Construction Of Two-dimensional Black Phosphorus-based Heparin Targeted Delivery Platform And Its Evaluation In Vivo And In Vitro

Background and Purpose:Unfractionated heparin?UH?with simple manufacturing process and relatively low cost is one of the most commonly used anticoagulants for the treatment and prevention of venous thrombotic diseases.However,compared with low molecular weight heparin and other new anticoagulants,it has the disadvantages of high risk of potential bleeding and short half-life in vivo.Nano-drug delivery platform has the characteristics of small size,surface modification and large specific surface area,which can change the pharmacokinetic characteristics of drugs and improve the bioavailability of drugs.It is the most studied new drug delivery method at present,and it is also one of the most challenging research hotspots in pharmacology.Black phosphorus nanosheets?BP NSs?,which is separated from macroscopic black phosphorus crystal,is a new type of two-dimensional?2D?material.Compared with traditional 2D materials such as graphene and transition metal dihalides,BP NSs has higher photothermal conversion efficiency,larger extinction coefficient and specific surface area.In addition,BP NSs has good biocompatibility and biodegradability because it is easy to be oxidized and degraded into non-toxic phosphite ions and phosphates under natural conditions.In order to change the disadvantage of UH and make it a cheap and effective drug,so as to better serve patients,it is necessary to improve its bioavailability and reduce the risk of drug use from the perspective of pharmacokinetics.To sum up,based on the new 2D material BP NSs,a delivery platform which can selectively transport UH to areas with high risk of thrombosis and increase local drug concentration may be a good way to improve the bioavailability of UH and reduce the risk of drug use.Methods:After a comprehensive comparison of various reported methods for the preparation of BP NSs,and combined with the objective conditions of our laboratory,we chose an improved organic solvent-assisted liquid phase stripping method with low cost,strong flexibility and convenient material collection to prepare BP NSs.Then the positively charged magnetic nanoparticles?Fe₃O₄-PEG-NH₂?and branched polyethyleneimine?PEI?were functionalized on the negatively charged BP NSs by the principle of electrostatic adsorption,and the magnetically mediated UH targeted delivery platform?PEI/Fe₃O₄@BP NSs?was obtained.Then a series of morphological and structural characterization of PEI/Fe₃O₄@BP NSs were carried out with various instruments,and its in vitro and in vivo characteristics and biosafety were studied comprehensively.Results:The hydrodynamic size of the BP NSs prepared by the improved organic solvent-assisted liquid phase stripping method is 353.8±11.8 nm.The hydrodynamic size of the PEI/Fe₃O₄@BP NSs constructed by Fe₃O₄-PEG-NH₂ and PEI functionalized modified BP NSs is 827.8±42.9 nm.Although,compared with BP NSs,the hydrodynamic size of PEI/Fe₃O₄@BP NSs is more than doubled due to agglomeration,the results of atomic force microscope show that its thickness does not increase significantly.In vitro,it has been found that PEI/Fe₃O₄@BP NSs has good photothermal conversion performance and remarkable magnetic orientation ability,the drug loading rate of UH is more than 450%,and the controlled release of UH can be achieved under the action of near-infrared laser(808 nm,1.0 W cm^-2).In addition,PEI/Fe₃O₄@BP NSs?PEI/Fe₃O₄@BP-UH NSs?containing UH has the ability to enhance the effect of near-infrared laser thrombolysis,and has lower cytotoxicity and erythrocyte hemolysis rate.In vivo studies have shown that PEI/Fe₃O₄@BP NSs has good magnetic targeting ability and excellent biosafety.The most important was the pharmacokinetic parameters of PEI/Fe₃O₄@BP-UH NSs combined with near infrared laser in the treatment group(C_max,t_max and AUC0?6h were 0.35±0.02 U/ml,1.26±0.08h and 6.91±0.58 U×h/mL respectively),and the pharmacokinetic parameters were 0.35U/ml and 0.02U/ml at 6h,respectively.The pharmacokinetic parameters of plain UH group(C_max and AUC0?6h were 0.52±0.02 U/ml and 3.92±0.51U×h/mL,respectively)were significantly improved as compared with those of control group,and the duration of the lowest effective drug concentration in the treatment group was significantly longer than that in the control group treated with intravenous injection of UH.Conclusions:Based on the above results,our magnetic-mediated near-infrared laser responsive UH targeted delivery platform can effectively improve the bioavailability of UH,and it is expected to become a new strategy for accurate prevention of venous thrombosis in certain areas with high risk of thrombosis,such as the lower extremities of bedridden paralyzed patients.