| In recent years,the therapy of immune checkpoint inhibitors（ICIs）has become one of the most promising strategies in the field of cancer treatment.Compared with chemotherapy and radiotherapy,the advantage of ICIs is that it can directly restore the exhausted host antitumor immune responses mediated by the tumor microenvironment.ICIS such as programmed death receptor-1/programmed death ligand-1（PD-1/PD-L1）inhibitors can block the co-inhibitory signal by blocking the binding of PD-1 and PD-L1 to rejuvenate the effector function of T cell.However,different patients have different responses to PD-1/PD-L1 inhibitors,and some patients relapse after treatment.So patients will develop primary and acquired resistance to PD-1/PD-L1 blockade therapy[1-3].Studies have found that epigenetic therapy can reverse the resistance of ICIS treatment in many ways.Each step of the immune process（1.Antigen presentation and T cell activation;2.T cells transport and infiltrate into the tumor;3.T cells recognize and remove tumor cells）can be regulated by epigenetic therapy[4],destroying the immunosuppressive state.Epigenetic therapy combined with immune checkpoint inhibitors can restore immune recognition and eliminate tumor to increase the clinical response rate.Epigenetic regulation drugs such as histone deacetylase inhibitor（HDACi）can stimulate anti-tumor immunity of both tumor cells and host immune cells at the same time,so as to enhance the body’s response to anti-PD-1 treatment[5,6].Therefore,the combination of HDACi and PD-1/PD-L1 inhibitors has received widespread attention.It is expected to exert the greater effect on tumor treatment.In immune checkpoint inhibitors,compared to PD-L1 monoclonal antibody that binds to membrane proteins only,siRNA-PD-L1 can down-regulate the expression of PD-L1 protein in membranes and cytoplasm by degrading endogenous messenger RNA（m RNA）,which has better potential for application.However,free siRNA is easily degraded by RNase during administration and circulation in the body.HDACis are usually poorly water-soluble and have a short half-life.For example,the half-life of vorinostat/suberoylanilide hydroxamic acid（SAHA）is only 0.8-3.9 h,which leads to restrictions on the route of administration and poor therapeutic effects.Therefore,the direct combined administration of these two kinds of drugs often leads to insufficient synergy and complex pharmacokinetic problems.It is necessary to construct a new type of drug co-delivery carrier system with modern pharmaceutical technology to realize the co-administration of HDACi and siRNA-PD-L1,playing a highly effective and synergistic antitumor effect.In this study,we synthesized an acid-activatable micelles co-loaded with SAHA and siRNA-PD-L1 for inhibiting immune escape and enhancing cancer immunotherapy.The micelles were composed of three independent components.The first component was a p H-responsive deblock copolymer polyethylene glycol-b-polyasparagine（PEG-b-P[Asp（DET）n],termed as PPD）,which was found to have remarkably low toxicity,facilitating its use for drug delivery.PPD can achieve co-loading and intracellular release through lysosomes because of the unique properties of the ethylenediamine unit（DET）integrated into the polyaspartamide side chains.PPD could co-load SAHA and siRNA-PD-L1,and release them in the cells through lysosomes.The second part was a pan HDACI vorinostat（SAHA）,DET could be covalently connected to SAHA through an ester bond to form polyethylene glycol-b-polyasparagine-vorinostat（PEG-b-P[Asp（DET-SAHA）n],PPDS）.The three part was siRNA-PD-L1,The PEG end of PPDS was hydrophilic,and the P[Asp（DET-SAHA）n]end was hydrophobic.DET in PPD presented the mono-protonated form at p H 7.4,therefore it could stably combine with negatively charged siRNA and self-assemble in water to form micelles,encapsulating siRNA in the core of the micelles to form the final complex siRNA@PPDS.Moreover,the acid-activatable anti-tumor micelles remained inert at physiological p H conditions and were activated upon internalization into lysosome of the tumor cells.Specifically,after siRNA@PPDS being taken up by the cell,DET was further protonated under the acidic environment（p H=5）of lysosomal,which leads to the larger size of micelles,and the micelles disintegrated,so the siRNA was released.At the same time,the lipase broke the ester bond between the PPD and SAHA,thus SAHA was also released.In addition,with the gradual protonation of DET,the p H in the lysosome decreased,chloride ions and H2O entered the lysosome under the action of the proton pump,causing the lysosome to swell and rupture,so the endocytosed drugs were released into the cytoplasm,showing their cooperating anti-tumor effects.In this paper,the anti-tumor effect of micelles siRNA@PPDS was investigated through cell experiments in vitro and tumor-bearing mice experiments in vivo.1.Synthesis,characterization of siRNA@PPDS micelles and the SAHA release in PPDS micelles in vitro.First,the amphiphilic block polymer PPD was prepared as the carrier,in which the diethylenetriamine（DET）was combined with the small molecule antitumor drug SAHA under the action of the condensing agent N,N carbonyl diimidazole（CDI）.The successful conjugation of PPD with SAHA was confirmed by NMR spectrum,which showed the appearance of benzene ring peak of SAHA atδ=7.0–7.5 ppm.siRNA-PD-L1 is negatively charged,and diethylenetriamine is positively charged at p H 7.4,so they can combine through positive and negative charges to form the target product siRNA@PPDS.The agarose gel electrophoresis assay was used to evaluate the loading capability of PPDS with siRNA.The migrations of PPDS bound siRNA were thoroughly blocked at the ratio of 40:1,compared to naked siRNA.Next,the particle size was examined by dynamic light scattering（DLS）measurements with a size of about 179.4±3.1 nm and a narrow size distribution with a polymer dispersibility index（PDI）of 0.238±0.006 at p H7.4.The particle size increased to 266.4±9.6 nm at p H 5 with a wide size distribution（PDI,of 0.380±0.031）,indicating the p H sensitivity of siRNA@PPDS micelles.Transmission electron microscope（TEM）images demonstrated that siRNA@PPDS micelles presented spherical morphology with a size of about 120 nm.The release of SAHA in PPDS was simulated in vitro.Considering that SAHA and PPD was connected by ester bond,we investigated the release profiles in two kinds of buffer solutions（with lipase or without lipase）at37°C using the dialysis method by simulating the normal physiological environment and the endosomal/lysosomal microenvironments in tumor cells.The result showed that in the presence of lipase,77.68%of SAHA was released from PPDS at 48 h,however,in the absence of lipase,only 38.14%of SAHA was released.The data verified that accumulative release behavior of SAHA was lipase-dependent.PPD can realize the loading and condition-responsive release of SAHA.2.In vitro anti-tumor effect of micelles siRNA@PPDSAt first,we studied the effects of micelles siRNA@PPD on tumor cells,including its cellular uptake ability and its ability to silence PD-L1 protein after being taken up by cells.Secondly,we studied the cytotoxicity of micelles PPDS and mechanism of their cytotoxicity,which includes cytotoxicity test,apoptosis test,cell cycle arrest test and western blot test.The results of cell uptake experiment displayed that siRNA@PPD micelles were more efficiently taken up by mouse melanoma（B16-F10）cells and mainly stayed in the cytoplasm after 12 h of incubation,confirming the efficient cellular uptake of siRNA@PPD micelles.The western blot experiment results showed that compared with siRNA-negative controls（siRNA-NC）,when the concentration of siRNA-PD-L1 was 100n M and200n M,the expression of PD-L1 protein was down-regulated to about 66%and 24%,respectively.Western blotting assay proved that siRNA@PPD micelles significantly suppressed PD-L1 expression in B16-F10 cells in a dose-dependent manner,indicating that siRNA@PPD micelles can protect siRNA-PD-L1 from entering the B16-F10 cells and perform its function of silencing PD-L1 protein.The results of cytotoxicity test showed that the polymer PPD was less toxic to both human hepatoma（Hep G2）cells and B16-F10 cells,while the inhibitory effect of both PPDS and free SAHA was strong and was dose-dependent after culturing for 48 h.For Hep G2 cells,the half maximal inhibitory concentration（IC50）of PPDS was 0.8091μM,which was lower than IC50 values of free SAHA（1.1290μM）.For B16 F10 cells,the IC50value of PPDS was 19.01μM,which was lower than IC50 values of free SAHA（23.76μM）.Thus,the PPD micelles are expected to be a biocompatible vector system applicable toward SAHA.The results of apoptosis test showed that both PPDS and free SAHA caused Hep G2 cells apoptosis in a dose-dependent manner,compared to control.Importantly,PPDS（1.25μM）showed apoptotic cell proportion of54.74%,which was higher than the apoptosis result of free SAHA（51.31%）.In parallel,PPDS（2.50μM）showed apoptotic cell proportion of 66.71%,which was higher than the apoptosis result of SAHA（62.32%）.The results of cell cycle arrest test showed that the B16-F10-cells treated with both free SAHA and PPDS were blocked in G1 phase,compared with the control（G1=70.89%）.As expected,the blocking effect of PPDS was better than free SAHA in a dose-dependent manner,respectively（PPDS,20μM or 40μM,G1=73.91%or 86.51%;free SAHA,20μM or 40μM,G1=72.50%or 84.01%）.The results of western blot experiments showed that,compared with the control group,when the concentration of SAHA in the free SAHA group was20μM and 40μM,the expression of Acetyl-Histone H3 protein increased by3.4 times and 5 times,respectively.When the SAHA concentration in the PPDS group was 20μM and 40μM,the expression of Acetyl-Histone H3protein increased 3.6 times and 7.2 times,respectively.These data illustrated that the promoting effect of PPDS was better than free SAHA in a dose-dependent manner,respectively.3.In vivo antitumor efficacy of micelles siRNA@PPDSTo evaluate the therapeutic effect of combined PD-L1 knock down（PD-L1 KD）and HDACi in vivo,an antitumor study was performed using C57BL/6 tumor-bearing mouse model by measuring tumor volume and survival period.The mice were randomly assigned to four groups and then treated with saline,PPDS,siRNA@PPD and siRNA@PPDS,respectively.The results showed that obvious tumor growth was observed in the mice treated with saline.Compared with saline group,siRNA@PPDS showed the most effective inhibition of tumor growth（P<0.01）,while siRNA@PPD or PPDS showed relatively slight inhibition of tumor growth（P>0.05 or P<0.05）,implying that the therapy combined PD-L1 KD and HDACi dramatically inhibited tumor growth,much more efficient than PD-L1 KD or HDACi alone.Furthermore,the statistical analysis of the mouse survival curve showed that compared with the control group or the siRNA@PPD group,the survival time of mice in the siRNA@PPDS group（P<0.05）was statistically different,also indicating that siRNA@PPDS showed remarkable antitumor activity.In addition,we evaluated the lung metastasis inhibition effect by observing metastatic lesions in mice bearing melanoma.The results showed that no lung metastatic lesions were found in melanoma-bearing mice treated with siRNA@PPDS,whereas the saline group had the most lung metastatic lesions followed by siRNA@PPD or PPDS.Moreover,the lung metastasis inhibition effect of siRNA@PPDS was further verified by H&E staining of the lungs,which was consistent with results of lung metastatic lesions.These findings indicated that the micelles-mediated combination of PD-L1 KD and HDACi could be a potential treatment for inhibiting tumor growth and lung metastasis,compared to PD-L1 KD alone.In summary,the experiment results of this paper proved that the successful synthesis of amphiphilic polymer carrier PEG-b-P[Asp（DET）n]（PPD）,the PPDS and the siRNA@PPDS.Next,the results of cell experiments in vitro have proved that the amphiphilic polymer carrier PPD exerted its advantages in drug loading and drug release.Compared with any free drug,drug-loaded micelles were easier to be taken up by tumor cells and performed their respective functions more efficiently.The experiment results of tumor-bearing mice in vivo proved that the anti-tumor effect of the combination of SAHA and siRNA-PD-L1 in vivo was stronger than that of any single agent,and it played a certain role in inhibiting the lung metastasis of melanoma.Therefore,we concluded that the combined application of HDACi drugs SAHA can enhance the effect of PD-1/PD-L1 immunotherapy,and is expected to become a potential anti-tumor treatment method. |
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