Synthesis and characterization of biopolymeric micelles, using in controlled delivery of modified anti-cancer drugs

Breast cancer is a major form of cancer, with a high mortality rate in women. Doxorubicin (DOX) has proven very effective for the treatment of breast cancer. Nevertheless, drawbacks such as cardiac toxicity, short half-life and low solubility in aqueous solution have hindered its application. Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. P-glycoprotein (P-gp), a plasma membrane protein, actively transports drugs out of the cells and decreases their intracellular accumulation, resulting in resistance to multiple anti-cancer drugs. Overexpression of P-gp is closely associated with MDR and the blocking of P-gp-mediated transport is known to reverse MDR. Blocking agents are generally called MDR modulators and the reversal of resistance by MDR modulators is expected to improve the outcome of cancer chemotherapy. To overcome MDR a chemotherapeutic agent, DOX and a P-gp inhibitor ᴅ-α-Tocopherol polyethylene glycol 1000 succinate or valspodar (TPGS 1000 or PSC 833) were co-loaded in the poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL–PEG–PCL) (PCEC) micelle to obtain DOX/TPGS 1000 or DOX/PSC 833 loaded polymeric micelles (PM). The biodegradable, biocompatible and amphiphilic PCEC triblock copolymers with different compositions were synthesized via ring opening polymerization of caprolactone in the presence of polyethylene glycol (PEG) as an initiator and stannous 2-ethyl hexanoate (Sn(Oct)2) as catalyst. The structure of triblock copolymers was characterized by proton nuclear magnetic resonance (1H NMR), fourier transform infrared spectroscopy (FT-IR), gel permeation chromatography (GPC). The thermal behavior of copolymers was characterized on differential scanning calorimeter (DSC). The self aggregation and micelle formation of the PCEC copolymers were investigated using fluorescence spectra by a spectro fluorophotometer. The critical micelle concentrations (CMC) of the polymers ranged from 0.000293-0.019202 mg/mL. Sol–gel–sol phase transition behaviors of aqueous PCEC solutions were studied using test tube-inverting method. Aqueous solution of the obtained PCEC copolymers underwent sol–gel–sol transition as temperature increased which was flowing sol at room temperature and then turned into nonflowing gel at body temperature. PCEC micelles were prepared by the nanoprecipitation technique. Briefly, 4 selected process parameters (molecular weight of PEG, PCL/PEG ratio, polymer concentration and solvent/water ratio) were adjusted at 2 different values for acetone or dimethylformamide (DMF) solvent. Experiments for each of the solvent with 36 experimental conditions, repeated twice, the particle size, zeta potentials and polydispersity index values of the micelles was determined by dynamic light scattering (DLS). The optimized micelle, which was prepared by acetone, with low polydispersity index of 0.75, the narrow monodispersed unimodal size distribution of 225 nm and zeta potential of -2.2 mV is significant. The micelles obtained exhibit a spherical shape as shown by scanning electron microscobe (SEM). After that DOX was loaded into PCEC micelles by nanoprecipitation method without using any surfactants and toxic organic solvent. In this work shaking speed, time of contact, amount of triethylamine (TEA) and %DOX were adjusted at 2 different values. Experiments for each of the 16 experimental conditions, repeated twice, the drug encapsulation efficiency (EE) and drug loading (DL) of the micelles was determined. The DOX amount of the micelles was measured by ultraviolet spectrophotometer (UV-Vis). The encapsulation efficiency (EE) calculated under these optimum conditions was found to be %78.98. Different P-gp inhibitors with DOX could be loaded high into the micelles. DOX/TPGS 1000 and/or DOX/PSC 833 loaded PCEC polymeric micelles were prepared for the first time. FT-IR analysis of the micelles showed that DOX, TPGS 1000 and PSC 833 was encapsulated into the micelles. The amounts of TPGS 1000 and PSC 833 were determined using the high performance liquid chromatography (HPLC). In formulations containing two active agents, the entrapment efficiency of DOX was found to be higher due to P-gp inhibitors. To investigate the controlled release of DOX phosphate buffered saline (PBS) was used as the release medium at pH 5.0, 6.5 and 7.4 at 37°C and the release kinetic was calculated. When analyzed the physical and chemical stability of various polymeric micelle formulations, the results show that the micelles are stable and can be stored safely for 15 days at a temperature of 5°C, proving that both co-encapsulated drugs can be retained in the system during this time. According to the results obtained DOX solubility can be increased with micelle formulations which contain a P-gp inhibitor together with the anti-cancer active agent. These results suggest that the co-delivery of DOX with TPGS 1000 or PSC 833 by PCEC micelles is very promising for cancer therapy. Key words: Biodegradable, poly(Ɛ-caprolactone)-poly(ethylene glycol)-poly(Ɛ-caprolactone), doxorubicin, P-gp, drug delivery system