Study On Structure Of Drug Delivery Of PSMA Conjugated PEG-PCLmicelles Targeted To Prostatic Cancer

Nowadays, researches on micellar nanoparticles preparation are increasing yearby year both domestically and abroad due to the advantages of micelles overconventional preparations. However, further application of micellar drug deliverysystem is limited by the inadequate drug delivered to the target site. Active targetingthat introduced certain ligands on the hydrophilic surface of nanoparticles that canbind specifically with the receptors overexpressed on tumor cells increases theintratumoral drug concentration, hence exerting anti-tumor effects in a more specificmanner.In this study, a prostate specific membrane antigen (PSMA) ligand（SMLP）wassynthesized successfully which was analyzed and characterized by using massspectrum (MS)、1H NMR. The highly effective hydrogen bonding between SMLP andPSMA guarantees the potential of SMLP as a prostate cancer targeting ligand. Underthis condition, we synthesized a series of mPEG-b-PCL and HOOC-PEG-b-PCLcopolymers with various block lengths by ring opening polymerization. The polymerswere characterized by infrared spectroscopy (FT-IR),1H NMR and gel permeationchromatography (GPC). The results of1H NMR showed that the ratio of block lengthsof the copolymers were controllable, moreover, the polymers showed narrowmolecular weight distribution with PDI less than1.2according to GPC tests. At last,SMLP was conjugated to the carboxyl group of PCL-b-PEG-COOH which was firstlyactivated by NHS to prepare PCL-PEG-NHS. The structure of the product was confirmed by using FT-IR、1H NMR. With docetaxel (DTX) as modal drug, twomethods in micelle preparation were compared: film-hydration and dialysis.Parameters like the length of hydrophobic block, the time and temperature ofhydration as well as time and power of ultrasonication were studied. The resultsshowed that the diameter of drug-loading micelles was about150nm in size withbroad distribution. The highest drug-loading content of micelles could reach8.9%(PCL12k-mPEG5k) and9.1%(PCL12k-PEG5k-SMLP). On the other hand, although thedrug loading content of micelles prepared by dialysis method showed no significantchange compared with those prepared by film-hydration method:8.2%forDTX-PCL12k-mPEG5k(DTX-PMs1) and8.4%for DTX-PCL12k-PEG5k-SMLP(DTX-PMs2), the particle size and size distribution decreased significantly:55nm indiameter and PDI less than0.05, which were in consistent with TEM results. Sodialysis was selected as the micelle preparation method in this study due to thesmaller size and narrower size distribution of prepared micelles meanwhile facilepreparation. According to the results of short-term stability assays, DTX-PMs1andDTX-PMs2showed better stabilities in drug loading. The critical aggregationconcentration (CAC) of a series amphiphilic copolymers were determined andcompared using pyrene as fluorescence probe, all these copolymers showed CACs inthe range of0.1-0.6mg/L which indicated the tendency of the copolymers inself-assemble, moreover, the copolymers constructing PMs1and PMs2showed lowerCAC values which means better thermodynamic stability over micelles composed ofother copolymers. The optimized formulations were determined to be DTX-PMs1and DTX-PMs2according to drug loading content, short-term stability and CAC. In invitro drug release tests, the drug release profile of DTX-PMs1and DTX-PMs2werecompared under different pH values, both the tested formulations released70%ofloaded drug in medium of pH7.4, showed better sustained release effects thanTaxotere, while the slightly increased release rates under pH5.5contributed to thedrug release in tumor tissue and endosome.The results of in vitro cell viability studies showed that blank micelles PMs1(non-targeting) and PMs2(targeting) exhibited no toxicity to LNCaP cells atconcentrations ranged from50-500μg/mL. This indicated good biocompatibility ofmicelles and the conjugation of SMLP had no interference to cells. For drug loadedmicelles incubated with LNCaP cells for48h and72h, the IC50of targeting andnon-targeting micelles showed significant difference in cell viability:48h:0.87±0.27vs13.48±1.03μg/mL;72h:0.02±0.008vs1.35±0.54μg/mL. This indicated that thecargo was taken up more effectively with the guiding of SMLP. Cellular uptakestudies were conducted to further verify the PSMA targeting property of DTX-PMs2and the results showed five-fold higher fluorescence intensity in LNCaP cellsincubated with DTX-PMs2micelles than DTX-PMs1micelles containing sameamount of coumarin-6for4h. All the above results exhibited the immense promise ofthe developed novel drug delivery system based on SMLP-PEG-b-PCL in treatingprostate cancer.