Study On Anti - Tumor Nano - Drug Delivery System Based On Novel Dendrimer

Nanoparticles drug delivery system is the effective method to improve the in vivo bioavailability of the hydrophobic drug. Drug carriers used to construct the nanoscale drug delivery system have developed rapidly to the practical application demand. As potential drug delivery vehicles, dendritic polymers have grasped more attention in recent years. This article introduced a series of novel codendrimers （PAMAM-co-OEG, co-D_x） derived from fourth generation PAMAM （G4.0） partially decorated with second generation OEG dendrons and their application for drug loading in order to obtain multifunctional drug characteristics. Based on these novel series amphiphilic codendrimers, drug-loaded polymicelles, nanoparticles and nanocrystals were prepared via physical encapsulation, and polyprodrug was synthesized via chemical conjugation. Then, the antitumor efficacy of these nanosacle drug delivery systems was evaluated. And there are four parts in detail:The first part of the graft has been carried out to explore the interactions between the series of codendrimers （co-D_x） from polyamidoamine （PAMAM） decorated by oligoethylene glycol （OEG） dendrons and drugs, these codendrimers present similar structures but different decoration degrees （DDs）. Firstly, codendrimer co-D64 as the model carrier was utilized to encapsulate a series of hydrophobic drugs with different functional groups. The drug-loading procedures were optimized by single factor exploration with resveratrol （Res）. From the results of drug-loading content （DLC） and entrapment efficiency （EE）, MTX （the EE was up to 68.7%） with acidic functional groups was observed as the most suitable drug for entrapment. Moreover, higher loading capacity and slower in vitro release of MTX were also observed in MTX/co-D64, co-D48, co-D32 and co-D₁6 polymicelles, which was attributed to the hydrophobic interactions and electrostatic interactions between MTX and co-D_x. Although these drug-loaded polymicelles presented similar particle size （90-110 nm）, morphology, thermal transition, and physical state, it was proved that codendrimers with lower DDs （co-D32 and co-D₁6） were the most suitable carriers to load MTX. This was due to the stronger hydrophobic and electrostatic interactions between the free amine groups of the codendrimers and MTX. The results from the drug release profiles indicated that the MTX-loaded polymicelles presented a biphasic release procedure, with an initial burst release followed by a slow release. All the MTX-loaded polymicelles showed sustained release for 48 h. The release kinetics was not controlled only by these interactions, but also the steric hindrance of the OEG dendrons. The 50% cellular growth inhibition （IC50） values of MTX/co-D64, co-D48, co-D32 and co-D₁6 polymicelles were determined as 13.9, 11.0,4.4 and 2.7 μg/mL v.s. free MTX 59.5 μg/mL, respectively. Overall, co-D₁6 with a moderate decoration degree could be a promising drug delivery system.In the second part, co-D₁6 was chosen as the most suitable drug carrier to prepare drug-loaded nanoparticles （MTX/co-D₁6 NPs and HK/co-D₁6 NPs） via antisolvent precipitation method augmented by ultrasonication. Based on the excellent hydrophility of co-D₁6, the MTX-loaded nanoparticles could be investigated easily with the high drug-loading content （-85.2%, w/w）. The MTX/co-D₁6 NPs possessed spherical morphology, nanoscaled particle size （approximately 182.4 nm）, and narrow particle size distribution. Release of MTX from MTX/co-D₁6 NPs showed a sustained release manner and completed within 48 h. Hemolytic evaluation indicated MTX/co-D₁6 NPs presented good blood compatibility, and the cytotoxicity of nanopartilces against breast cancer cells （IC50:7.9μg/mL v.s.25.1）μg/mL, p< 0.001) in vitro and antitumor efficacy in vivo were enhanced significantly compared to MTX injection （p< 0.001）. The in vivo study in 4T1 bearing nude mice after intravenous injection of Dir coating MTX/co-D₁6 NPs has revealed that, compared with free drug, the average fluorescence intensity of MTX/co-D₁6 NPs in the tumor showed 2.2 times higher. According to the higher drug-loading content, enhanced anti-tumor efficacy, and appropriate particle size, MTX/co-D₁6 NPs as the drug delivery systems could have potential application for cancer chemotherapy in clinic. The drug-loading content of HK/co-D₁6 NPs was～60% （w/w）, and the nanoparticles exhibited uniform spherical morphology with nanoscaled particle size （approximately 128.6 nm） and high Zeta potential value （about 63.9 mV）. Release of HK from HK/co-D₁6 NPs in PBS （containing 0.5% SDS） showed a sustained release manner and completed within 120 h, and the release curve was followed the Higuchi model （R2 = 0.9661）. The cytotoxicity of nanoparticles against 4T1 cells （IC50: NPs-2.2 μg/mL v.s. free drug-5.7 μg/mL, p< 0.001） in vitro and antitumor efficacy in vivo were enhanced significantly compared to HCPT injection （p< 0.001）. The HK/co-D₁6 NPs as the drug delivery systems could also been applied for cancer chemotherapy in clinic.Based on co-D₁6, drug-loaded nanocrystals （HCPT/co-D₁6 NCs and DTX/co-D₁6 NCs） were prepared via antisolvent precipitation method augmented by ultrasonication in the third part. The HCPT-loaded nanocrystals were investigated with as high drug-loading content as 90%（w/w）, the particle size of HCPT/co-D₁6 NCs was about 168.6 nm with uniform rod structure morphology and the Zeta potential was approximately 24.2 mV. HCPT was existed as the crystalline compound in the nanocrystals. Release of HCPT from HCPT/co-D₁6 NCs completed within 48 h, and the cytotoxicity of the nanocrystals against HepG2 cells （IC50:8.7 μg/mL v.s.16.7 μg/mL, p < 0.001） enhanced significantly in vitro. The antitumor efficacy in vivo of HCPT/co-D₁6 NCs of low dose （1.25 mg/kg） was improved significantly compared to HCPT injection of high dose （5 mg/kg） （60% v.s.46%, p< 0.01）. The in vivo study in H22 bearing mice after intravenous injection proved that, in contrast to injections, nanocrystals exhibited a remarkably enhanced biodistribution in each tissues (heart （88.14-fold AUC0-24h）, liver （61.56-fold AUC0-24h）, spleen （19.95-fold AUC0-24h）, lung （84.88-fold AUC0-24h）, renal （244.11-fold AUC0-24h）, brain （2.50-fold AUC0-24h） and tumor （3.02-fold AUC0-24h）. The in vivo study in 4T1 bearing nude mice after intravenous injection of Dir coating nanocrystals has revealed similar results. The average fluorescence intensity was high compared with injection at different time in the mice, and also in in vitro tissues at 24 h after administration. Above all, HCPT/co-D₁6 NCs were expected to become a more effective treatment of tumor drug delivery systems. The DTX-loaded nanocrystals were investigated with as high drug-loading content as 74%（w/w）. The particle size of DTX/co-D₁6 NCs was about 252.0 nm with uniform rod structure morphology and the Zeta potential was approximately 25.5 mV. DTX was existed as the crystalline compound in the nanoparticles. Release of DTX from DTX/co-D₁6 NCs completed within 192 h, and the cytotoxicity of nanoparticles against 4T1 cells （IC50:0.7 μg/mL NCs v.s.3.8 μg/mL Sol.） enhanced significantly in vitro, which indicated the effective of the nanocrystals in vivo.In the last part, the polyprodrug PGV was synthesized from co-D₁6 and vincristine （VCR） via the pH-sensitive imine bond. Dynamic light scattering and transmission electron microscopy demonstrated that PGV self-assembled into spherical aggregates with a mean diameter of approximately 110 nm in aqueous solutions. The characteristic of the polyprodrug was studied, PGV demonstrating a good capacity for the loading of VCR, exhibiting drug-loading content （DLC） as high as 32%（reversible conjugation）. The cytotoxicity of PGV was evaluated in vitro. PGV showed time-dependent cytotoxic effect for MCF-7 cells in vitro, the IC50 values of 24 h,48 h,72 h and 96 h were 155.6, 33.9,4.2 and 1.6 μg/mL, respectively, but the IC50 values were still relatively high compared with free VCR due to the sustained release property. Specifically, PGV behaved as prodrugs, releasing VCR at a nearly constant rate during a long period of 16 days, with no burst release in pH 5.5 PBS. All of these properties revealed that PGV has good potential to be used for i.v. injectable anticancer drug release, especially in long-acting interventional chemotherapy.In summary, on the basis of the previous synthesis of a series of codendrimers named co-D_x, this study explored the applicability of co-D_x as drug carriers for micelles at first, and the codendrimer co-D₁6 was found as the most suitable drug carrier. Then, the codendrimer co-D₁6 was applicated to prepare nanoscale drug delivery systems of high drug loading content such as MTX or HK nanoparticles and HCPT or DTX nanocrystals. The in vitro and in vivo antitumor effects of the drug delivery systems were investigated deeply. Apart from physical encapsulation of drugs, co-D₁6 could also couple vincristine through pH sensitive chemical bond to prepare PGV, which realized the pH sensitive drug release behavior. In a word, co-D_x could carry different types of lipid soluble drugs not only by physical entrapment but also through chemical coupling in order to obtain drug-codendrimer nanoaggregates. co-D_x was a newly medical polymer material with broad application prospects.