Study On The Antitumor Effects Of Doxorubicin Dimeric Prodrugs And Its Nanotherapeutics

Based on the tumor-specific microenvironment,researchers have developed a variety of stimuli-responsive nanomedicines that achieve specific release of drugs at the tumor site,thus efficiently eliminating tumor cells without damaging normal cells,providing new insights for tumor treatment.However,due to the existence of multiple endogenous stimuli in the tumor microenvironment and intertumoral heterogeneity of chemical species,the selection of suitable linkers for the construction of efficient nanomedicines has become a crucial issue in current research.In addition,the abnormal tumor mechanical microenvironment not only promotes tumorigenesis,development,and metastasis,but also hinders the delivery of drugs to the tumor site,which seriously restricts the effectiveness of tumor therapy.Therefore,the construction of reasonable stimuli-responsive nanomedicines and the modulation of the tumor mechanical microenvironment can further improve the antitumor effects of nanomedicines.To this end,five different stimuli-responsive doxorubicin dimeric prodrug nanoparticles(DOX2-CC NPs,DOX2-SS(SH)NPs,DOX2-SS NPs,DOX2-Bor NPs and DOX2-DMA NPs)were firstly prepared in this study to investigate the impact of the linkers on antitumor effect.And the concept of precise and traceless linkers was proposed for construction of stimuli-responsive nanomedicine.Further,based on the DOX2-SS prodrug,FDINs were prepared to reveal the potential mechanism by which photothermal modulation of the tumor mechanical microenvironment affects drug delivery and cancer stem cells.FDINs-mediated photothermal effect improved the tumor mechanical microenvironment by inhibiting cancer-associated fibroblasts and degrading extracellular matrix,normalizing the tumor vessels,and thus improving drug delivery and cancer stem cell elimination efficiency for enhanced tumor therapeutic effects.The main research contents and findings are as follows.(1)Five stimuli-responsive DOX dimeric prodrugs,linked by stable carbon chains(DOX2-CC),amide disulfide(DOX2-SS(SH)),carbamate disulfide(DOX2-SS),borate ester(DOX2-Bor)and dimethylmaleamide(DOX2-DMA),were prepared and were able to selfassemble into nanoparticles without the addition of carriers.In vitro fluorescence recovery and drug release experiments showed that DOX2-SS NPs,DOX2-Bor NPs and DOX2-DMA NPs could release DOX and recover DOX fluorescence by disrupting the linkers after incubation with buffer containing 10 mM GSH,10 mM H2O2 or pH 5.0,and the cumulative drug release after 72 h incubation was 44%,69% and 44%,respectively.In contrast,no DOX fluorescence recovery was determined in DOX2-CC NPs after incubation with various stimuli.The degradation products of DOX dimeric prodrug nanoparticles were characterized by high-performance liquid chromatography and high-resolution mass spectrometry,and revealed that DOX2-SS NPs,DOX2-Bor NPs and DOX2-DMA NPs could release primitive DOX after incubation with the corresponding stimuli,respectively.However,DOX2-SS(SH)NPs could only release DOX-SH in buffers containing 10 mM GSH,whereas DOX2-CC NPs were unable to release DOX or its derivatives after incubation with various stimuli.Therefore,this study confirms that the linkers not only influence the release rate and cumulative drug release of DOX dimeric prodrug nanoparticles,but also alter the degradation products of them.(2)In vitro cellular experiments demonstrated that DOX2-SS(SH)NPs,DOX2-SS NPs,DOX2-Bor NPs and DOX2-DMA NPs could selectively eliminate tumor cells,while DOX2-CC NPs were incapable to release DOX and showed low cytotoxicity in tumor cells and normal cells.Compared with the DOX2-SS(SH)NPs that released DOX-SH,DOX2-SS NPs,DOX2-Bor NPs and DOX2-DMA NPs produced the primitive DOX,and exhibited higher cytotoxicity.The 4T1 and H22 tumor models were constructed to investigate antitumor effect of DOX dimeric prodrugs,and DOX2-Bor NPs inhibited the most tumor growth in orthotopic 4T1 tumors,while DOX2-SS NPs and DOX2-DMA NPs had better antitumor effects in subcutaneous H22 tumor,indicating that the linkers play a vital role in the antitumor effect of DOX dimeric prodrug nanoparticles.Finally,the detection of intracellular glutathione(GSH)and hydrogen peroxide levels inside tumor cells indicated that the ability of tumor cells to maintain intracellular redox levels was a key factor contributing to the difference in antitumor activity of DOX dimeric prodrug nanoparticles.(3)The polymer with active tumor targeting ability,HES-FA,was synthesized based on hydroxyethyl starch(HES)and folic acid(FA)and used for stabilizing DOX2-SS/IR780NPs(DINs)to produce stable and homogeneous DOX2-SS/IR780@HES-FA(FDINs)with diameter of 148 nm.In vitro drug release experiments showed that disulfide bond in DOX2-SS endowed nanoparticles with a reduction-responsive drug release function in GSHoverexpressed tumor cells.In vitro cellular uptake assays showed that the interaction between FA and FA receptor could enhance the uptake of FDINs by tumor cells.In vitro cytotoxicity assays demonstrated that apoptosis was the main mechanism of tumor death caused by FDINs.Moreover,FDINs exhibited higher cytotoxicity to tumor cells compared to normal cells.(4)In subcutaneous 4T1 tumor model,FDINs exhibited prolonged blood circulation time,efficient tumor targeting and enhanced drug accumulation at tumor sites.In orthotopic4T1 tumor model,FDINs-mediated photothermal effect depleted tumor-associated fibroblasts and extracellular matrix related components,decreased tumor stiffness and solid stress in tumor site,normalized tumor blood vessels,overcame tumor hypoxia,enhanced drug accumulation at the tumor site,and eliminated cancer stem cells(CSCs).Therefore,FDINs remodeling tumor mechanical microenvironment significantly improved antitumor effect of nanomedicine in both subcutaneous and orthotopic 4T1 tumor models.In summary,five stimuli-responsive DOX dimeric prodrug nanoparticles were constructed to investigate the influence of linkers on the antitumor effect of dimeric prodrug-based nanomedicines.And HES-FA was used to improve the stability of dimeric prodrug nanoparticles,prolong blood circulation time,and enhance tumor accumulation.Moreover,FDINs-mediated photothermal effect modulated the tumor mechanical microenvironment,which improved drug delivery and CSCs elimination efficiency,thus enhancing antitumor effects.This study provides new insights for the design of stimuliresponsive nanomedicines and new strategies for tumor therapy against stroma-and CSCsrich solid malignancies.