Targeted Multidrug Resistance Reversal In Tumor Based On PEG-PLL-PLGA Polymer Nano Drug Delivery System

Part I Biocompatibility Assessment of PEG-PLL-PLGA Nanoparticles In Vitro and In VivoObjective The present study was designed to evaluate the biocompatibility of polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid copolymer (PLGA) (PEG-PLL-PLGA) nanoparticles (NPs) before clinical application.Methods PEG-PLL-PLGA, the synthesis of biodegradable material PEG, PLL and PLGA, followed by loading doxorubicin (DNR), was prepared to act as a novel nano drug delivery system (NDDS). The current study applied some tests to assess the safety of PEG-PLL-PLGA. Cell apoptosis of DNR-PEG-PLL-PLGA and intracellular accumulations of DNR were determined by flow cytometry (FCM) assay. Hemolysis test and micronucleus (MN) assay were carried out to estimate its blood toxicity and genotoxicity. DNR-PEG-PLL-PLGA was injected into mice through tail vein to calculate the median lethal dose (LD50). Blood was taken by removing the eyeball of mice to evaluate the influence of DNR-PEG-PLL-PLGA in hepatic and renal functions. Furthermore, the pathologic changes of heart, liver, spleen, lung and kidney were observed in PEG-PLL-PLGA treated mice by hematoxylin-eosin (HE) of histopathological examination.Results The surface morphology of PEG-PLL-PLGA was spherical shape and dispersed uniformly under transmission electron microscope (TEM). The polydispersity index (PI) was 0.085±0.017 (n= 3), and the average diameter was about 229±15 nm detected by laser particle size analyzer. The Zeta potential was -20.1±1.6 mV. Different concentrations of PEG-PLL-PLGA were grade 1 which belonged to no cytotoxicity. FCM showed that the relative fluorescence intensity (RFI) of DNR was 4.27±2.06,and RFI of DNR-PEG-PLL-PLGA was 4.39±1.89. Both DNR-PEG-PLL-PLGA and DNR treated groups significantly increased the intracellular DNR concentration (P<0.05) as compared with the control group. The apoptotic rate induced by DNR-PEG-PLL-PLGA and the fluorescence intensity of intracellular DNR demonstrated that DNR-PEG-PLL-PLGA could be taken up by the mouse fibroblast cells (L929 cells). Hemolysis test and MN assay demonstrated that the PEG-PLL-PLGA has no obviously blood toxicity and genotoxicity. LD50 of DNR-PEG-PLL-PLGA was 464.4 mg/kg (the relative concentration of DNR was 23.22 mg/kg based on the loading efficiency), the 95% confidence interval was 399.00-541.80 mg/kg. The LD50 results showed that DNR-PEG-PLL-PLGA had a wide safe scale. Compared with the negative control group, there were no significant pathological changes of organizational structure and cell morphology in DNR-PEG-PLL-PLGA group. The results of acute toxicity testing revealed that DNR-PEG-PLL-PLGA group had no hepatic and renal toxicity in mice.Conclusions Therefore, PEG-PLL-PLGA appeared to be highly biocompatible and safety in vitro and in vivo that could be suitable for further application in the treatment of tumor as NDDS.Part Ⅱ Targeted Multidrug Resistance Reversal in Tumor Based on PEG-PLL-PLGA NanoparticlesObjective The research investigated the reversal of multidrug resistance (MDR), and the biodistribution of a new class of PEG-PLL-PLGA followed by loading DNR and tetrandrine (Tet) that targeted leukemia cells in nude mice model via a surface bound tranferrin (Tf).Methods The PEG-PLL-PLGA followed by loading DNR and Tet was synthesized as previously reported using a double-emulsion method. The carbonyldiimidazole (CDI) group spontaneously reacted with Tf to form Tf-PEG-PLL-PLGA, freezing high-speed centrifugation and gel permeation chromatography protein to detect the crosslinking rate were prepared to synthesis DNR-Tet-Tf-PEG-PLL-PLGA (PEG-PLL-PLGA abbreviation as NPs, DNR-Tet-Tf-NPs) drug-loaded nanoparticles. A total of 36 nude mice were subcutaneously injected in the right forelimb with 1× 107 K562/A02 cells/0.2 mL per nude mice, and subcutaneous injected site developed grain sized tumors. When the tumor size reached 75-150 mm3, the mice were randomly divided into six groups:Group A, control group, mice were treated with 0.9% normal saline 0.2 mL; Group B, DNR; Group C, DNR and Tet; Group D, DNR-NPs; Group E, DNR-Tet-NPs; Group F, DNR-Tet-Tf-NPs. These nude mice were administered with their treatment regimen intravenously by tail vein on every other day. The anti-tumor effect treated with drug was observed and the volume of tumor growth was calculated through tumor inhibition rate (IR). After 14 days treatment, the mice were sacrificed by cervical dislocation, and heart, liver, spleen, lung, kidney and tumor tissues isolated from each group, the internal structure of the organization was examined by microscope. Expression of Bax, Bc1-2, Caspase3 and Survivin was detected by using immuno-histochemical staining (streptavidin-perosidase, SP) method. P-glycoprotein (P-gp), MRP, NF-κB, transferrin receptor (TfR) mRNA and protein expression observed by quantitative real-time PCR (qRT-PCR) and Western blot to evaluate the effect of multimodal treatment on overcoming MDR. The concentration of DNR in each tissue was calculated using HPLC for subsequent determinations of its distribution in tissues from the different groups. FCM analysis of co-labelled with the near-infrared fluorescent (NIRF) dye, NIR797 was used to demonstrate the near-infrared imaging distribution of NIR797-labeled-NPs and NIR797-labeled-Tf-NPs in nude mice.Results Injection of DNR-Tet-Tf-NPs into nude mice bearing MDR leukemia cells K562/A02 xenografts was shown to inhibit tumor growth, the tumor inhibition rate was highest in the DNR-Tet-Tf-NPs group compared with the other five groups which had the significant difference (P＜O.05). These results indicated that DNR-Tet-Tf-NPs group with targeted anti-tumor effect and reversal of MDR function. Each group of heart, liver, spleen, lung and kidney had no significant pathological changes. Moreover, no chemotherapeutic drug-induced histological changes and tumor metastasis could be seen under histopathological examination. However, contemporaneous immuno-histochemical analysis of tumor tissue showed the targeted nanoparticles induced apoptosis in tumor cells. Expression of Bax, Caspase-3 in DNR-Tet-NPs and DNR-Tet-Tf-NPs groups compared to the control group and DNR group, positive cells markedly increased. The opposite result was observed in Bcl-2, Survivin expression, positive cells obviously decreased. The results showed the strongest Bax, Caspase-3 expression but the weakest Bcl-2, Survivin expression in DNR-Tet-Tf-NPs group. Targeted tumor cells exhibited a marked increase in TfR expression, with noticeable decreases in P-gp, MRP and NF-κB, as assessed by qRT-PCR and Western blot analysis. Moreover, in HPLC experiment, the DNR concentration of tumor tissue was higher after injection of DNR-Tet-Tf-NPs than DNR-Tet-NPs (P<0.05). Through the Caliper IVIS (In Vivo Imaging System) of the major organs and part of tumors found that a large number of NIR797-labeled nanoparticles mainly gathered at the tumor site, after the injection of 72 h, the strongest signal reached the tumor location. Drug accumulation at the tumor site of NIR797-labeled DNR-Tet-Tf-NPs group by NIRF imaging was significantly higher than in the NIR797-labeled DNR-Tet-NPs group suggesting that Tf modified nanoparticles have targeted anti-tumor effect.Conclusions In summary, DNR-Tet-Tf-NPs could provide a specific and effective means to target nano drugs to MDR tumor cells.