Preparation And Application Of Stimuli-responsive Polymer Microspheres For Delivery Of Anti-cancer Drugs

Objective: Cancer is the first killer in our country. Generally, traditional chemotherapy drugs cause severe side-effect, which is originated from their lack of selectivity to the cancer cells. Therefore, it’s necessary to create intelligent drug carriers which could carry the anticancer drugs to target tumor cells in cancer research. Recently, the preparation and application of multi-stimuli responsive drug carriers has aroused much concern. However, there are two biggest challenges in this field. One is how to prepare these drug carriers in a highly controllable way. The other is how to precisely switch on and off the release of the encapsulated drug molecules in response to a combination of environmental changes, such as p H value, temperature, redox potential and light.Methods: In this study, a novel triple-stimuli responsive polymer microsphere P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA with uniform size and superior quality was prepared by a two-stage distillation-precipitation polymerization with subsequent surface modification with folic acid(FA). Firstly, P(MAA-co-BAC) microspheres were synthesized by distillation-precipitation polymerization with methacrylic acid(MAA) as monomer and N,N’-bis(acryloyl)cystamine(BAC) as crosslinker. Secondly, the core-shell polymer microspheres P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC) were synthesized by the same method with P(MAA-co-BAC) microspheres as the core, N-isopropylacrylamide(NIPAAm) and glycidyl methacrylate(GMA) as comonomers and BAC as crosslinker. Thirdly, the tumor targeted molecule FA was conjugated onto the surface of the microspheres by ring opening reaction of epoxy groups and amidation. Finally, P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres were prepared.Next, the resultant microspheres generated from each step were characterized comprehensively. Transmission electron microscope(TEM) was used to evaluate their surface morphology, size and size distribution. FT-IR and UV-vis spectra were used to determine their molecular structure. Elemental analysis was used to measure their nitrogen content and determine the FA content in the FA-conjugated microspheres. Then the laser particle size analyzer was used to evaluate their thermo-sensitivity and physical stability by analyzing their hydrodynamic size, size distribution and Zeta-potential under different temperatures in water.Redox-induced degradation performance of the FA-conjugated microspheres was characterized via their turbidity change under different temperatures and redox conditions, which was monitored by a UV-visible spectroscopy at the wave-length of 630 nm at predetermined intervals of time. Doxorubicin hydrochloride(DOX) was chosen for the investigation of the drug loading and controlled release behavior of the FA-conjugated microspheres. WST-1 assay was used to study the cytotoxicity of the FA-conjugated microspheres before and after loading DOX. Then the laser confocal fluorescence microscopy and flow cytometry were used to investigate their delivery capability of DOX in vitro and molecular targeting property. These studies would evaluate the potential and advantage of the FA-conjugated microspheres as a anticancer drug delivery system.Results:All of the results of TEM, FT-IR spectrum, UV spectrum, elemental analysis and laser particle size analyzer showed that the P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres were prepared successfully. TEM images showed that the microspheres generated from each step were all monodisperse spheres with uniform size and smooth surface. The average particle size of the FA-conjugated microspheres was 159 nm and the average shell thickness was about 12 nm. The result of elemental analysis indicated that the FA content in the FA-conjugated microspheres was around 13.05%. The result of the laser particle size analyzer demonstrated that the microspheres generated from each step were still monodisperse spheres with a narrow size distribution in water. Also seen as the result, the Dh of the P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres before and after loading DOX both decreased considerably when the environmental temperature was increased from 25 to 37 ℃, which proved that these microspheres were endowed with significant thermo-sensitivity. In addition, the Zeta-potential of the microspheres with or without DOX loading were-23.03 m V and-29.06 m V, respectively, which indicated that the physical stability of these microspheres could be maintained by electrostatic interactions even with a high drug loading capacity.The degradation experiment demonstrated that the FA-conjugated microspheres degraded almost 70% within 4 h in 10 m M GSH buffer solution at 37 ℃, while they were very stable in absence of GSH. In a word, the decomposition of these microspheres was performed by a GSH triggered reduction of BAC crosslinkers in the polymer networks. The drug loading experiment showed that the DOX loading capacity of the P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres increased significantly with the increasing of the initial DOX concentration and leveled off with a saturated loading capacity of 208.0% and the corresponding encapsulation efficiency reached as high as 85.4%. In vitro drug release experiment showed that DOX-loaded P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres exhibited a p H/temperature/redox multi-stimuli responsive drug release characte.WST-1 assay indicated that the polymer microspheres had no obvious toxicity to MCF-7 cells even at a high concentration of 2000 μg m L-1. After loading DOX, the cytotoxicity of these microspheres was very similar to that of free DOX. The result of the laser confocal fluorescence microscopy and flow cytometry indicated that the FA-conjugated microspheres could delivery DOX into MCF-7 cells with much higher efficiency than the microspheres without FA conjugation. These results were well in accordance with the expectation that the FA-conjugated microspheres could be more efficiently bound and internalized by FR expressing cells MCF-7 presumably via receptor-mediated endocytosis.Conclusion:The P(MAA-co-BAC)/P(NIPAAm-co-GMA-co-BAC)-FA microspheres with controllable particle size and appearance, high stability, redox-induced degradability, p H/temperature/redox multi-stimuli responsive drug release character, low cytotoxicity and precise molecular targeting properties will probably become a promising vector for delivery of anti-cancer drugs for future applications in chemotherapeutics.