconductors, metals, magnetic oxides, encapsulated molecules, while the shell can be useful to stabilize the core and make compatible the core and the environment. It is also possible to change the charge, functionality or reactivity of the surface. This is especially important for medical purposes, e.g. for drug delivery applications. We will focus in core-shell nanoparticles composed of a magnetic core encapsulated in an organic (i.e., polymeric) or inorganic shell. Those nanoparticles are based on the use of a magnet to locate them in the proximity of a tumor (active targeting). At this location the nanoparticles can release the conjugated drug which is associated to the magnetic core or an alternating magnetic field is used to induce heat in the magnetic nanoparticles and produce ablation of the tumoral tissue (hyperthermia). In all these applications the coating of the particles is a very important issue. It helps to make the particles biocompatible, preventing aggregation and the degradation of the metallic core, and reducing the extent of
21 G. Kickelbick, L.M. Liz-Marzn, Core-shell nanoparticles, in Encyclopedia of Nanoscience and Nanotechnology, H.S. Nalwa (Ed.), American Scientific Publishers, 2004, Vol. 2, pp. 199-220. 11 clearance by the reticuloendothelial system. Moreover, the outer coating surface of the particles can be functionalized to allow the binding of drugs or biomolecules to the system to cause a therapeutical action or to get local targeting. The magnetic core is composed of 3d metals (iron, cobalt, iron/cobalt alloys, iron/platinum alloys, iron/nickel alloys, and their oxides). The shell is composed of polymeric materials (i.e., dextrane, albumin, starch, etc.) or inorganic materials (ie., silica, graphite and gold). Inorganic, amorphous silica is biocompatible, non-toxic, and posses hydroxyl surface groups (4.6 OH/nm 2 , though this value strongly depends on the synthesis temperature) which provide intrinsic hydrophilicity and allow surface attachment by covalent linkages of specific drugs or biomolecules. Also, amorphous silica is a heat-resisting material, with a low specific gravity, high surface area and good mechanical strength. In addition, the isoelectric point of magnetite ferrofluids is reached at pH close to 7. Aqueous ferrofluids therefore flocculate in the pH range of 5 to 9 and are stable only under highly acidic or basic conditions. On the other hand, the isolectric point of silica is reached at pH 2-3 and therefore silica nanoparticles are negatively charged at the pH of the blood. As consequence, dispersions of silica particles in biological media are stable and prevent magnetite agglomeration. However, the magnetic nanoparticles used for MRI are based on a magnetic core encapsulated in an organic shell (generally dextrane), for example superparamagnetic or ferromagnetic iron oxide nanoparticles are widely used as negative contrast agents in MRI for oral and parental administration (i.e., Feridex, Endorem or Resovist, GastroMARK or Lumirem, Sinerem, etc). Currently, magnetic nanoparticles are commercially used to treat tumors by hyperthermia and thermoablation (Magforce Nanotechnologies AG, Germany) and by using the magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix , France, Alnis BioScience, USA). The TNT System (Triton Biosystems) consists of polymer-coated iron oxide nanoparticles tagged with an antibody, and an external magnetic field is used to kill diseased cells. Pre-clinical animal models test not shown any side effects in healthy tissue when using silica or carbon encapsulated iron and iron oxide nanoparticles. -Silica coated nanoparticles A versatile method for the preparation of core-shell nanomaterials is the growth of the shell material on pre-existing cores by chemical methods. This has been used often for the deposition of silica shells on various nanoparticles, including metallic, 22 semiconductor, 23 or magnetic 24,25 materials. A schematic view of the
22 L.M. Liz-Marzn, P. Mulvaney, The assembly of coated nanocrystals, J. Phys. Chem. B 2003, 107, 7312-7326. 12 synthetic steps followed for silica encapsulation of magnetic nanoparticles in solution is shown in the figure below, comprising coprecipitation of Fe(II) and Fe(III) hydroxides, slow deposition of sodium silicate for thin silica shells, and shell growth in ethanol.
For example, it has been recently demonstrated 26 that semiconductor quantum dots can be assembled on the surface of silica-coated magnetic nanoparticles, so that single particles containing magnetic and luminescent functionalities can be obtained through relatively simple chemical processes.
23 M.A. Correa-Duarte, M. Giersig, L.M. Liz-Marzn, Stabilization of CdS Semiconductor Nanoparticles Against Photodegradation by a Silica Coating Procedure, Chem. Phys. Lett. 1998, 286, 497-501. 24 M.A. Correa-Duarte, M. Giersig, N.A. Kotov, L.M. Liz-Marzn, Control of Packing Order of Self- Assembled Monolayers of Magnetite Nanoparticles with and without SiO2 Coating by Microwave Irradiation, Langmuir 1998, 14, 6430-6435. 25 Y. H. Deng, C. C. Wang, J. H. Hu, W. L. Yang and S. K. Fu, Investigation of formation of silica-coated magnetite nanoparticles via solgel approach, Colloids Surf. A: Physicochem. Eng. Aspects, 262 (2005) (1-3), pp. 87-93. 26 V. Salgueirio-Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marzn, M. Farle, Composite Silica Spheres with Magnetic and Luminescent Functionalities, Adv. Funct. Mater. 2006, 16, 509-514.
EFTEM Green SiO 2 Red Fe EFTEM (708 eV) Fe L 3 Edge EFTEM (22 eV) SiO2 Plasmon
HRTEM images and EFTEM color map showing the Iron (red) atomic distribution (in this case forming the Fe 3 O 4 nanoparticles), and the silicon (green) distribution, as part of the shells. The silica coating was provided by the sol-gel method. (M. Arruebo, R. Fernndez-Pacheco et al. publishing) 13
The assembly of nanoparticles on the surface of silica particles can be achieved trough a technique based on the alternate deposition of oppositely charged species, 27,28 using polyelectrolytes as molecular cement, and can be implemented for the incorporation of drugs or other biomolecules within the nanoparticles. Additionally, the nature of the polyelectrolytes can be tailored, so that they are sensitive to external stimuli, such as pH or temperature, to trigger drug encapsulation and release. Combination of these techniques can be useful to fabricate multifunctional nanoparticles including a magnetic core (for external manipulation), a luminescent, intermediate shell (for labelling) and an external polyelectrolyte shell (for drug encapsulation). The various steps can be schematically represented as shown in the figure below. A B C A B C
-Carbon coated iron nanoparticles Carbon coated iron and iron oxide nanoparticles are suitable for biomedical applications. 29 The inert carbon encapsulation, provide a way to make biocompatible, functionalize and also gives the possibility to adsorb and desorb therapeutical agent as doxorrubicine. 30 These nanoparticles are obtained by two procedures: by the discharge arc method designed by Krtschmer-Huffman in
27 F. Caruso, R. A. Caruso, H. Mhwald, Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating, Science 1998, 282, 1111-1114. 28 V. Salgueirio-Maceira, F. Caruso, L.M. Liz-Marzn, Coated Colloids with Tailored Optical Properties, J. Phys. Chem. B 2003, 107, 10990-10994. 29 Kuznetsov, A. et al , J. Mag. Mag. Mat. 194, 22 (1999). 30 De Teresa J.M., Marquina C., Algarabel P.A., Morelln L,.Fernandez-Pacheco R. Ibarra M.R. Interantional Journal of Nanotechnology (2005) Schematic view of the synthesis of multifunctional nanoparticles. First, magnetic cores are encapsulated in a silica shell, onto which luminescent quantum dots are assembled. Subsequently, a second silica shell is deposited and polyelectrolyte/biomolecule multilayers are deposited. 14 1990, 31 or by high energy ball mill grinding. 32 The Krtschmer method uses a cylindrical chamber, in which there are two graphite electrodes: a stationary anode containing 10 microns starting iron powders, and a moveable graphite cathode. An arc is produced between the graphite electrodes in a helium atmosphere. The graphite electrode is sublimed and builds up a deposit on the inner surface of the chamber. In the material collected from this deposit we found: carbon nanostructures, amorphous carbon and iron and iron oxide nanoparticles encapsulated in graphitic layers. High energy grinding is performed in a ball mill. A suspension of iron micrometric powders and graphite powders in ethanol is grounded for several hours to obtain a viscous solution that is dried up to obtain the final product as fine iron carbon powders.
-Gold coated iron nanoparticles In many cases gold could be the ideal coating material because of its well- known optical properties, 33 easy chemical functionalization with thiolated organic molecules 34 and high stability of the gold-coated nanoparticles in solutions of physiological pH. Recently, gold-coated nanoparticles of about 60 nm in size with
31 Kratschmer W. et al., Nature 347, 354 (1990) 32 Tapolsky, G. et al, Eur. Cells and Materials Vol. 3 Suppl. 2, 12 (2002). 33 T.A. Taton et al. Science 289, 1757 (2000) 34 M. Brongesma, Nat. Mater. 2, 296 (2003)
HRTEM images and EFTEM color map showing the iron (green) atomic distribution (in this case forming the Fe 3 O 4
nanoparticles), and the carbon (red) distribution, as part of the shells. (R. Fernandez- Pacheco et al.) 15 an iron oxide core of 9 nm have been synthesized by a wet-chemical method in two steps. 35 However, metallic iron is expected to be a better core magnetic material than iron oxides for drug delivery and biosensor applications because it is magnetically softer and its saturation magnetization is about a factor two larger than that of iron oxides. Unfortunately, successful preparation of Fe/Au core-shell nanostructures has been a lasting scientific challenge. Several works have reported the synthesis of non-well structural characterized and/or non-long term stabilized nanoparticles, until a novel method combining wet chemistry for synthesis of the Fe cores and laser irradiation of Fe nanoparticles and Au powder in liquid medium has been very recently proposed 36 . In this CONSOLIDER project we aim to study the synthesis of gold-coated metallic and oxidized iron nanoparticles and their feasibility for drug-delivery applications. Au/(-Fe 2 O 3 , Fe 3 O 4 ) core-shell nanoparticles will be prepared by reduction of Au 3+ onto the Fe oxide surfaces of previously synthesized nanoparticles (co-precipitation and further oxidation) using a modification of iterative hydroxylamine seeding procedure 37 . We intend to get a good control of the Au shell thickness, since this layer is responsible of the resulting optical properties, total size and colloidal stability of the core-shell nanoparticles. Gold-coated metallic iron nanoparticles will be synthesized following a two steps method. First, Fe cores will be obtained by thermal decomposition of iron pentacarbonyl in the presence of oleic acid 38 which acts as a surfactant, and further particle precipitation by addition of ethanol. In the second step, we will explore two techniques to achieve gold coating of the iron cores. In the first method, Fe and Au nanoparticles will be dispersed together in a water-based solution, which will be irradiated with a pulsed laser beam in order to promote the selective fusion of the gold nanoparticles and ulterior condensation onto the Fe cores, as described in reference 36 . The second method is based on sonochemistry, which is an alternative technique that can be used for the production of coated particles 39 . Power ultrasound effects provoke chemical changes due to cavitation phenomena involving the formation, growth and implosive collapse of bubbles in liquids.
35 J.L. Lyon et al., Nano Letters 4, 719 (2004) 36 J. Zhang et al., J. Phys. Chem. B 110, 7122 (2006) 37 K. J. Brown et al., J. Chem. Mater. 12, 306 (2000) 38 D. Farrell et al., J. Phys. Chem. B 107, 11022 (2003) 39 V.G. Pol, A. Gedanken and J. Caldern-Moreno, Chem. Mater. 15, 1111 (2003) 16 Sonication of a solution containing a gold precursor in the presence of (metallic/oxidized) iron cores provides an alternative means of trapping gold nanometer seeds, which produce active supported heterogeneous catalysis giving rise to the formation of core-shell nanoparticles. A careful structural characterization of the obtained core-shell nanostructures will be carried out to determine the quality and continuity of the resulting gold coatings which ensure the long-term stability of the iron core.
Work Plan 1.1.1 -Optimization of core-shell nanoparticles with high magnetic response Until now, silica and carbon coated magnetic nanomaterials have been mostly restricted to iron oxides, since these present a better chemical compatibility with the silica shell. The magnetic response of such core-shell nanoparticles can be improved by using other magnetic materials, such as metals (Co, Ni) or alloys (CoPt 3 , FePt) in the case of silica. The formulation of such novel core-shell materials is expected within the duration of the project. -Development of novel multifunctional core-shell nanoparticles The process outlined above for the incorporation of multiple functionalities in a single particle can be extended to other materials. For instance, magnetic cores can be combined with metallic shells, which can be continuous or assemblies of smaller nanoparticles. In either case, the morphology can be tailored for high absorption coefficients in the visible or NIR. -Drug encapsulation in multifunctional nanoparticles For the described multifunctional nanoparticles, drug encapsulation is envisaged via wrapping with polyelectrolyte multilayers. As mentioned above, such multilayers can be designed so that they are sensitive toward pH or temperature, and this can be used to trigger encapsulation and release.
1.1.2.-Matrix-like nanoparticles -Nanoparticles based on polymeric matrices Natural occurring polymers and synthetic polymeric (dendrimers, dendritic polymers and micelles) are used to deliver an adsorbed or encapsulated drug. Their advantages are their biodegradability in many cases and their well-known chemistry. These advantages have made that different research groups and 17 pharmaceutical companies have invested all their efforts in developing drug- conjugated nanoparticles for drug delivery. Hence, interdisciplinary research at the interface of polymer chemistry and the biomedical sciences has produced the first polymer-based nanomedicines for the diagnosis and treatment of cancer. These water-soluble hybrid constructs, designed for intravenous administration, fall into two main categories: polymerprotein conjugates or polymerdrug conjugates. Polymer conjugation to proteins reduces immunogenicity, prolongs plasma half-life and enhances protein stability. Polymerdrug conjugation promotes tumor targeting through the enhanced permeability and retention (EPR) effect and, at the cellular level following endocytic capture, allows lysosomotropic drug delivery. The successful clinical application of polymerprotein conjugates (PEGylated enzymes and cytokines) and promising results arising from clinical trials with polymer-bound chemotherapy (i.e., doxorubicin, paclitaxel, camptothecins) has provided a firm foundation for more sophisticated second-generation constructs that deliver the newly emerging target-directed anticancer agents (e.g. modulators of the cell cycle, signal transduction inhibitors and antiangiogenic drugs) in addition to polymerdrug combinations (i.e., endocrine- and chemo-therapy). First-generation technologies include antibodydrug conjugates, for example Mylotarg (http://www.pharmacist.com/pdf/mylotarg.pdf), and also several polymer conjugates carrying either low-molecular-weight drugs or proteins. Besides these drug-polymer conjugates, biodegradable polymers such as polyesters and polyanhydrides and their copolymers with hydrophilic segments (e.g. PEO, PPO) have been efficiently used for the nanoencapsulation and delivery of drugs. These polymers have the specific advantage of being degraded by a hydrolitic mechanism which leads to the erosion of the nanomatrix and subsequent delivery of the associated drug. Moreover, these polymers have a long safety record as shown by the fact that they are major components of several marketed formulations (Lupon depot, Decapeptil, Gliadel..among others). Recently, the attention has been directed to the use of copolymers of poly(lactic acid/glycolic acid) with PEG. This is due to the ability of these copolymers to organize forming PEG-coated PLGA nanoparticles. This PEG coating is critical for drug targeting purposes since it provides the nanoparticle with long-circulating properties in the blood stream as well as targeting capabilities (PEG can be linked to peptide or antibody molecules).
18 -Polipeptide matrix nanoparticles Polypeptide nanoparticles will be exploited to entrap drugs and obtain a controlled release. Moreover, the entrapped drug will be protected from degradation. Two kind of polypeptide nanoparticles will be explored: polypeptide dendrimers and SAP-based nanoparticles. In the first group, the covalent assembly of the multiple peptide chains will provide a matrix nanoparticle, with the ability to entrap the drug, either covalently or non-covalently. In the case of SAP-based nanoparticles, the assembly of different copies will be achieved due to the non-covalent self- assembly properties of sweet-arrow peptide (SAP).
i) Polypeptide dendrimers Dendrimers are nanoparticles that are creating great interest. Dendrimers are synthetic, highly branched, monodispersed macromolecules. As their molecular size increases, they adopt a spherical shape that has a vacant inner core that can encapsulate drug molecules, and a highly functionalized surface that can be derivatized with a ligand. Biocompatible dendrimers are obvious candidates for drug delivery applications. A particularly interesting class of biocompatible dendrimers is obtained when peptides are the base of the dendrimeric structrure. Polypeptide dendrimers have been previously described, 39 and the pioneering work of the group of J. Tam has demonstrated the application of lysine dendrimers as immunogens. 40 In the present project we plan to focus our attention on polyproline dendrimers a particular class of polypeptide dendrimers. Proline is singular among the 20 genetically coded amino acids in that it contains both a cyclic backbone as well as a secondary, as opposed to primary, -amino group. These structural features impart unique stereochemical properties to proline. Polyproline oligomers exist in two distinct conformations. In organic solvents, they adopt a conformation known as polyproline I, a right-handed helix in which all peptide bonds are cis-oriented ( = 0 ). 41 In aqueous solvents, they adopt the conformation known as polyproline II, a left- handed helix in which all peptide bonds are trans-oriented ( = 180 ). 42 The transition from polyproline I to polyproline II implies a considerable increase in the long dimension of the helix that changes from 1.9 to 3.1 per residue.
39 L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Royo, F. Albericio. Chem. Rev., 2005, 105, 1663- 1681. 40 Nardelli, B.; Lu, Y. A.; Shiu, D. R.; Profy, A. T.; Tam, J. P. Immunology 1992, 148, 914-920. 41 Traub, W.; Shmueli, U. Nature 1963, 198, 1165-1166. 42 Cowan, P. M.; McGavin, S. Nature 1955, 176, 501-503 19
This unique conformational plasticity of polyproline chains allows to modulate the dendrimer properties by changing the length of the branches in response to the environment. In this way, drugs could be trapped in organic solvents, where polyproline chains adopt a polyproline I conformation, and released under physiological conditions in which polyproline spacers form more extended polyproline II helices. A more classical alternative to this non-covalent drug entrapment could be attachment by covalent bond formation with the building blocks of the dendrimer. One of the characteristics of dendrimers is the facility to modulate its properties by modification of the dendrimer surface. 43 This surface modification would be used either for covalent attachment of the drug or, even more interesting, to functionalize the dendrimer surface with vector peptide sequences able to target the nanovector to a given tissue, and especially to a tumoural tissue (see point 1.2.). The biocompatibility of peptide structures and the possibility of cellular
43 Lee, J. W.; Ko, Y. H.; Park, S. H.; Yamaguchi, K.; Kim, K. Angew. Chem., Int. Ed. 2001, 40, 746-749 polyproline I polyproline III 20 internalization of polyproline structures 44,45 are additional potential benefits of this approach.
ii) SAP- based nanoparticles Sweet Arrow Peptide (SAP), VRLPPPVRLPPVRLPP, is an amphipathic Pro-rich cell-penetrating peptide, i.e., it has the special ability to cross the cell membrane. Because of its amhiphathic character, SAP was expected to self-assemble. In aqueous media SAP adopts polyproline II structure, with hydrophobic residues pointing to one face of the helix and hydrophilic ones facing to the opposite (see figure). The self-assembling properties of SAP were proven by circular dichroism (CD) and transmission electron microscopy (TEM). TEM micrographs of the replicas showed fibrils of 16 (3) nm width and variable length.
In the present project, nanoparticulate molecular materials of SAP will be obtained by top down methods. In order to covalently attach the drug, a percentage of the arginines of the SAP sequence, (VRLPPP) 3 , will be replaced by lysine or serine in order to bind carboxylic containing-drugs to the SAP-based nanoparticles through amide or ester bonds, respectively.
44 L. Crespo, G. Sanclimens, B. Montaner, R. Prez-Toms, M. Royo, M. Pons and F. Albericio, E. Giralt. Peptide Dendrimers Based on Polyproline Helices. J. Am. Chem. Soc., 2002, 124, 8876-8883. V R L P P P V R L P P P V R L P P P 21 Apart from preventing the drug degradation in the biological media and to offer a controlled release, SAP-based nanoparticles will have the unique ability to internalize the entrapped drug inside the cell. 46,47,48
Workplan 1.1.2 a) Synthesis of polyproline dendrimers with reactive functional groups at the surface. b) Covalent attachment of antitumoral drugs to polyproline dendrimers. We will focus on doxorubicin (as a proof of concept) and on antiangiogenic drugs. c) Non-covalent entrapment of antitumoral drugs in polyproline dendrimers. We will exploit the conformacional plasticity of the polyproline branches in response to changes in solvents polarity. The effect of the size of the drug will be also studied. Again, we will focus on antiangiogenic drugs. d)Synthesis of SAP-based nanoparticles. Monomeric SAP will be prepared using well established solid-phase peptide synthesis methodologies. SAP-based nanoparticles will be obtained by top down methods (use of critical superfluids) from the nanofibrilar structures formed in highly concentrated SAP aqueous solutions. e)Synthesis of functionalized SAP-based nanoparticles. Lysine- and serine- containing SAP monomers will be prepared by solid-phase synthesis. Attachment of antiangiogenic drugs will be done in solution by reaction with the amino- and the hydroxy- groups of lysine and serine side-chains, respectively. Then, drug-loaded nanoparticles will be prepared as described above. In an alternative procedure that will also be explored, the drug will be attached in solution to preformed lysine- or serine-containing SAP-based nanoparticles.
45 Farrera-Sinfreu, Josep; Giralt, Ernest; Castel, Susanna; Albericio, Fernando; Royo, Miriam. Cell- Penetrating cis-g-Amino-L-Proline-Derived Peptides. J. Am. Chem. Soc., 2005, 127, 9459-9468. 46 S. Pujals, J. Fernndez-Carneado, C. Lpez-Iglesias, M. J. Kogan and E. Giralt, BBA-Biomembranes, 2006 , in press. 47 [9 J. Fernandez-Carneado, M. J. Kogan, S. Pujals, and E. Giralt, Biopolymers 2004, 76 196-203. 48 10 J. Fernandez-Carneado, M. J. Kogan, S. Castel, and E. Giralt, Angewandte Chemie, Int. Ed. 2004, 43,1811-1814.