The Softness Of Tumour-cell-derived Microparticles Regulates Their Drug-delivery Efficiency

Insufficient drug accumulation in tumour and systemic toxicity are the major causes of traditional chemotherapy failure in clinic.Although nanotechnology-based drug-delivery systems are capable of improving the permeability and retention of chemotherapeutic drugs,they still face the challenges of insufficient accumulation at the tumour site,and suboptimal extravasation and subsequent penetration into tumour parenchyma.It is difficult for nanotechnology-based drug-delivery systems to target tumour-repopulating cells(TRCs)which located within the hypoxic compartment and far from the tumour vessels in solid tumours.Therefore,ideal nanotechnology-based drug-delivery systems targeting TRCs should overcome the sequential physiological barriers in vivo and be efficiently internalized by TRCs.Many cell types release extracellular vesicles in response to stimuli,including exosomes and microparticles(MPs).These vesicles hold great potential as drug-delivery systems due to high biocompatibility,intrinsic targeting properties and low immunogenicity.The size of MPs is 100-1,000nm,which are a heterogeneous population.It was reported that chemotherapeutic microparticles derived from normal cancer cells can kill mass of tumour,resulting in excellent antitumour efficacy in clinical trials.Based on the understanding that the biomechanical properties of nanoparticles such as softness has a profound impact on the in vivo processing,we constructed softer chemotherapeutic microparticles derived from TRCs(3D-MPs),which were selected by three dimensional soft fibrin gels(90Pa).It is confirmed that 3D-MPs are also softer and more deformable than MPs derived from differentiated tumour cells cultured in convitional rigid two-dimensional plastic(2D-MPs),and such softness confers MPs the enhanced ability to deliver anticancer drugs by enhanced tumour accumulation,easy extravasation and penetration into tumour tissue,and efficient internalization into TRCs by in vitro and vivo experiments.Therefore,MPs loaded drugs can play an excellent antitumor effect.It also shows that the cytoskeleton-related protein cytospin-A plays a critical role in the regulation of 3D-MPs softness.These results will provide an insight into developing softness-tailorable nanotechnology-based drug-delivery systems,and also the basis of novel chemotherapeutic MPs for targeting TRCs.Main research contents and results are as follows:(1)The characteristics and antitumour effects of drug-packaging 3D-MPsH22 and B16-F10 TRCs were selected by soft three dimensional(3D)fibrin gel(90 Pa)culture system.The TRCs were expanded in conventional 2D plates in serum-free medium.It is confirmed that doxorubicin and 5-fluorouracil were effectively packaged by 2D-and 3D-MPs followed by ultraviolet irradiation.The drug loading capacity for both 2D-and 3D-MPs were 0.06μg of DOX per μg protein and 0.04μg of 5-FU per μg protein,respectively.The diameters of drug-packaging MPs were from 510 to 540nm,and zeta potentials were from-25 to-30mV.DOX@2D-MPs or DOX@3D-MPs has a 7 day stability of in physiological conditions.In addition,DOX@2D-MPs and DOX@3D-MPs showed p H-responsive sustained drug-release profiles,which released more drugs in acidic environments.Compared with free DOX,DOX@2D-MPs and high Doxil,DOX@3D-MPs exhibited the strongest inhibitory effect in large or normal tumour size H22 tumour-bearing mice or in B16-F10 melanoma lung metastatic model.Moreover,5-FU@3D-MPs also showed highly efficient anticancer activity in H22 tumour-bearing mice.However,no significant toxicity was observed in DOX@3D-MPs-treated group compared with that in high dosage of Doxil-treated group.Meanwhile,no tumours were formed in mice after intravenously injected 2D-or 3D-MPs into mice,suggesting that 3D-MPs as a drug carrier are safe.(2)The process of 3D-MPs in vivoDOX@3D-MPs achieved the highest AUC(area under the curve)value which is about 38.9,3.1-and 6.7-fold relative to free DOX,DOX@2D-MPs and Doxil,respectively.These suggest that 3D-MPs efficiently mediate DOX accumulation in tumour tissue.The immunohistochemistry slice results showed that DOX was strongly colocalized with tumour vessels in DOX,DOX@2D-MPs and Doxil treated groups,but weakly colocalized with tumour vessels in the DOX@3D-MPs treated group.In a mice dorsal window chamber model and EGFP transgenic zebrafish model,the results show that DOX extravasated quickly from the blood vessels and distributed in the extravascular regions over time in the DOX@3D-MPs treated group.In vitro,we used soft 3D fibrin gel tumour spheroid model to validate the penetration.As a result,the fluorescence intensity of DOX in the DOX@3D-MPs group was much stronger than that in other groups.The in vitro incubation assay showed that DOX was more efficiently taken up by H22 TRCs or H22 cells in the DOX@3D-MPs treated group.Correspondingly,DOX@3D-MPs treatment resulted in the strongest cytotoxicity against H22 TRCs and H22 cells at lower DOX concentration.However,DOX@3D-MP and DOX@2D-MP exhibited less cytotoxicity against RAW264.7 macrophages and ECV304 cells.The results of animal experiments showed that the strongest DOX fluorescence was detected in the tumour cells and side population cells in tumour tissues,but a little in tumour macrophages,stromal cells and other immune cells.DOX@3D-MPs exhibited strongest cytotoxicity against tumour cells and TRCs but less cytotoxicity to tumour macrophages and endothelial cells.The mechanism of cell killing has been analyzed by intracellular tracing that 3D-MPs and DOX entered cancer cells together and colocalized with lysosomes,followed by DOX translocation to the nuclei over time and killed cells.(3)The effect of softness on the process of 3D-MPs in vivoIt was found that the Young’s modulus of 3D-MPs was about 1/3 of 2D-MPs by atomic force microscope(AFM)and the sizes of 3D-MPs were significantly larger under low osmotic conditions but significantly smaller under high osmotic conditions than that of 2D-MPs,these results show that 3D-MPs has good deformability.The experiments in vivo and in vitro show that 3D-MPs enhanced tumour accumulation,easy extravasation and tumour penetration,and efficient internalization by tumour cells and side population cells compared with 2D-MPs after being intravenously injected into H22 tumour-bearing mice.Jasplakinolode(Jasp;an agent for actin polymerization and stability)treatment resulted in significantly reduced tumour accumulation and penetration of 3D-MPs,as well as cellular uptake by tumour cells and side population cells,while latrunculin A(LatA;an agent for actin depolymerization and instability)treatment significantly enhanced tumour accumulation and penetration of 2D-MPs,as well as cellular uptake by tumour cells and side population cells.We also noticed that fewer 3D-MPs were taken up by RAW264.7 marcophages.However,Jasp treatmen could increase the uptake of 3D-MPs by macrophages,while LatA treatment decreased the uptake of 2D-MPs by macrophages.These results indicated that softness affected the process of 3D-MPs in vivo,and provided basis for elucidation that drug loading 3D-MPs had better process in vivo and anti-tumour effect.(4)Molecular mechanism regulating the softness of 3D-MPsWe then investigated the mechanism throung which TRCs generate soft MPs.We analysed the protein expression profiles of 2D-and 3D-MPs using isobaric tags for relative and absolute quantification(i TRAQ)-based proteome analysis.The expression of cytospin-A in 3D-MPs was lower than in 2D-MPs,as evidenced by western blot.We transfected H22 cells with cytospin-A small interfering(siRNA)to knock down cytospin-A expression.Knocking down cytospin-A decreased the Young’s modulus of 2D-MPs(2D-MPs cytospi-A siRNA)nearly to 3D-MPs and the secreted MPs became softer.It is found that 2D-MPs cytospi-A siRNA enhanced tumour accumulation,extravasation and tumour penetraton,as well as uptake by tumour cells and side population cells in H22 tumour-bearing mice.The 2D-MPs cytospi-A siRNA significantly increased DOX in tumour accumulation,extravasation and tumour penetration,and uptake by tumour cells and side population cells in H22 tumour-bearing mice intravenously administrated with DOX@2D-MPs cytospin-A siRNA.Correspondingly,DOX@2D-MPs cytospin-A siRNA showed significantly stronger antitumour activity similar to DOX@3D-MPs.Furthermore,smaller colony numbers and colony sizes were formed when central tumour cells of H22 tumour-bearing mice administrated with DOX@2D-MPs cytospin-A siRNA were seeded in soft 3D fibrin gels,revealing the stonger TRC-killing efficacy.These results demonstrated that cytospin-A plays a key role in regulating the softness of MPs.By inhibiting the expression of cytospin-A in cells,the MPs produced is softer,which changes the process in vivo and enhances the anti-tumor effects of drugs.The research in this paper will provide a new idea for the construction of a new type of nano drug-delivery system with adjustable soft and hardness,and provide a basis for the development of a new type of high-efficiency targeted TRCs drug-delivery system.