Studies On PH-responsive And Stepwise-targeting Nanoparticles For Delivery Of SiRNA To M2 Phenotype Tumor Associated Macrophages(TAMs)

Cancer immunotherapy,which is aimed to stimulate a robust host immune response against tumor cells and enhance antitumor immunity in tumor microenvironment,has become a clinical method for cancer treatment.In recent years,cancer immunotherapy has become a new method for cancer treatment after surgery,radiotherapy and chemotherapy.It is now appreciated that most solid tumors are abundantly populated with TAMs,and they are closely related to tumor growth,development and metastasis.TAMs can be devided into M1 phenotype and M2 phentype.M1 phenotype TAMs are able to produce copious amounts of pro-inflammatory cytokines,acyivate the immune response,kill microorganisms and tumor cells.M2 phenotype TAMs are generally considered to be able to promote tune inflammatory responses,accelerate angiogenesis,tissue remodeling,tumor cell activation,metastasis and immunosuppression.As the tumor cells could secrete factors like IL-10,CSF-1 and TGF?,most of the TAMs show an M2 like phenotype.Killing M2 TAMs and promoting the conversion of M2 TAMs to an immunogenic and antitumor phenotype are new approaches in cancer immunotherapy.Thus,M2 TAMs are considered to be a promising target for treating patients with malignant tumors.At present,immunotherapy based on M2 TAMs mainly includes monoclonal antibody,small molecular inhibitor and siRNA Among these strategies.siRNA has drawn increasing attention due to its highly specific gene regulation by selectively knocking down target genes.To modulate TAMs to an antitumor phenotype,siRNA has the potential to achieve macrophage-specific and pathway-specific cell-signaling modulation.Furthermore,siRNA has the ability to target the pathologic site where drugs are invalid.Recently,some delivery systems including dendrimers,cationic complexes,micelles and other nonviral vectors have been developed for the delivery of siRNA to M2 TAMs.These vehicles were intended to specifically and effectively deliver siRNA to TAMs with minimized side effects.However,as macrophages widely distribute in human tissues,the non-specific delivery of siRNA to macrophages and systemic interference with macrophage behavior may lead to unintended side effects including autoimmune manifestations.Therefore,highly potent and specific delivery vehicles for siRNA are required to facilitate siRNA in reaching their target cells.In this study,a pH-responsive stepwise-taregting delivery system was established.The nanoparticles accumulate at the tumor tissue through both the enhanced permeation and retention effect(EPR effect)and transcytosis of the first targeting material.Under the mildly acidic tumor microenvironment,the pH-responsive first targeting material falls off.The second targeting material of the inner core then exposes and targets M2 TAMs through binding on the receptors over-expressed on these cells.In this study.NGR peptides were chosen as the first targenting ligand,CMCS as the pH sensitive material.CMCS-PEG-NGR(CPN)was synthezed as the first targeting pH-responsive material.Mannose was chosen as the second targeting ligand.PEI was used to condense siRNA.Mannose-PEI(M-PEI)was synthesized as the second targeting material.The positive charged inner core(M-PEI/siRNA,MPR)was fabricated.Negatively charged CPN coated on MPR to fabricate pH-respnsive stepwise-targeting CPN/M-PEI/siRNA(CMPR)through electrostatic interaction.Negative control siRNA was selected as the model gene to fabricated PEI/siRNA(PR),MPR,CPN/PEI/siRNA(CPR)and CMPR.The preparation process,physical and chemical propertites,stability,cellular uptake and intracellular distribution of the preparations was investigated,respectively.The pH sensitivity of CPN was verified.The targeting ability of MPR and CMPR in vivo was characterized.The preliminary safety evaluation was explored.The main research methods and results as as follows:1.Construction and characterization of first targeting nanoparticlesIn this part,first targeting pH-responsive material CPN was synthezed.Negative control siRNA was selected as the model gene.PR and CPR were prepared by incubated at room temperature.Agsrose gel retardation assay was carried out to expore the proper weight ratio of PEI to siRNA and weight ratio of CPN to siRNA,respectidvely.The morphology of the optimized PR and CPR was characterized by transmission electron microscope(TEM).The particle sizes and zeta potential were detected by Malvern Zetasizer Nano ZS-90.Stability of PR and CPR was irnvestigated by agsrose gel retardation assay.Characteristic peaks of CMCS,PEG and NGR appeared in the 1H NMR spectrum of the product,which confirmed the successful synthesis of CPN.The optimized weight ratios of PEI to siRNA and CPN to siRNA were 2:1 and 4:1,respectively.TEM images of both PR and CPR displayed a type of analogous spherical shape.Size measurements showed an average 83.7±4.9 um diameter of PR and 154.5±2.1 nm of CPR.Zeta potentials of PR and CPR were 20.97±1.53 mV and-14.10±0.90 mV,respectively.The PDI of both PR and CPR were less than 0.2.Within 12 h,both PR and CPR could protect siRNA against RNase A and serum.2.Construction and characterization of pH-responsive stepwise-targeting nanoparticlesIn this part,second targeting material M-PEI was synthezed.Negative control siRNA was selected as the model gene.MPR and CMPR were prepared by incubated at room temperature.Agsrose gel retardation assay was carried out to expore the proper weight ratio of M-PEI to siRNA and weight ratio of CPN to siRNA,respectidvely.The morphology of the optimized MPR and CMPR was characterized by TEM.The particle sizes and zeta potential were detected by Malvern Zetasizer Nano ZS-90.Stability of MPR and CMPR was investigated by agsrose gel retardation assay and Malvern Zetasizer Nano ZS-90.The determination method of siRNA in PBS with different pH values was established.The release of siRNA from CMPR in PBS with pH 5.0,6.5 and 7.4 was tested using a dialysis bag diffusion technich.The isoelectric point of CPN was detected by acid-base titration assay.Characteristic peaks of MPITC and PEI appeared in the 1H NMR spectrum of the product,which confirmed the successful synthesis of M-PEI.The IR spectrum further confirmed the synthesis of M-PEI.The optimized weight ratios of M-PEI to siRNA and CPN to siRNA were 2:1 and 4:1,respectively.TEM images of both MPR and CMPR displayed a type of analogous spherical shape.Size measurements showed an average 93.2±4.1 nm diameter of MPR and 146.4±6.9 nm of CMPR.Zeta potentials of MPR and CMPR were 16.400.92 mV and-17.00±0.10 mV,respectively.The PDI of both PR and CPR were around 0.2.Within 12 h,both MPR and CMPR could protect siRNA against RNase A and serum.Furthermore,CMPR was more stable than MPR in 10%plasma.The results of in vitro release showed that the cumulative release of siRNA from CMPR under pH 5.0 and pH 6.5 conditions were significantly higher than that under pH 7.4 condition.The releasese of siRNA was pH sensitive.The isoelectric point of CPN was detected to 6.33.3.Targeting evaluation of pH-responsive stepwise-targeting nanoparticles in vivo and in vitro and their preliminary safety evaluationIn this part,macrophages were labled with F4/80 antibody,M2 TAMs with CD206 antibody,siRNA with Cy3 or Cy5.The uptake of PR and MPR were investigated confocal laser scanning microscopy images were taken on murine peritoneal macrophages.Fluorescence co-localization and quantitative analysis of siRNA and CD206 was investigated.Competitive binding assay of MPR was carried out on murine peritoneal macrophages through confocal laser scanning microscopy.The uptake of CMPR on murine peritoneal macrophages under pH 6.5 and pH 7.4 conditions were investigated.Fluorescence imaging assays in vivo and in vitro were carried out to investigate the biodistribution of CMPR.Cyto-section study was carried out to explore the uptake pf PR,MPR and CMPR by the cells in the tumor tissue.Finally,hemolytic and histological analysis was conducted to verify the primary safety of PR,MPR,CPR and CMPR.Results of cell uptake showed that there was no significant difference in the uptake of PR and MPR by M2 TAMs after incubation with the nanoparticles for 1 h or 4 h.Results of the competitive binding assay showed that the co-localization efficiency of siRNA and CD206 in M2 TAMs was 1.63 times higher than that of the suppressed group after incubated with MPR for 1 h.There was no significant difference between the two groups when the cells incubated with MPR for 4 h.The co-localization efficiency of siRNA and CD206 under pH 6.5 was 1.40 higher than that under pH 7.4.Fluorescence imaging assays indicated that the fluorescenceintensity of tumor was 2.96×108 in CMPR group was 4.65 times stronger than that in the free siRNA group(6.36×107).Results of cyto-section study showed that MPR and CMPR were mainly uptaken by M2 TAMs compared with free siRNA and PR.Hemolytic test implied that PR,MPR,CPR and CMPR were safe to injection without hemolysis.Histological analysis illustrated that there were no pathological change of organs of mice which were administrated with PR,MPR,CPR and CMPR.The primary safety of the nanoparticles was good.In this study,CPN and M-PEI were synthezed as the first targeting pH-responsive material and the second targeting material,respectively to construct a kind of pH-responsive and stepwise-targeting nanoparticles CMPR.CMPR increased thier accumulation at the tumor tissue through the first targeting ability.And the increased accumulation in M2 TAMs was achieved by the second targeting.CMPR have good stability and biocompatibility,which have the great potential for further exploitation.This study provides a new idea and new method for the development of delivery systems for the delivery of siRNA to M2 TAMs.