Study On The Antitumor Of T7-modified Ph-Redox Dual Sensitive Polyamidoamine As A Co-delivery Carrier For SiRNA And Doxorubicin

Objective: In this paper,an active-targeted,p H and redox-sensitive polyamidoamine complex was used as a carrier and simultaneously loaded with doxorubicin(DOX)and PKM2-si RNA to construct a tumor microenvironment responsive targeted co-delivery system.We examined the characteristics of the carrier through a series of experimental indicators.Meanwhile,its in vitro and in vivo anti-tumor effects and mechanisms were also studied and evaluated.Methods: Based on the 5th generation PAMAM(G5.0 PAMAM)as the scaffold,the bifunctional polyethylene glycol(HS-PEG-COOH)is linked via the crosslinker SPDP to the terminal amino group of PAMAM to form a disulfide bond with reductive sensitivity,and the other end is coupled with the active targeting peptide T7 to form T7-PEG-SS-PAMAM(T7-PSSP);The doxorubicin(DOX)was loaded into the hydrophobic cavity of PAMAM via physical encapsulation to obtain T7-PSSP/DOX complexes.Subsequently,PKM2-si RNA was adsorbed onto T7-PSSP/DOX to form the codelivery system T7-PSSP/DOX/si RNA.The structure of polymer-gene complex was characterized by 1H-NMR,NICOMP particle size and potential analyzer.The stability and redox release behavior of the complex were investigated by the agarose gel electrophoresis.MTT assay,Flow Cytometry(FCM)and Confocal Microscopy(CLSM)were used to evaluate the safety,cell uptake,intracellular co-localization and lysosomal escape of the carrier.After administration,the expression of PKM2 and related proteins were detected by RT-PCR and Western Blot.The dialysis method and MTT were applied to evaluate drug release behavior and the synergistic inhibitory effect in vitro.At last,we established hepatocarcinoma model of Hep G2 and used live-imaging technique to investigate the differences in the distribution of the complex in nude mice.The tumor growth was monitored during DOX and si RNA co-administration.Compared to other groups,T7-PSSP/DOX/si RNA significantly reduced tumor volume in Hep G2 cells-bearing nude mice,showing synergistic effects of DOX and PKM2 si RNA.H&E staining also indicated that the most potential therapeutic effects of T7-PSSP/DOX/si RNA on the tumor without distinct damages to normal tissues.Rusults:(1)The cationic carrier T7-PSSP was successfully synthesized.The carrier has a well-rounded and uniform particle size.After the si RNA is loaded,the particle size is 119.8±0.76 nm with a positive charge of 8.92±0.38 m V.The MTT assay proves that it has good biocompatibility because the survival rate of cell is more than 90% when the concentration is at a safe range(0~100 ?g/m L).(2)Agarose gel experiments showed that T7-PSSP has a higher compression ability against si RNA.Anti-RNase and serum protection experiments also showed that the carrier can well protect the si RNA from degradation,the in vitro storage assay at 4°C showed that the particle size of the carrier complex did not change significantly during the 28 days,indicating that T7-PSSP/si RNA has a good stability.Besides,T7-PSSP/si RNA can efficiently release the carried si RNA in the high GSH condition to further show its release behavior has reduction sensitivity.(3)In vitro uptake experiments showed that the modification of T7 peptide can significantly increase the uptake of si RNA in Hep G2 cells compared to the non-targeted group.RT-PCR and Western Blot analysis showed that the T7-PSSP/si RNA group had the highest gene down-regulation and protein silencing levels.The results of in vitro pharmacodynamics showed that the metabolic level of cells was obviously affected:the level of intracellular ATP and lactic acid production decreased,the migration rate of cells weakened and the rate of apoptosis increased significantly.(4)The co-localization of DOX and si RNA in the cytoplasm were indicated the simultaneous delivery of si RNA and DOX after incubating with T7-PSSP/DOX/si RNA.With the prolongation of incubation time,the fluorescence of DOX and FAM-si RNA in cells gradually increased,indicating DOX and si RNA can accumulated efficiently in cell.DOX signal gradually transferred from the cytoplasm to the nucleus,while almost of si RNA still located in the cytoplasm,thus improving synergistic therapeutic efficacy in their each targeted region.From the release curve,it can be seen that T7-PSSP/DOX/si RNA has obvious acid and reductive sensitivities which can rapidly release the drug under the stimulation of intracellular environment.Our results showed that by simultaneously delivering DOX and PKM2-si RNA into cancer cells,the PKM2-si RNA can silence the PKM2 m RNA with effect and significantly suppress the cell metabolism and substantially enhance the antic-ancer action of DOX.T7-PSSP/DOX/PKM2-si RNA has stronger ability to inhibit the growth of Hep G2 cells than T7-PSSP/DOX.Western Blotting was also used to examine the mechanism of its co-inhibition of cell growth.We found that when PKM2-si RNA was loaded alone,the expression of intracellular tumor suppressor protein P53 and apoptotic protein Bax increased,and the expression level of both proteins further increased at the time of co-loading.We come to a conclusion that DOX and PKM2-si RNA can exert a synergistic effect on this signal pathway.(5)Fluorescence imaging of nude mice and tissues showed that our carrier can help si RNAs have longer retention time in vivo than naked si RNA.Naked si RNAs were quickly excreted from kidney,while complexes were localized in kidney,liver and tumor.Besides,T7-PSSP can rapidly deliver si RNA to the tumor site than PSSP.In vivo pharmacodynamic experiments showed that the co-delivery system T7-PSSP/DOX/si RNA has the best antitumor effect.H&E analysis indicated that T7-PSSP/DOX/si RNA exhibited almost no toxicity to normal organs,which was a great improvement in safty compared with free DOX.Conclusions: The synthesized T7-PSSP polymer exhibited a higher encapsulation efficacy for DOX and si RNA,and can effectively release DOX and si RNA under the stimulation of tumor cell microenvironment.The complex can efficiency target the tumor site and silence the PKM2 protein while up-regulating the tumor suppressor protein P53 and the apoptosis protein Bax,thus increasing the sensitivity of cells to DOX and exerting a synergistic anti-tumor effect.Therefore,T7-PSSP is expected to become an efficient biological environment-responsive drug/gene co-delivery carrier.