Studies On Integrin Mediated Curcumin Loaded Micelle Delivery Systems

Curcumin (Cur), bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, is a low molecular weight polyphenol compound derived from the rhizome of the plant Curcuma longa. Recently, it is reported that curcumin has a wide range of pharmacologic actions such as anti-inflammation, anti-human immuno-deficiency virus, anti-microbial, anti-oxidant, anti-parasitic, anti-mutagenic and anti-cancer with low or no intrinsic toxicity. In spite of the wide spectra of pharmacologic properties, the application of curcumin in clinic has been hampered due to low solubility in aqueous solution and rapid degradation at physiological pH.Therefore, the improvement in stability, solubility and bioactivity of curcumin will be needed. Recent studies show that inclusion of hydrophobic drugs into polymeric micelles is one of the most attractive alternatives. Amphiphilic block copolymers can form nano-sized aggregates with core-shell structure which can solubilize poorly water soluble drugs and thus improve their bioavailability and protect from inactivation in biological media. In this study, PEG-PLA, as one of biocompatible and biodegradable materials, was used as a delivery carrier to improve the solubility of Cur in aqueous solution.Surface modification of PEG-PLA has been actively studied. In recent years, the arginine-glycine-aspartic acid peptide (RGD) can specifically recognize and bind with αvβ3and avP5integrin receptors which are important biomarkers overexpressed in sprouting tumor vessels and most tumor cells. It has been shown that RGD-modified nanoparticle could be a promising drug delivery system for targeting tumor therapy via RGD peptide specifically targeting to integrin receptors overexpressed on tumor-cell-surface and tumor neovasculature endothelial cells. In the present study, we developed a RGD peptide conjugated polymeric micelle system for Cur, aiming at strengthening the drug’s antiangiogenic efficacy, and improving the antitumor efficacy. For this purpose, we synthesized an RGD targeting amphiphilic copolymer, RGD-PEG-PLA, and prepared Cur loaded micelles using thin-film hydration method with modification. The physicochemical and biological properties such as cellular uptake and the in vitro cytotoxicity of the micelles were studied. In particular, the efficacy of Cur-RPP in vivo was investigated. The major findings were as follows:1. RGD-PEG-PLA conjugate was synthesized by the reaction between biodegradable N-hydroxysuccinimide activated polyethylene glycol-polylactic acid (NHS-PEG3000-PLA2000) and RGD tripeptide. The amide bond was generated. The HNMR and elemental analysis were used to prove the successful conjugation of RGD to PEG3000-PLA2ooo.2. Preparation of Cur-RPP:Cur loaded mixed micelles were prepared by thin-film hydration method with modification (ultrasound-constant temperature oscillation). A central composite design(CCD) with two factors at five levels was used to optimize the formulation. Before using the design, a number of preliminary experiments were conducted to identify the control factors. The optimized Cur-RPP was prepared with the ratio of RGD-PEG3000-PLA2000and MPEG2ooo-PLA2ooo=1:9, with high drug loading (4.70±0.03)%and the encapsulation efficiency (86.34±0.77)%. Under TEM, the micelles were spherical shape with smooth surface, the average particle size was about20nm, the in vitro release followed a first-order kinetics model. The results of DSC and PXRD showed that Cur was in crystalline form existing in micelles.3. The antitumor activity evaluation of Cur-RPP in vitro and in vivo:The hemolysis study showed that the micelle hemolysis rate was less than5%when-RPP was given by the route of injection. The cytotoxicity in vitro of drug loaded micelles was evaluated on HUVEC and B16cells using the MTT method. The Cur-RPP significantly increased the cytotoxicity of Cur on HUVEC with the IC50values reduced from16.66μg/mL to5.24μg/mL, and on B16with the IC50value reduced from14.20μg/mL to7.29μg/mL. The study of cellular uptake of Cur-RPP on HUVEC cells showed that the RGD could increase the intake of integrin receptors over-expressed cell. In vivo antitumor studies showed that continuous administration of30mg/kg could significantly reduce the tumor volume in mice.It was concluded from above results that RGD-targeted Cur-loaded micelles (Cur-RPP) could significantly improve the solubility of Cur,and be exploited as a potential new drug delivery system for Cur.