Synthesis and characterization of poly(ε-caprolactone)-block-poly[N-(2-hydroxypropyl)methacrylamide] micelles for drug delivery.

Amphiphilic block copolymers based on HPMA and e-CL were synthesized by ring-opening polymerization of e-CL followed by RAFT polymerization of HPMA. A copolymer composed of 34 kDa PHPMA and 8.5 kDa PCL associated into micelles with CMC of 5.4 μg · mL−1. A novel retinoid, 3-Cl-AHPC-OMe, was incorporated into micelles with 25 wt.-% loading by dialysis method. The effective diameter of drug loading micelles was 117 nm. Incubation of micelles in PBS at 37 °C indicated 86 wt.-% of the drug was released after 96 h. Cytotoxicity studies performed with C4-2 prostate cancer cells showed the IC50 dose was 1.96 μM after 72 h of incubation, whereas the micelles without drug showed no cytotoxicity. Keywords: block copolymers, drug delivery systems, micelles, reversible addition fragmentation chain transfer (RAFT) Introduction Polymeric micelles are some of the most studied anticancer drug delivery systems.[1] They self-assemble from amphiphilic block copolymers in aqueous solution into nano-aggregates composed of a hydrophobic core and a hydrophilic shell.[2–4] Their advantages include simple preparation, high drug loading capacity, suitability for loading hydrophobic drugs, and accumulation in solid tumors due to enhanced permeability and retention (EPR) effect. Their main challenges are colloidal stability and an appropriate drug release profile.[1] The micelle core is formed by the hydrophobic block. Its chemical structure and molecular weight are important factors that determine the stability, drug loading capacity and drug release profile of micelles.[2] Polyesters, such as poly(e-caprolactone) (PCL)[5,6] and polylactide,[7] polyamino acids like poly(aspartic acid) and poly(β-benzyl-L-aspartate),[8] and poly(propylene oxide)[9] have been widely used as hydrophobic blocks. Poly(ethylene glycol) (PEG) has been the most frequently used hydrophilic shell-forming block,[3] while poly(N-vinyl-2-pyrrolidone) has also been repeatedly used.[10] PEG provides steric stabilization and minimization of non-specific protein interactions.[11] It is used in several products that have been approved by the Federal Drug Administration (FDA).[12] However, the lack of PEG functionality, the accelerated blood clearance (ABC) effect of PEGylated liposomes,[13] and the detection of anti-PEG antibodies[14] encouraged search for PEG alternatives. Poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) is a hydrophilic, biocompatible and non-immunogenic water-soluble polymer intensively studied as drug carrier.[15] Like PEG, PHPMA modification has shown to increase blood-circulation times. Examples include semitelechelic PHPMA-modified nanospheres[16] and acetylcholinesterase modified at multiple points with PHPMA.[17] Recently, various micelles were designed with hydrophilic shells formed by PHPMA.[18–24] Interestingly, Hennink’s laboratory used lactate modified PHPMA as the hydrophobic block and PEG as the hydrophilic block to prepare thermosensitive, biodegradable micelles.[25] The multifunctionality of PHPMA demonstrated advantages in the design of different architectures.[2] Lele and Leroux reported the synthesis of amphiphilic triblock[19] and star-shaped[20] copolymers containing PHPMA as a hydrophilic block and PCL, a biodegradable polymer, as the hydrophobic block. Doxorubicin and amphotericin B were loaded into the triblock based micelles (1–4 wt.-%), whereas indomethacin was incorporated into the star-shaped micelles (5–12 wt.-%). Researchers[20,21] used traditional free-radical polymerization to prepare PHPMA blocks. Consequently, the polydispersity of the PHPMA block will be >1.5 and might affect the control of size distribution of the final micelles. In this work, we explore the potential of a novel micellar drug carrier system based on a diblock copolymer of PCL and PHPMA. The block copolymer was synthesized by a combination of ring-opening polymerization (ROP) of e-caprolactone (e-CL) followed by “living” reversible addition-fragmentation chain transfer (RAFT) polymerization of HPMA. Using this approach, the molecular weight and polydispersity can be controlled. Polymeric micelles were formed by self-assembly of copolymers forming a hydrophobic PCL core and a hydrophilic PHPMA shell. To evaluate the feasibility of the micelles for drug delivery, a hydrophobic drug, (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic methyl ester (3-Cl-AHPC-OMe; not used in polymeric micelle-based drug delivery systems before), was chosen and physically incorporated into the hydrophobic micelle core. The micelles were characterized by pyrene assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), and stability measurements. In vitro cytotoxicity of the 3-Cl-AHPC-OMe delivery system was evaluated on the C4-2 prostate cancer cell line.