Core-shell Structure Nanocarriers Co-loaded Doxorubicin And Gene For Cancer Combination Therapy

Cancer is one of the most intractable diseases which influence the life-time of human.Tumorigenesis is a complex process involving multiple pathological pathways and complicated microenvironments changes.Combination therapy instead of monotherapy has been widely accepted in clinical.In recent years,combination of chemotherapy and gene therapy has emerged as a preferred method of treatment for cancer.Combination of chemotherapeutic drug and gene has advantages such as reducing the dosage of drug,decreasing side effects and increasing treatment efficiency.However,it is difficult to co-delivery of chemotherapeutic drug and gene cause of the differences in the physical properties of small molecule chemical drugs and macromolecular gene drugs.Co-delivery of chemotherapeutic drug and gene is thus a serious challenge in combination therapy.Efficient co-delivery strategy was expected not only to co-load the given chemotherapeutic drug and gene,but also to deliver the drug and gene to the predetermined targeting sites,even if the given chemotherapeutic drug and gene have different targets.Thus,reasonable combination of chemotherapeutic drug and gene may bring about synergistic effect in caner therapy.Lately,core-shell structure nanoparticles were emerged as potential co-delivery carriers for combination of chemotherapy and gene therapy due to its superiorities in multilayer ordered structure for loading and controlled release behavior.Two kinds of core-shell structure nanoparticles were developed and the feasibilities in combination of chemotherapy and gene therapy were evaluated.The first is for co-delivery of drug and gene to the same cell(tumor cells)and the second is for co-delivery of drug and gene to tumor cells and M2-type tumor-associated macrophages(M2-type TAMs),separately.The drug and gene were co-loaded in the core-shell nanoparticles by different method to meet different delivery requirements.Tumor cells targeting core-shell structure co-delivery nanoparticles(HDDN)have tumor microenvironment pH and enzyme dual-responsive properties.It was designed to improve the tumor cells targeting efficiency and to decrease the toxicity of chemotherapeutic drug to non-tumor tissue.The xenotype cells targeting core-shell structure co-delivery nanoparticles(CDMPR)were designed to co-load chemotherapeutic drug and gene,then deliver chemotherapeutic drug to tumor cell and gene to M2-type TAMs,which was named xenotype cells stepwise targeting co-delivery strategy.The delivery process of CDMPR was investigated in vitro and in.vivo to verify the xenotype cells stepwise targeting co-delivery feasibility.The main researches and results were shown as follows:Part I Dual-responsive core-shell structure nanoparticles for targeting co-delivery of doxorubicin(DOX)and gene to tumor cellsChapter 1.The cationic drug loading material doxorubicin-4-hydrazinobenzoic acid-polyetherimide(DOX-HBA-PEI,DHP)was synthesized and the structure of DHP was proved by 1H NMR and FTIR.The drug loading amount of DHP was 13.90± 1.87%.The accumulated release of DOX from DHP at pH 5.0 was more than that at pH7.4,which exhibited the pH-responsive release behaviors.Compared with PEI,the HBA-PEI exhibited non-toxicity(p<0.01).The IC50 of DHP on HepG2 and B16 cell lines were 0.12 ± 0.01 ?g/mL and 0.36 ± 0.25 ?g/mL,respectively.This result proved that chemical modification of DOX did not influence the antituomr efficiency.Chapter 2.The dual-responsive core-shell structure nanoparticles were assembled by the electrostatic effect.The core DHP/DNA(DDN)was formed fabricated through the electrostatic interaction between DHP and DNA.When the ratio of DHP to DNA was larger than 30:16,it could form the inner core.After tested the transfection efficiency and the characteristic of the core,DDN at 30:4 was considered as the prescription of the inner core and it would be used for preparing HA/DDN(HDDN).The particle size of DDN was 77.83 ±12.96 nm and the zeta potential of DDN was 22.6 ±2.86 mV.The HDDN was fabricated by absorption of negative charged hyaluronic acid(HA)on the surface of the positive charge DDN.The optimal prescription of HDDN was the ratio of HA to DNA at 70:8.The particle size of HDDN was 148.3±3.88 nm and the zeta potential of HDDN was-18.1±2.03 mV.The HDDN exhibited the round morphology and well dispersibility.Furthermore,HDDN could protect the DNA from being degraded by DNase and serum.It has the enzyme-triggered release and disassembly behaviour.Chapter 3.HepG2 and B16 were selected as CD44 positive cell lines and NIH3T3 was chose as CD44 negative cell line.The cellular uptake of HDDN in B16 and HepG2 cells were 70%which was higher than that in NIH3T3 cells(p<0.05).This result proved that HDDN had tumor cells targeting ability.The internalization mechanism of HDDN was through CD44-mediated endocytosis effect.The IC50 value of HDDN on HepG2 and B16 cell lines were 13.88±1.77 ?g/mL and 3.68±0.56?g/mL,respectively.Furthermore,The transfection efficiency of HDDN in 10%serum was equal to that in the absence of serum while the transfection of DDN was significantly decreased in the presence of 10%serum.The efficient co-delivery of DOX and pEGFP was proved by fluorescent image taken by laser confocal microscope.Part 2 Stepwise targeting core-shell structure nanoparticles for co-delivery of DOX and siRNA to xenotype cells in chemo-immunotherapyChapter 4.The tumor tissue targeting material O-carboxymethyl-chitosan-poly(ethylene glycol)-aspargine-glycine-arginine(CMCS-PEG-NGR,CPN)and M2-type TAMs targeting material mannose-PEI(MPEI)were synthesized and confirmed by 1H NMR.The drug loading shell material CMCS-DOX-PEG-NGR(CDPN)were synthesized and characterized.The drug loading of CDPN was 16.48%and the isoelectric point of CDPN was 6.53.Chapter 5.Stepwise targeting core-shell structure nanoparticles were developed to co-delivery of DOX and siRNA to xenotype cells.The inner core MPEI/siRNA(MPR)was fabricated through the electrostatic interaction between MPEI and siRNA.When the ratio of MPEI to siRNA was larger than 1:1,MPR could be assembled.MPEI to siRNA at 2:1 was considered as the optimal prescription of MPR and it was used for preparing CDPN/MPR(CDMPR).The CDMPR was fabricated by absorption of negative charged drug loading material CDPN on the surface of the positive charge MPR.The optimal ratio of CDPN to siRNA was 8:1.The particle size of MPR was 62.3±1.4 nm and the zeta potential of MPR was 27.1 ±2.2 mV.The particle size of CDMPR was 168.5±1.0 nm and the zeta potential of CDMPR was-9.0±2.3 mV.The CDMPR exhibited the round morphology and good dispersibility.CDMPR showed the pH responsive charge reversal ability which led to the pH-responsive detachability of CDPN from MPR if the microevirment pH value was exactly that range.CDMPR could protect siRNA from being degraded by RNase in circulation.The accumulated release of DOX from CDMPR was 52.92%±0.83%at pH 6.5,which was significantly higher than that at pH 7.4(17.95%±0.77%)(p<0.01).These results proved the pH-sensitive drug release behavior of CDMPR.Chapter 6.The cellular uptake study proved the M2-type TAMs targeting ability of MPR compared with PR.The internalization of CDMPR was pH depended.The cellular uptake of CDMPR on RAW264.7 cell lines at pH 6.5 was higher than that at pH7.4,which was consistent with the isoelectric point of CDPN.The cellular uptake test was carried out using co-incubation of M2-type RAW264.7 cells and Hepa1-6 cells transwell model to investigate the xenotype targeting ability of CDMPR.The Cy3-siRNA could be delivered to M2-type RAW264.7 cells at 1 h and DOX could be internalized by the Hepal-6 cells at 4 h.The fluorescence of siRNA was not emerged in the tumor cells.IKK?-siRNA was used as model gene.The results showed that CDMPR has the potential to transfect siRNA into RAW264.7 cells and decrease the expression of IKK? protein in RAW264.7 cells(p<0.05).Comparing with other groups,the CDMPR could decrease the amount of M2-type RAW264.7 and increase the M1-type RAW264,7 in polarization assay(p<0.05).CDMPR could polarize M2-type RAW264.7 cells to M1-type effectively.MTT assay results proved that CDMPR exhibited the dose-dependent cytotoxicity in vitro.Chapter 7.The delivery process of CDMPR in vivo was investigated on Hepal-6 tumor bearing mice.The drug distribution studies in tumor tissue exhibited that CDMPR was internalized by M2-type TAMs and then DOX were release from M2-type TAMs and internalized by tumor cells.In this sense,M2-type TAMs exhibited as drug-reservoir.The siRNA was delivered to M2-type TAMs precisely.The CDMPR was proved to be accumulated at tumor tissue by the live imaging assay.The antitumor efficiency and TAMs polarization ability of CDMPR were verified on Hepal-6 tumor bearing mice.Comparing with mixture administration,CDMPR exhibited better antitumor efficiency and TAMs polarization ability(p<0.05).CDMPR could enhance the efficiency of chemo-immunotherapy.The HE stain assay proved that CDMPR was preliminary safety in non-tumor tissue.