Design of Novel Biodegradable Crosslinked Stealth Polymeric Nanoparticles Using the Macromonomer Approach by Dispersion Polymerization.

摘要

Nanoparticle fabrication with poly(lactide) is limited to the dispersion of preformed polymers which makes it difficult to attach targeting moieties to the surface of the particle and to introduce crosslinked networks. We have used the macromonomer method to prepare crosslinked, drug-loaded PLA-PEG (stealth) nanoparticles using free-radical polymerization with the capacity to attach other molecules to the nanoparticle surface to make it multifunctional.;P(LLA-HEMA) macromonomer was prepared by ring-opening polymerization of L-lactide and characterized. A pH-sensitive crosslinking agent was also synthesized and characterized. Crosslinked nanoparticles were fabricated using different proportions of macromonomer, initiators, crosslinking agent and stabilizer. Particle size distribution, polydispersity index and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS). Confirmation of nanoparticle formation was by SEM. Optimization studies were carried out to minimize particle size (nm) as a function of the composition of the formulation variables using statistical experimental design (D-optimal mixture design). Model validity was checked by diagnostic plots. Model verification was done by comparing particle sizes of suggested solutions synthesized in the laboratory with the predicted particle sizes. The release profile of paclitaxel-loaded nanoparticles was determined by HPLC. In vitro cytotoxicity studies were carried out using a sensitive ATP assay in selected breast and ovarian cancer cell lines. Uptake studies were done by confocal microscopy to determine cellular internalization.;1HNMR and FT-IR spectra confirm the synthesis of the macromonomer and crosslinking agent. Formation of nanoparticles was confirmed by SEM. The statistical model (Scheffe Polynomial) reveals that the crosslinking agent and stabilizer are the terms which minimize particle size. The predicted sizes compare favourably with the data obtained in the laboratory. Drug release studies reveal that encapsulated drug is released over 7 days. In vitro cytotoxicity assay show that the blank nanoparticle is biocompatible with no toxicity for the duration of the assay compared to medium-only treated controls and that the paclitaxel-loaded nanoparticle formulation exhibit similar cytotoxicity compared to free drug in solution against the cancer cell lines tested. It was also demonstrated that the nanoparticles are rapidly internalized by MCF-7 cancer cells within one hour probably by non-specific endocytosis. The stealth nanoparticles are suitable for the design of controlled delivery systems for bioactive agents.