Preliminary Study Of IRGD Modified PLGA/CS Nanoparticles With Core-shell Structure

Carmustine(1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU), a chemotherapeutic alkylating agent, is one of the most frequently used chemotherapy drugs in the clinical treatment of glioma. But it is easy to emerge drug resistance in the clinical application, which is associated with O6-methylguanine-DNA-methyltransferase (MGMT) as reported. O6-benzylguanine (BG) can directly deplete MGMT levels to reverse the drug resistance. Our previous study prepared drug-loaded PLGA/CS nanoparticles with core-shell structure by emulsion solvent evaporation method. PLGA served as the core of NPs carrying BCNU, while CS was used as a coating shell loading BG. Compared with original drug, nanoparticles containing both BCNU and BG could significantly prolong the residence time of drugs in vivo, improve the ability to cross the BBB, and enhance the antitumor activity of BCNU. But the release behavior of two drugs was not ideal, and the ability of targeting delivery need to be further improved.The av integrin is over expressed on the surface of many tumors’ cells (including glioma cells) and their neovascular endothelial cells. One cyclic peptide called internalizingRGD GRGD, CCRGDK/RGPDC) has high affinity with the integrin. After binded to the receptor, it can be hydrolysised by proteolytic enzymes in the tumor. The sequence of one fragment was RGDK/R, it can internalize into tumor cells through neuropilin-1(NRP-1). In this study, we intended to prepare an active targeting drug delivery system modified by iRGD. We improved the method for preparing nanoparticles and connected the targeting peptide to the surface of nanoparticles for further investigation.Based on the HPLC analytic method of BCNU and BG that had been established, the stability of the two drugs in different conditions was studied. MTT method was used to investigate the cytotoxicity of BCNU and sensitizing effect of BG. The results showed that, the temperature and pH value both have great influence on the stability of BCNU, it could improve the stability of BCNU through decreasing the temperature and pH value. Cytotoxicity experiments showed that BG could significant sensitize the cytotoxicity of BCNU in all three kinds of cells, the degree was determined by the expression level of MGMT.PLGA/CS nanoparticles were prepared by spontaneous emulsification solvent diffusion method (SESD). First, different factors were investigated respectively, including the molecular weight of PLGA, the concentration of PLGA, the concentration of PVA and the concentration of CS, etc. Then the optimal prescription was obtained by the orthogonal test. The stability of BCNU in nanoparticles and in vitro release behavior were studied and results were compared with the nanoparticles prepared by emulsion solvent evaporation method which was mastered by our research group before. The optimal prescription’s diameter and zeta potential were 121.6 ± 3.3nm and 32.3±4.1 mV, the drug loading efficiency of BCNU and BG was 1.86±0.17% and 6.82±1.15%, respectively. These were close to the original prescription. In vitro stability study showed that the degradation rate constants’ ratio of BCNU in original prescription and optimal prescription was 1:8. The optimal prescription significantly improved the stability of the BCNU. As shown in vitro release experiment, the release of BG is close to 100% at 4 h, while no more than 65% of BCNU released at 24 h. It indicated that BG could release from the NPs prior to BCNU, and BCNU had good sustained-release ability, which achieved the desired objective.In this study, iRGD was conjugated to the nanoparticles by Maleimide-polyethyleneglycol-N-Hydroxysuccinimide (MAL-PEG-NHS). First, PEG was connected to CS by reaction. Then iRGD was conjugated to PLGA/CS-PEG nanoparticles (PEG-NPs) through the reaction with the Maleimide group. BCA protein assay kit and the X-ray photoelectron spectroscopy both proved that the peptide had successful conjugated to the nanoparticles. The results of characterization experiments showed that, PEG-NPs and PLGA/CS nanoparticles had almost the particle size, zeta potential and drug loading efficiency of PEG-NPs were 122.5±2.89 nm,32.7±6.66 mV and 1.82±0.11%, respectively. Which were close to PLGA/CS NPs. While these of iRGD-PEG-NPs were 126.3±3.67 nm,34.3±5.51 mV and 1.50 ±0.04%, the drug loading efficiency was a little lower. Transmission electron microscopy (TEM) images demonstrated that the nanoparticles had regular spherical shape with core-shell structure.Experiments were carried out to study the stability of BCNU in nanoparticles in both PBS and plasma. The degradation rate constants’ratio of original drug, BCNU in PEG-NPs and iRGD-PEG-NPs in PBS was 13.46:1:1.04, in plasma was 18.39:1 1.13. It showed that the nanoparticles could significantly improve the stability of BCNU in both PBS and plasma. The result of in vitro release experiment showed that, the release of BG in PLGA/CS NPs, PEG-NPs and iRGD-PEG-NPs was all close to 100% at 4 h, while no more than 68% of BCNU released at 24 h in all three kinds nanoparticles. The release behavior of these nanoparticles was almost the same.The C6, F98 and U87 cells were chosen as models to study the cytotoxicity of drug-loaded nanoparticles by MTT method. After being encapsulated into the NPs, the antitumor activity of BCNU was significantly improved. When BG coexisted with BCNU in the NPs, the cytotoxicity of C6, F98 and U87 were improved 2.3,4.0 and 1.7 folds respectively. The iRGD could enhance the cytotoxicity through modifying on the surface of the drug-loaded NPs. The cellular uptake was evaluated by fluorescence microscope and flow cytometry. The results showed that cellular uptake increased with the rise of the NPs’concentration. NPs modified by iRGD could significantly improve the internalization ability. Tumor spheroid experiment was carried out to study the penetration ability of NPs. The nanoparticles modified by iRGD could penetrate more easily than ordinary nanoparticles.This study constructed one kind of core-shell nanoparticles modified by iRGD, which encapsulated both BCNU and BG. BG could release from the NPs prior to BCNU, and BCNU had good sustained-release ability. NPs significantly improved the stability of BCNU and vitro antitumor ability. The ability of cell uptake and penetration was also enhanced. It indicated that the nanoparticles could effectively improve the ability of targeting, penetration and antitumor.