Development of Novel Polymeric Micelles as Drug Delivery Systems for Curcumin

Abstract

Curcumin is a natural yellow phenolic compound which presents in many kinds of herbs, especially in turmeric (Curcuma longa L.). It has been reported in many studies that curcumin is a natural antioxidant and anticancer agent. However, its pharmaceutical applications as therapeutic agent are limited because of its poor aqueous solubility and chemical instability under alkaline condition. Therefore, the aims of this present study were to develop and explore the advantages of novel polymeric micelles composed of block copolymers of methoxy poly(ethylene glycol) (mPEG) and N–(2–hydroxypropyl) methacrylamide (HPMA) modified with aromatic benzoyl side groups as nanocarriers for solubility, stability, and anticancer activity enhancement of curcumin. In first stage of study, the physicochemical characteristics and biological activity of curcumin were investigated. The antioxidant activity of curcumin in comparison with three important natural antioxidants, namely gallic acid, ascorbic acid, and xanthone, were performed on free radical scavenging action. The results indicated that the activities of these compounds were dose–dependent. The 50% effective concentration (EC50) of curcumin was found to be 11 µg/mL (30 µM). Curcumin showed significantly higher antioxidant activity than ascorbic acid and xanthone but less than gallic acid, indicating that antioxidant capacity of curcumin is higher than most of the other natural antioxidants which are ascorbic acid and xanthone. The solubility study showed that curcumin has a poor solubility in water, hexane, and toluene, but it is well soluble in polar aprotic solvent and alcohols. The kinetic degradation of curcumin under various conditions (pH, temperature, and dielectric constant of the medium) was investigated. To fully solubilize curcumin and to prevent curcumin precipitation that occurs when low volume fractions of co–solvent are present, a 50:50 (v/v) buffer/methanol mixture was used as standard medium to study its degradation kinetics. The results showed that the degradation of curcumin followed first order kinetics. It was further shown that an increasing pH, temperature, and dielectric constant (or increasing aqueous buffer volume fraction) of the medium resulted in an increase in degradation rate. Curcumin showed rapid degradation due to autoxidation in phosphate buffer pH = 8.0 with a rate constant of 280 × 10-3 h-1, corresponding with a half–life (t1/2) of 2.5 h. Dioxygenated bicyclopentadione was identified as the final degradation product as evidenced from liquid chromatography electrospray ionization mass spectroscopy (LC–ESI–MS) analysis.Polymeric micelles have shown great promises in solubilization, stabilization, and delivery of hydrophobic drugs for cancer treatment. Therefore, the amphiphilic block copolymers, ω–methoxy poly(ethylene glycol)–b–(N–(2–benzoyloxypropyl) methacrylamide (PEG–HPMA–Bz, molecular weight of PEG = 5000 Da, molecular weight of HPMA–Bz block from 18000 – 43800 Da), were synthesized. One polymer with molecular weight of 28300 Da had the lowest polydispersity index and was obtained in a high yield. This polymer was used in this study and compared with different aromatic substituted micelle forming polymers, (ω–methoxypoly(ethylene glycol)–b–(N–(2–hydroxypropyl) methacrylamide dilactate) (PEG–HPMA–DL)which has dilactate side chains and ω–methoxy poly(ethylene glycol)–b–(N–(2–benzoyloxy) methacrylamide)–co–(N–(2–lactoyloxypropyl) metha-crylamide) (PEG–HPMA–Bz–L) which has monolactate (75%) and benzoyl (25%) side groups. A simple micellar formation was used by a fast heating method for PEG–HPMA–DL and PEG–HPMA–Bz–L, whereas a nanoprecipitation method was used to obtain micelles of PEG–HPMA–Bz. The average particle sizes of curcumin–loaded polymeric micelles ranged from 46 to 85 nm, and the micelles showed spherical shapes. The PEG–HPMA–Bz micelles with benzoyl side groups (100%) gave the best solubili-zation and the curcumin solubility increased up to 2 mg/mLat a polymer concentration of 10 mg/mL. Fourier transform infrared spectroscopic analysis, x–ray diffraction patterns, and differential scanning calorimetry confirmed that curcumin was successfully entrapped in the micelles. The PEG–HPMA–DL micelles without aromatic benzoyl group almost fully released curcuminin20 days, whereas PEG–HPMA–Bz and PEG–HPMA–Bz–L released in the same time (27% and 50% of their curcumin loading, respectively). This release profile pointed a greater affinity of curcumin for polymeric micelles with aromatic groups. More importantly, curcumin in PEG–HPMA–Bz, PEG–HPMA–Bz–L and PEG–HPMA–DL micelles was about 300, 8, and 2 times, respectively, more stable than curcumin in phosphate buffer pH = 8.0. Curcumin–loaded PEG–HPMA–Bz micelles also showed the best stabilization of curcumin (80% of curcumin remaining) with the similar particle size as the first day (50 – 60 nm) after storage at 4, 30, and 40°C for 90 days, whereas curcumin–loaded PEG–HPMA–Bz–Land PEG–HPMA–DL micelles were less stable. The greater loading capacity, slower sustained release, and greater stabilization of the PEG–HPMA–Bz micelles are probably due to 𝜋–𝜋 stacking between the aromatic groups of curcumin and benzoyl groups of the polymer.Moreover, the interaction of proteins and all three polymeric micellar systems investigated in this thesis did not cause aggregation of the polymericmicelles. It was demonstrated that all three curcumin–loaded polymeric micelles showed no toxicity towards normal cells (red blood cells and peripheral blood mononuclear cells (PBMCs)), but provided good cytotoxic effects against human ovarian carcinoma cells (OVCAR–3), human cervical adenocarcinoma (HeLa), human colorectal adenocarci-noma (Caco–2), human breast adenocarcinoma (MCF–7), human lymphoblastic leukemia (Molt–4), and human chronic myelogenous leukemia (K562). Additionally, curcumin–loaded polymeric micelles were effectively internalized into K562 cells as demonstrated by fluorescence microscopy. It was shown that curcumin–loaded polymeric micelles induced cell cycle arrest at G2/M phase in K562 cells, and they also suppressed Wilms’ tumor 1 protein levels to 43 – 47%. In conclusion, the results in this study suggest that the enhanced solubility and stability of curcumin by loading into PEG–HPMA–Bz micelles is a promising nanodelivery system to provide suitable nanocarriers for further pharmaceutical and clinical development for cancer therapy.