Study Of Size-controllable Nano-drug Delivery System For Overcoming Double Barriers

Although conventional nano drug delivery systems have the advantages of improving drugs half-life,reducing side effects,improving the compliance of drug users,it is a huge challenge in terms of transing tumor-barriers.The main barriers affecting efficient entry of nanomedicine into tumor cells are vascular barriers and extracellular matrix(ECM)barriers.Therefore,how to overcome the barriers where nano drug delivery systems reanch the site has been drawn wide attention.For the tumor vascular barriers,the effect of reversibly dilating blood vessels is applayed to enhance permeability of nanoparticles to tumor blood vessels,and at the same time,the small particle size effect is used to overcome the ECM barriers,thus to overcome this two barriers of the tumor and enhance the efficacy of the nano drugs.Based on above questions,a strategy was presented to utilize reversible vasodilatation effect of nitric oxide(NO)and size-controllable characteristic of ultrasound(US)responsive liposome(URL)to construct a non-destructive nanomedicine,which is able to cross both obstacles simultaneously.In this work,URL was built as a carrier via forming a gas layer between lipid bilayer to encapsulate small particles PAMAM@DOX(PD,~10 nm)and NO donationnitrosoglutathione(GSNO),constructing a URL/PD/GSNO with both NO production and size-controllable drug delivery system.Under ultrasound stimulation,GSNO decomposed and generated a large amount of NO when drug delivery system arrive at the tumor.Own to its nature of fat-souluble small molecule,NO rapidly diffused and acted on the vascular smooth muscle of the tumors,thus resulting in tumor vascular vasodilatation and “opening” the tumor vascular barriers;Moreover,the URL lipid bilayer was destroyed,which leading to release of small nanoparticles PAMAM@DOX sharply and efficiently and overcoming the tumor ECM barriers.Under the reversible vasodilator action of NO allowed more small particles PAMAM@DOX to extravasate through endothelial gap and penetrate into tumor deep.Although,the structure of different types of tumor blood vessels is quite different.We selected MCF-7 human breast cancer(with significant EPR effect)and MiaPaCa-2 human pancreatic cancer(without significant EPR effect)with significantly different EPR(Enhanced Permeability and Retention effect,EPR)effects to evaluate the effect of the drug delivery system for overcoming double barriers.We prepared the ultrasound responsive liposome by the thin film dispersion method.Small particles PAMAM@DOX and NO donation-nitrosoglutathione were encapsulated into liposome to build nano drug delivery system of URL/PD/GSNO.Transmission electron microscopy verified that the final preparation of URL was about 420 nm.Ultrasound imaging confirmed that gas layer was encapsulated in URL.Assessment of the ability of PAMAM@DOX crossing ECM barrier in vitro showed that the PAMAM@DOX+US group could successfully arrive at the depth of tumor ECM.The results of ultrasound-sensitive drug release showed that the release rate of DOX from URL was reached to 76% at pH5.5 condition within 72 h incubation.The MCF-7 cell line was chosen as tumor model.Cellular uptake test revealed that after incubation with PAMAM@DOX for 1 h,PAMAM@DOX mainly localized in cytosol;However,after incubation with PAMAM@DOX for 2 h,we observed that a considerable quantity of DOX were located in the cell nucleus for producing antitumor effect.Lysosome co-localization experiments showed that PAMAM@DOX could be taken up by cells and located in acidic lysosome,and intracellular DOX release induced by endosomal escape distinctly increased,thus effectively killing cells.Cytotoxicity assessment revealed that URL acted as a drug carrier showed high safety and little toxicity to MCF-7 cell;The rate of inhibition was 72.8% and the apoptotic rate was 78.8% when inhibition with PAMAM@DOX for 48 h.The MCF-7 tumor-bearing nude mice and MiaPaCa-2 tumor-bearing nude mice were chosen as tumor models.The results of organizational distribution and NOmediated vascular barriers leakiness showed that in the case of MCF-7 tumor model,there was no significant difference in tumor tissues fluorescence signal between URL+US and URL/GSNO+US groups,while there was a distinct difference in MiaPaCa-2 tumor model,which the probe uptake of tumor in URL/GSNO+US group was strongly higher than URL+US group.The situation implied that,NO,triggered by ultrasound,possessed a potential effect on improving the vascular barriers leakiness and was better in the human pancreatic cancer MiaPaCa-2 with low permeability.After ultrasound treatment,the drug concentration of URL/PD/GSNO in both tumor models increased significantly.These results confirmed that the sizecontrollable characteristic of URL in combination with vasodilatory effect of NO allowed much more anti-tumor drugs to extravasate through the tumor vasculature and penetrate into tumor deep.Experiments in vivo showed that in MCF-7 and MiaPaCa-2 tumor models,the anti-tumor effect of URL/PD/GSNO+US was 69.6% and 75.1%,respectively.In conclusion,our study provide a new strategy for the clinical treatment of cancer in the future of simultaneously overcome tumor vascular barriers and the extracellular matrix barriers.