Study On Multifunctional Guanidination PAMAM For Co-delivery Of SiRNA And DOX To Treat Breast Cancer

Objective:Since the previous construction of pH/redox sensitive nanocarrier PEG-S-S-PAMAM?PSSP?based on the Generation 5 PAMAM,PEG-S-S-PAMAM-BGD?PSSPBG?was developed by further modifying with biguanidine?BGD?based on the Generation 5 PAMAM in this study,in order to co-delivery of chemotherapeutic agent doxorubicin?DOX?and gene agent STAT3 small interfering RNA?STAT3-siRNA?.And,the in vitro and in vivo anti breast tumor effect and the relevant molecular mechanisms were evaluated.Methods:Three polyamidoamine PAMAM-BGDs?PBG?with different biguanidine modification ratios?25,50,100 BGDs per PAMAM?were synthetised,then the antitumor drug DOX was physically loaded into the hydrophobic cavity of polymers to obtain the DOX-loaded complexes(PBG₂₅/DOX,PBG₅₀/DOX,PBG₁₀₀/DOX).The effects of biguandination on DOX uptake was studied by using flow cytometry,and the optimal biguanidine modification ratio PBG was determined to be PBG₁₀₀/DOX.The PEG was respectively attached to the selected PBG and PAMAM through redox-cleavable disulfide bond,yielding PSSPBG and PSSP conjugates.Then,DOX was loaded into the hydrophobic core through physical encapsulation,forming PSSPBG/DOX and PSSP/DOX complexes.The particle size,Zeta potential,drug loading,encapsulation efficiency,cell uptake and in vitro release profiles were evaluated.And the siRNA binding capacity,serum stability,transfection efficiency,transport mechanism,lysosomal escape ability and cell membrane integrity were also studied.RT-PCR and Western Blot were used to detect the expression level of STAT3 gene and protein.MTT and Western blot were used to investigate the in vitro antitumor effect and antitumor molecular mechanisms,respectively.The MCF-7 tumor-bearing nude mice model was established to study the in vivo antitumor effect,and the pathological changes of tumor and the main organs were evaluated by H&E staining.Results:The ¹H-NMR results confirmed successful synthesis of biguanidine?BG?modified PAMAM conjugates.Flow cytometry assay results showed that the cell uptake efficiency was enhanced as the degree of BG modification increased.The particle sizes of all the PBG/DOX complexes were about 40⁵0 nm,and had good drug loading and entrapment efficiency.Then,PSSPBG?PBG with best cell uptake efficiency modified with PEG through disulfide bonds?and PSSP?PAMAM modified with PEG through disulfide bonds?were successfully synthesized and characterized by ¹H-NMR.Experiments showed that the Zeta potential reduced after biguanidination.MTT assay demonstrated biguanidination can further increase the safety of the carrier.PSSPBG/DOX complex showed obvious pH/redox sensitivity.Compared to PSSP/siRNA complex,biguanidine modified PSSPBG/siRNA complex has higher cell uptake.PSSP/siRNA and PSSPBG/siRNA complexes were both observed to achieve good lysosomal escape ability by laser confocal microscopy.Uptake mechanism study proved that in addition to traditional clathrin mediated endocytosis pathway,PSSPBG/siRNA and PSSP/siRNA nanoparticles could also directly penetrate the cell membrane by inducing the instability of MCF-7 cell membrane,and release the drug under the high redox microenvironment of cytoplasm.Besides,compared with PSSP/siRNA,PSSPBG/siRNA showed higher ability to penetrate the cell membrane.Compared with PSSP/STAT3-siRNA transfected cells,PSSPBG/STAT3-siRNA transfected cells had lower expression of STAT3 and Bcl-2 gene at mRNA and protein levels.Also,the apoptosis rate of PSSPBG/STAT3-siRNA transfected cells was increased.The cytotoxicity results prevealed that PSSPBG/DOX complex with biguanidine had lower IC₅₀value compared with PSSP/DOX complex,and co-delivery of STAT3-siRNA and DOX group had the lowest IC₅₀value.Live-imaging experiments of nude mice showed that PSSP and PSSPBG conjugates could effectively accumulate at tumor sites and maintain a long time relatively.The results of in vivo antitumor activity showed that PSSPBG/DOX had better antitumor effect compared with PSSP/DOX,andtheco-deliveryofSTAT3-siRNAandDOXgroup?PSSPBG/DOX/STAT3-siRNA?had obviously enhanced antitumor effect than that of DOX alone group.Finally,H&E staining showed that PSSPBG/DOX/siRNA could significantly reduce the side effects of DOX.Conclusion:In this study,biguanidine modified PSSPBG conjugate with pH/redox dual sensitivity was successfully constructed based on G5 PAMAM.The nanoparticles had homogeneous size distribution,high encapsulation efficiency and drug loading.And in vitro drug release activity showed that it displayed apparently redox and pH dual sensitive drug release profile.The PSSPBG conjugate showed good biocompatibility,enhanced cell uptake,high siRNA loading and effective gene delivery ability,which greatly decresed the expression of certain carcinogenic gene and proteins.The PSSPBG conjugate can also be used for co-delivery of siRNA and DOX to improve the anti-tumor effect.This antitumor drug delivery system combines gene therapy with traditional chemotherapy,which would have further research value in the field of cancer treatment.