STERICALLY STABILIZED LIPOSOMES AS A POTENT CARRIER FOR SHIKONIN

Konstantinos N. Kontogiannopoulos 1, Andreana N. Assimopoulou 1, Vassilios P. Papageorgiou 1,*

Organic Chemistry Laboratory, Chemical Engineering Department, Aristotle

University of Thessaloniki, 54124 Thessaloniki, Greece.

Corresponding author. Tel: +30 2310996241, Fax: +30 2310996252, email address: vaspap@eng.auth.gr

Abstract Asdasdas

Keywords: Alkannin; Naphthoquinone; Pegylated Liposomes; Sterically Stabilized Liposomes; cancer

Abbreviations Hp Chi-aDDnSs A S MCRnSs EPC DPPC DSPC DSPE-mPEG2000 Hyperbranched polymers Chimeric advanced Drug Delivery nano Systems Alkannin Shikonin Modulatory Controlled Release nano Systems Egg phosphatidylcholine Dipalmitoyl phosphatidylcholine Distearoyl phosphatidylcholine -(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 dipalmitoyl-en-glycero-3-phosphoethanolamine CHOL PBS SLS PDI Cholesterol Phosphate buffer saline pH 7.4 Sodium lauryl sulfate Polydispersity index

1. Introduction
Over the past few decades, increasing attention has been given to drug targeting in order to reduce side-effects and improve therapeutic efficacy by preventing undesired drug localization in healthy tissue sites and decreasing rapid degradation or elimination of drugs (Han et al., 2007; Vyas et al., 2006; Yousefi et al., 2009). Among a variety of targeted drug carrier systems, liposomes have been studied extensively because of their capability to accommodate a large variety of drugs, alongside their good biocompatibility, low toxicity and lack of immune system activation or suppression. In general, an optimized system is consisted of nanoliposomes, which possess a long circulation lifetime. Such liposomes will circulate sufficiently long to accumulate at sites of disease, such as tumors, as a result of the leaky vasculature and reduced blood flow exhibited by the diseased tissue (Drummond et al., 2008; Fenske & Cullis, 2005; Yang et al., 2007).

Liposomes were one of the first nanoparticulate drug delivery systems to show increased delivery of low molecular weight anticancer agents to solid tumors. Liposomes with diameters in the range of 100 nm can accumulate in solid tumors via the enhanced permeability and retention (EPR) effect (Maeda et al., 2000), which occurs when nanoparticulates extravasate from the circulation into tumors through gaps in the vasculature endothelium (Ishida et al., 2009; Jain, 2001). The ability of liposomes to localize in solid tumors via the EPR effect partly depends on their long circulating properties, which can be achieved by grafting polyethylene glycol (PEG) to the surface of the liposomes (pegylated liposomes or sterically stabilized liposomes-SSL) (Papahadjopoulos et al., 1991). Anticancer agents encapsulated in SSL have shown increased efficiency and lower toxicity in treatment of solid tumors by achieving higher accumulation in tumor tissue but limited accumulation in healthy organs (Gabizon et al., 1994; Vaage et al., 1993). Doxorubicin-containing SSL (DXRSSL), Doxil/Caelyx, has been approved for clinical use (Engels et al., 2007). Alkannin and Shikonin (A/S; Figure 1) are chiralpair of naturally occurring isohexenylnaphthazarins. They are found in the external layer of the roots of at least a hundred and fifty species that belong mainly to the genera Alkanna, Lithospermum, Echium, Onosma and Anchusa of the Boraginaceae family (Papageorgiou et al., 1999; Papageorgiou et al., 2006).

Figure 1: The chiral pair alkannin and shikonin that possess major biological activity.

Alkannin, Shikonin and their derivatives were originally introduced and established as wound healing agents by Prof. Papageorgiou. A wound healing pharmaceutical ointment is already commercially available under the trademark HELIXDERM and the medical devices HELIXFILM, HELIXGEL and

HELIXSPRAY (wound healing collagen film, gel and spray respectively) are under development. Further biological investigations over the last 35 years have shown that A/S are potent pharmaceutical substances with a wellestablished wide spectrum of antimicrobial, antiinflammatory and antioxidant activity (Papageorgiou, 1980; Papageorgiou et al., 1999; Papageorgiou et al., 2006; Papageorgiou et al., 2008). Extensive scientific research has been conducted the last years on cancer chemotherapy, focusing on A/S effectiveness on several tumors and mechanism(s) of antitumor action (Chen et al., 2002; Komi et al., 2009; Lee et al., 2008; Papageorgiou et al., 1999; Papageorgiou et al., 2006; Yang et al., 2009; Yao & Zhou, 2010; Zeng et al., 2009). The scarce aqueous solubility of A/S (0.00002M) (He, 2009) is a barrier for their oral and internal administration, since they cannot be easily dissolved and further absorbed from the receptor. A/S are also oxidized (Cheng et al., 1995), polymerized (Assimopoulou & Papageorgiou, 2004a, b; Papageorgiou et al., 2002) and internally metabolized (Meselhy et al., 1994a; Meselhy et al., 1994b). Regarding the toxicity of the active compounds, alkannin was found to bear a LD50 of 3 g/kg in mice and less than 1 g/kg in rats, when administered orally in a feeding study (Majlathova, 1971). Shikonin, on the other hand, was found to be rather more toxic to mice by

intraperitoneal administration, with a LD50 of 205 mg/kg (Hayashi, 1977). In addition, during in vivo testing (mice, intraperitoneal administration), shikonin showed toxicity at dosages higher than 15 mg/kg/day (Sarcoma180) and at 10x5 mg/kg/day (L-1210) (Sankawa et al., 1977). Both the solubility and instability matters could be overcome by delivering A/S through a drug delivery nano-system, such as a liposomal formulation, which could furthermore enhance their antitumor activity through toxicity decrease and targeted delivery. The purpose of this work was to prepare and characterize shikoninloaded liposomes as a new drug delivery system for shikonin, in order to reduce side effects of the free drug, to enhance selectivity against cancer cells and to protect shikonin from internal biotransformations (Meselhy et al., 1994b; Meselhy et al., 1994a). In this context, three new pegylated liposomal formulations of shikonin were prepared and characterised in terms of their physicochemical characteristics, pharmacokinetics and stability and also compared to the corresponding conventional liposomes. Characterization of all liposomal formulations prepared (both conventional and pegylated) was performed in terms of particle size distribution, potential, entrapment efficiency and release profile of the entrapped drug. Finally, a stability study was performed at 4oC for a 28 days period in order to examine the physicochemical and pharmacological stability of the prepared formulations (both conventional and pegylated). This research is a continuation study of the authors on exploiting the biological properties of A/S and other naphthoquinones through the preparation of DDSs, such as microcapsules (Assimopoulou et al., 2003; Assimopoulou & Papageorgiou, 2004d), cyclodextrins (Assimopoulou & Papageorgiou, 2004c), liposomes

(Kontogiannopoulos et al., 2011a), chimeric advanced drug delivery nanosystems (combining dendritic and liposomal technology) (Kontogiannopoulos et al., 2011b) and electrospun fiber mats (Kontogiannopoulos et al., 2011c).

2. Materials and Methods

2.1 Materials
Shikonin was used after purification from a commercial sample (Ikeda Corporation, Tokyo, Japan), by silica gel column chromatography (gradient mixtures of n-hexane: dichloromethane: chloroform) followed by recrystallization (n-hexane), according to the procedure proposed by Prof. Papageorgiou (Assimopoulou et al., 2008) (purity obtained 100%). Dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), distearoyl phosphatidylcholine (DSPC) and -(Carbonyl-methoxypolyethyleneglycol 2000)-1,2 dipalmitoyl-en-glycero-3-phosphoethanolamine (DSPE-mPEG2000) were purchased from Genzyme Pharmaceuticals (Cambridge, USA). Cholesterol (CHOL), phosphate buffer saline pH 7.4 (PBS), sodium lauryl sulfate (SLS), dialysis sacks (molecular weight cut off 13000) and Sephadex G75 were purchased from Sigma Aldrich (St. Louis, USA). All organic solvents were of analytical grade and were purchased from SigmaAldrich (St. Louis, USA). Water used in all experiments was of HPLC grade.

2.2

Preparation of shikonin-loaded pegylated liposomes

Initially, the lipids together with the cholesterol were dissolved in

chloroform:methanol 2:1 (v/v) at constant molar ratios 13:1 lipid/DPSE-mPEG2000 (mol/mol) and 4.5:1 lipid/CHOL (mol/mol). Shikonin was diluted in the same organic solvent (in a different flask) and added to the above mixture under stirring in a 50 mL roundbottom flask at 30:1 lipid/drug molar ratio (mol/mol). The organic solvent was slowly removed under reduced pressure using a rotary evaporator (EYELA Rotary Vacuum Evaporator N-N Series, Digital Water bath SB651, Tokyo, Japan), forming a thin film of the lipid on the inner side of the flask. The flask containing the lipid film was left overnight under vacuum, for the removal of organic solvent traces. The lipid film was then hydrated with 10 mL PBS (pH 7.4) for 1 h, in water bath above the main phase transition temperature (Tm) of the lipids (45oC for EPC lipids, 51oC for DPPC lipids and 65oC for DSPC lipids) in order to prepare multilamellar vesicles

(MLVs). The system was vortexed at 1500 rpm using an IKA MS2 Minishaker (IKA Works, Inc, Wilmington, USA) for 10 min. Small unilamellar vesicles (SUVs) were prepared from the resultant liposomal suspension (MLVs), after sonication for two 5 min periods interrupted by a 5 min resting period, using a probe sonicator (amplitude 0.7; pulser 50%; Heat Systems Ultrasonics Inc., Sonicator W375 Cell Disruptors). The resultant vesicles were allowed for 30 min to anneal any structural defects. Non-encapsulated shikonin was removed by passing the liposomal suspensions through a Sephadex G75 column that was swollen with water overnight.

2.3 Characterization of shikonin-loaded pegylated liposomes

2.3.1 Particle size measurement and -potential Size and -potential of liposomes are crucial parameters that indicate their physical stability. The hydrodynamic diameter of all liposomal formulations was measured by light scattering. 50 L of each liposomal formulation were 60 -fold diluted in PBS (pH 7.4) immediately after preparation and z-average mean and potential were measured. Measurements were made at 25oC and at a 90 angle in a photon correlation spectrometer (Malvern ZetaSizer Nano S, Malvern Instruments, Malvern, UK) and analyzed by the CONTIN method (MALVERN software).

2.3.2 Determination of entrapment efficiency To remove the nonencapsulated shikonin, liposomal suspensions were passed through a Sephadex G75 column prior to the determination of the entrapment efficiency. The percentage of shikonin incorporated into liposomes was estimated by UVvis spectrophotometry (UV-Vis Hitachi U1900, Hitachi High-Technologies Corporation, Tokyo, Japan) at the characteristic wavelength of shikonin (516 nm). 0.5 mL of each liposomal formulation in PBS were suspended in 2.5 mL of methanol to destroy the liposomal structure, releasing the drug into the organic phase. Absorbance of the organic phase was measured and shikonin concentration was determined using the following shikonin calibration curve in methanol: Drug Concentration (mg/mL) = 0.0485 x Absorbance - 0.00009 ; (R2=0.99992) (1)

The entrapment efficiency was calculated using the following equation: Entrapment Efficiency (%) = (Fi/Ft) x 100 (2)

where Fi is the amount of shikonin incorporated into shikonin-Hp complexes and Ft is the initially added amount of shikonin.

2.3.3 In vitro drug release The release profile of shikonin from all liposomal formulations was studied in (PBS+1% SLS) at 37oC. 3 mL of each sample were placed in dialysis sacks (molecular weight cut off 13,000; Sigma-Aldrich). Dialysis sacks were inserted in 20 mL (PBS+1% SLS) in shaking water bath (Selecta) set at 37oC. Aliquots of samples (3 ml) were taken from the external solution at specific time intervals and that volume was replaced with fresh release medium in order to maintain sink conditions. The amount of shikonin released at various times, up to 72 h, was determined using UV vis spectrometer at max=518 nm with the aid of the following calibration curve of shikonin in the release medium: Drug Concentration (mg/mL) = 0.04837 x Absorbance - 0.00004 ; (R2=0.99998) (3)

The cumulative percentage of drug release was calculated and plotted versus time using the equation: % Cumulative Drug Releasedt = Drug Releasedt / Total Entrapped Shikonin x 100 (4)

2.3.4 Stability studies All liposomal formulations (both conventional and pegylated) were tested for their stability by means of drug leakage, mean particle size, polydispersity index (PDI), and potential. Specifically, immediately after preparation liposomes formulations were placed in glass vials and stored at 4oC for 28 days. Aliquots of samples were taken at specific time intervals and mean particle size, potential and entrapment efficiency were measured as described earlier.

2.4 Statistical analysis

Results are shown as mean value standard deviation (S.D.) of three independent experiments. Statistical analysis was performed using Students t-test and multiple comparisons were done using one-way ANOVA. P values <0.05 were considered statistically significant. All statistical analyses were performed using SPSS 14.0.

3. Results and Discussion

The distinct features of pegylated liposomes such as reduced uptake by the reticulo-endothelial system, favourable pharmacokinetics (long circulating time, slow clearance rate, small volume of distribution), reduced accumulation in healthy tissues and, most importantly, preferential tumour uptake owing to their ability to extravasate through the hyperpermeable tumour vasculature are best illustrated by PEGylated liposomal doxorubicin (Caelyx, Doxil, Myocet) (Engels et al., 2007). In the present study pegylated (or sterically stabilized liposomes) are used for the first time, as drug delivery system for shikonin, using three types of lipids (EPC, DPPC and DSPC).

3.1 Particle size measurement and -potential

Fdsfsdf Fsdfa

3.2 Entrapment efficiency

Dsfdsf Dsfasdf

10

3.3 In vitro drug release

Fsdfasd Dsfasdf

3.4 Stability studies

Sadsad Dsad

11

4. References
Assimopoulou, A.N., Papageorgiou, V.P., Kiparissides, C., Synthesis and release studies of shikonin-containing microcapsules prepared by the solvent evaporation method, Journal of Microencapsulation, 20 (5), 2003, 581596. Assimopoulou, A.N., Papageorgiou, V.P., Study on polymerization of the pharmaceutical substances isohexenylnaphthazarins, Biomedical Chromatography, 18 (8), 2004a, 492-500. Assimopoulou, A.N., Papageorgiou, V.P., Study on isohexenylnaphthazarins polymerization in alkaline media, Biomedical Chromatography, 18 (8), 2004b, 508-522. Assimopoulou, A.N., Papageorgiou, V.P., Encapsulation of isohexenylnaphthazarins in cyclodextrins, Biomedical Chromatography, 18 (4), 2004c, 240-247. Assimopoulou, A.N., Papageorgiou, V.P., Preparation and release studies of alkannincontaining microcapsules, Journal of Microencapsulation, 21 (2), 2004d, 161173. Chen, X., Yang, L., Oppenheim, J.J., Howard, O.M.Z., Cellular pharmacology studies of shikonin derivatives, Phytotherapy Research, 16 (3), 2002, 199-209. Cheng, H.W., Chen, F.A., Hsu, H.C., Chen, C.Y., Photochemical decomposition of alkannin/shikonin enantiomers, International Journal of Pharmaceutics, 120 (2), 1995, 137-144. Drummond, D.C., Noble, C.O., Hayes, M.E., Park, J.W., Kirpotin, D.B., Pharmacokinetics and In Vivo Drug Release Rates in Liposomal Nanocarrier Development, Journal of Pharmaceutical Sciences, 97 (11), 2008, 4696-4740. Engels, F.K., Mathot, R.A.A., Verweij, J., Alternative drug formulations of docetaxel: a review, Anti-Cancer Drugs, 18 (2), 2007, 95-103. Fenske, D.B., Cullis, P.R., 2005, Entrapment of small molecules and nucleic acidbased drugs in liposomes, in: Duzgunes, N. (Ed.), Liposomes, Pt E, pp. 7-40. Gabizon, A., Catane, R., Uziely, B., Kaufman, B., Safra, T., Cohen, R., Martin, F., Huang, A., Barenholz, Y., Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes, Cancer Research, 54 (4), 1994, 987-992. Han, H.D., Lee, A., Hwang, T., Song, C.K., Seong, H., Hyun, J., Shin, B.C., Enhanced circulation time and antitumor activity of doxorubicin by comblike polymer-incorporated liposomes, Journal of Controlled Release, 120 (3), 2007, 161-168. Hayashi, M., Pharmacological studies on crude plant drugs, Shikon and Tooki (II) Shikonin and Acetylshikonin, Nippon Yakurigaku Zasshi, 73 (2), 1977, 193-203. He, J., Complex of shikonin and beta-cyclodextrins by using supercritical carbon dioxide, Journal of Inclusion Phenomena and Macrocyclic Chemistry, 63 (3-4), 2009, 249-255. Ishida, T., Shiraga, E., Kiwada, H., Synergistic antitumor activity of metronomic dosing of cyclophosphamide in combination with doxorubicin-containing

12

PEGylated liposomes in a murine solid tumor model, Journal of Controlled Release, 134 (3), 2009, 194-200. Jain, R.K., Delivery of molecular and cellular medicine to solid tumors, Advanced Drug Delivery Reviews, 46 (1-3), 2001, 149-168. Komi, Y., Suzuki, Y., Shimamura, M., Kajimoto, S., Nakajo, S., Masuda, M., Shibuya, M., Itabe, H., Shimokado, K., Oettgen, P., Nakaya, K., Kojima, S., Mechanism of inhibition of tumor angiogenesis by betahydroxyisovalerylshikonin, Cancer Science, 100 (2), 2009, 269-277. Kontogiannopoulos, K.N., Assimopoulou, A.N., Dimas, K., Papageorgiou, V.P., Shikoninloaded liposomes as a new drug delivery system: physicochemical characterization and in vitro cytotoxicity, European Journal of Lipid Science and Technology, 113 (9), 2011a, 1113-1123. Kontogiannopoulos, K.N., Assimopoulou, A.N., Hatziantoniou, S., Karatasos, K., Demetzos, C., Papageorgiou, V.P., Chimeric advanced drug delivery nano systems (chi-aDDnSs) for shikonin combining dendritic and liposomal technology, International Journal of Pharmaceutics, in press, DOI: 10.1016/j.ijpharm.2011.09.031, 2011b. Kontogiannopoulos, K.N., Assimopoulou, A.N., Tsivintzelis, I., Panayiotou, C., Papageorgiou, V.P., Electrospun fiber mats containing shikonin and derivatives with potential biomedical application, International Journal of Pharmaceutics, 409 (1-2), 2011c, 216-228. Lee, H.J., Magesh, V., Nam, D., Lee, E.O., Ahn, K.S., Jung, M.H., Kim, D.K., Kim, J.Y., Kim, S.H., Shikonin, Acetylshikonin, and Isobutyroylshikonin Inhibit VEGF-induced Angiogenesis and Suppress Tumor Growth in Lewis Lung Carcinoma-bearing Mice, Yakugaku Zasshi-Journal of the Pharmaceutical Society of Japan, 128 (11), 2008, 1681-1688. Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K., Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, Journal of Controlled Release, 65 (1-2), 2000, 271-284. Majlathova, L., Futterungsversuch mit Alkannin an Mausen, Food / Nahrung, 5 (5), 1971, 505508. Meselhy, M.R., Kadota, S., Tsubono, K., Kusai, A., Hattori, M., Namba, T., Shikometabolins A, B, C and D, novel dimeric naphthoquinone metabolites obtained from shikonin by human intestinal bacteria, Tetrahedron Letters, 35 (4), 1994a, 583-586. Meselhy, M.R., Kadota, S., Tsubono, K., Hattori, M., Namba, T., Biotransformation of shikonin by human intestinal bacteria, Tetrahedron, 50 (10), 1994b, 30813098. Papageorgiou, V.P., Naturally occurring isohexenylnaphthazarin pigments: a new class of drugs, Planta Medica, 38 (3), 1980, 193203. Papageorgiou, V.P., Assimopoulou, A.N., Couladouros, E.A., Hepworth, D., Nicolaou, K.C., The chemistry and biology of alkannin, shikonin, and related naphthazarin natural products, Angewandte Chemie International Edition, 38 (3), 1999, 270-300. Papageorgiou, V.P., Assimopoulou, A.N., Kyriacou, G., Determination of naturally occurring hydroxynaphthoquinone polymers by size-exclusion chromatography, Chromatographia, 55 (7-8), 2002, 423-430. Papageorgiou, V.P., Assimopoulou, A.N., Samanidou, V.F., Papadoyannis, I.N., Recent advances in chemistry, biology and biotechnology of alkannins and shikonins, Current Organic Chemistry, 10 (16), 2006, 21232142.

13

Papageorgiou, V.P., Assimopoulou, A.N., Ballis, A.C., Alkannins and Shikonins: A new class of Wound Healing Agents, Current Medicinal Chemistry, 15 (30), 2008, 32483267. Papahadjopoulos, D., Allen, T.M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.D., Woodle, M.C., Lasic, D.D., Redemann, C., Martin, F.J., Sterically stabilized liposomes-improvements in pharmacokinetcis and antitumor therapeutic efficacy, Proceedings of the National Academy of Sciences of the United States of America, 88 (24), 1991, 11460-11464. Sankawa, U., Ebizuka, Y., Miyazaki, T., Isomura, Y., Otsuka, H., Shibata, S., Inomata, M., Fukuoka, F., Anti-tumor activity of shikonin and its derivatives, Chemical & Pharmaceutical Bulletin, 25 (9), 1977, 2392-2395. Vaage, J., Donovan, D., Mayhew, E., Abra, R., Huang, A., Therapy of human ovarian-carcinoma xenografts using doxorubicin encapsulated in sterically stabilized liposomes, Cancer, 72 (12), 1993, 3671-3675. Vyas, S.P., Rawat, M., Rawat, A., Mahor, S., Gupta, P.N., Pegylated protein encapsulated multivesicular liposomes: A novel approach for sustained release of interferon alpha, Drug Development and Industrial Pharmacy, 32 (6), 2006, 699707. Yang, H.J., Zhou, P., Huang, H.B., Chen, D., Ma, N.F., Cui, Q.Z.C., Shen, S.X., Dong, W.H., Zhang, X.Y., Lian, W., Wang, X.J., Dou, Q.P., Liu, J.B., Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo, International Journal of Cancer, 124 (10), 2009, 2450-2459. Yang, T., Cui, F.D., Choi, M.K., Cho, J.W., Chung, S.J., Shim, C.K., Kim, D.D., Enhanced solubility and stability of PEGylated liposomal paclitaxel: In vitro and in vivo evaluation, International Journal of Pharmaceutics, 338 (1-2), 2007, 317326. Yao, Y., Zhou, Q., A novel antiestrogen agent Shikonin inhibits estrogen-dependent gene transcription in human breast cancer cells, Breast Cancer Research and Treatment, 121 (1), 2010, 233-240. Yousefi, A., Esmaeili, F., Rahimian, S., Atyabi, F., Dinarvand, R., Preparation and In Vitro Evaluation of a Pegylated Nano-Liposomal Formulation Containing Docetaxel, Scientia Pharmaceutica, 77 (2), 2009, 453-464. Zeng, Y., Liu, G., Zhou, L.M., Inhibitory effect of acetylshikonin on human gastric carcinoma cell line SGC-7901 in vitro and in vivo, World Journal of Gastroenterology, 15 (15), 2009, 1816-1820.

14