Synthesis And Characterization Of Novel Stimuli-Responsive Nanoparticle For Tumor-Targeted Drug Release

Polymeric nanoparticles as novel drug delivery systems have attracted increasing attentions in biomedical application in recent years. On the basis of numerous previous research work, we designed and synthesized a series of novel poly(N-isopropylacrylamide)-based thermoresponsive polymeric nanogels/micelles as intelligent drug carriers, and investigated their thermoresponsive properties, controlled release behaviors and in vitro tumor targeting behaviors in detail.Chapter 1 presents a detailed review of recent progress of synthesis of intelligent polymeric nanopaticles, drug delivery systems and tumor targeting.In Chapter 2, a series of biocompatible and stimuli-sensitive poly(N-isopropylacrylamide-co-propyl acrylic acid) (P(NIPAAm-co-PAAc)) nanogels were synthesized by emulsion polymerization. In addition, polyethyleneimine (PEI) was further grafted to modify the PNIPAAm-based nanogels. The P(NIPAAm-co-PAAc)-g-PEI nanogels exhibited good thermosensitivity as well as pH sensitivity. Transmission electron microscopy (TEM) showed that the P(NIPAAm-co-PAAc)-g-PEI and P(NIPAAm-co-PAAc) nanogels displayed well dispersed spherical morphology. The mean sizes of the nanogels measured by dynamic light scattering (DLS) were from 100 nm to 500 nm at different temperatures. The cytotoxicity study indicated P(NIPAAm-co-PAAc) nanogels exhibited a better biocompatibility than both PNIPAAm nanogels and P(NIPAAm-co-PAAc)-g-PEI nanogels although all the three kinds of nanogels did not exhibit apparent cytotoxicity. The drug-loaded nanogels, especially the PEI-grafted nanogels, showed temperature-trigged controlled release behaviors, indicating the potential applications as an intelligent drug delivery system.In Chapter 3, multifunctional and thermosensitive poly(N-isopropylacrylamide-co-propyl acrylic acid-co-hydroxyethyl methacrylate) (P(NIPAAm-co-PAAc-co-HEMA)) nanogels were prepared by miniemulsion polymerization. The mean sizes of the nanogels measured by dynamic light scattering (DLS) varied from 120 to 400 nm with an increase in temperature. Transmission electron microscopy (TEM) showed that the nanogels displayed well-dispersed spherical morphology. The nanogels were conjugated by human transferrin (Tf) and the coupling of transferrin molecules with nanogels was verified by the UV-vis spectroscopy. The cytotoxicity study indicated that the nanogels did not exhibit apparent cytotoxicity. Fluorescence spectroscopy analysis as well as confocal laser scanning microscopy (CLSM) was used to confirm that the Tf-conjugated nanogels could specifically bind to A549 tumor cells. In addition, the Tf-conjugated nanogels loaded with Doxorubicin (Dox) could efficiently release the drug inside the cell, suggesting that the Tf-conjugated nanogels are useful drug carriers for tumor cell targeting.In Chapter 4, both arginine-glycine-aspartic acid (RGD)-containing peptide and transferrin (Tf) were conjugated to the thermosensitive poly(N-isopropylacrylamide-co-propyl acrylic acid) (P(NIPAAm-co-PAAc)) nanogel to prepare a dual targeting drug carrier. The obtained nanogel was characterized in terms of fluorescence spectroscopy, UV-vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). In order to track the dual-ligand conjugated nanogel, fluorescein isothiocyanate (FITC) was further conjugated to the nanogel. Cell internalization experiment showed that the dual-ligand conjugated nanogels exhibited obviously enhanced endocytosis by HeLa cells as compared with non-tumorous cells (COS-7 cells). The drug loaded dual-ligand conjugated nanogel could be transported efficiently into the target tumor cells at 37℃and thereafter the anti-tumor effect was enhanced significantly, suggesting that the dual-ligand conjugated nanogel has great potential as tumor targeting drug carriers.In Chapter 5, stimuli-sensitive diblock copolymer, poly(N-acroyloxysuccinimide)-b-poly(N-isopropylacrylamide) (PNAS-b-PNIPAAm) was synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization. Using the terminal carboxyl group of the diblock copolymer to initiate the ring-opening polymerization ofε-caprolactone (CL), the amphiphilic PNAS-b-PNIPAAm-b-PCL triblock copolymer was further synthesized. The triblock copolymer was characterized by NMR, IR and SEC-MALLS. In order to enhance the internalization to tumor cells, biotin was introduced into the triblock copolymer. The LCST of biotinylated PNAS-b-PNIPAAm-b-PCL was about 35.3℃. The formation of micellar aggregates (MAs) self-assembled from biotinylated PNAS-b-PNIPAAm-b-PCL was confirmed by CMC and TEM. The cell viability study demonstrated that the MAs have a low cytotoxicity. The anti-tumor drug doxorubicin (Dox) was loaded in the MAs, and in vitro release behavior of Dox showed the MAs exhibited thermosensitive drug release. The confocal microscopy studies confirmed that, with pretreatment of biotin-transferrin, the self-assembled MAs could specifically bind to tumor cells, indicating that the multifunctional MAs could be used as a promising drug carrier for tumor targeting.In Chapter 6, taking advantage of the geometric compatibility,α-βcyclodextrin dimer is designed via "click" chemistry to connect the hydrophilic and hydrophobic segments to form self-assembled noncovalently connected micelles (NCCMs) through host-guest interactions. The targeting property of NCCMs is switched off before reaching the tumor sites and switched on after removing the PEG segment in the tumor sites, which is called "tumor-triggered targeting". With de-shielding of PEG segment, the drugs loaded in NCCMs could be released rapidly due to the thermo-induced phase transition.