Inhibition Of The Intrinsic Coagulation Pathway Improves Tumor-targeted Drug Delivery And Anticancer Effects Of Cationic Solid Lipid Nanoparticles

Background and objective:Solid lipid nanoparticles(SLNs)represent a class of colloid micelle composed of natural or synthetic lipids being solid at room temperature with the drug loading in lipid core,of which the average diameter ranges from 40 to 1000 nm in the submicron.As a carrier system,cationic SLNs(c SLNs)hold great promise for controlled release,targeted delivery and good biocompatibility.Therefore,c SLNs can improve the therapeutic efficacy of chemotherapeutic drugs in cancer,while diminishing side effects to healthy tissue,which are promising in clinical application.The blood half-life and biodistribution of c SLNs,the two major factors determining the efficacy of targeted drug delivery,are affected mostly by the magnitude of surface charge and the density of surface-coated poly(ethylene glycol)(PEG).Under in vitro conditions,positive charge and low PEG density have been shown to facilitate nanoparticle-based drug-delivery by promoting cellular uptake and distribution in subcellular compartments.However,under in vivo conditions,increased positive charge or reduced PEG density will reduce the blood circulation time,and then reduce the efficiency of drug delivery.The exact mechanism is unclear yet.Now most studies focus on the effect of surface charge and PEG density on the interaction of c SLNs with plasma protein.The interaction of c SLNs with the major blood cellular components has been overlooked.Whether or not the interaction between c SLNs and blood cellular components may affect the blood circulation time and biodistribution of c SLNs remains unclear.Therefore,from the view of blood cells,our study investigates the effect of surface charge and PEG density on the in vivo drug delivery of c SLNs,and explores the blood clearance mechanism of c SLNs with high surface charge and low PEG density.At last,we raise new stratedy enhancing the efficiency of drug delivery of c SLNs with high surface charge and low PEG density,which will provide important reference for clinical application of c SLNs.Methods:We prepared 9 kinds of c SLNs with similar size by emulsification,which differed in surface charge and PEG density.Firstly,We focused on these 9 kinds of c SLNs to detect the effect of hemolysis on red cells using spectrophotometer,activation on platelets using flow cytometry and adsorption of serum protein qualitatively by SDS-PAGE and quantitatively by BCA kit.Then we chose 5 representative c SLNs to conduct in vivo experiment.We intravenously injected these 5 c SLNs into mice to measure the concentration change of red cells,white cells and platelets in vivo using auto-analyzer.We found platelets were most sensitive component.So we further measured the activation and binding effect of 5 c SLNs on platelet in vivo by flow cytometry.At the same time,we observed the thrombus formation in vivo by H&E histopathological analysis and measured the coagulation pathway involved in the thrombus formation.We found that c SLNs induced thrombus formation by intrinsic coagulation pathway.Finally,we propose a strategy to enhance the therapeutic efficiency of c SLNs by using of inhibitor of intrinsic coagulation-heparin,which could inhibit activation of platelets,intrinsic coagulation and thrombus formation.We established the breast tumor model in situ and chose c SLN3-3 as paclitaxel(PTX)delivery carrier,which induced most serious coagulation.We compared the half-life in circulation,accumulation in tumor and therapeutic efficiency of c SLN3-3-PTX with or without heparin pretreatment to evaluate the feasibility of this strategy.Results1.As surface charge increased and PEG density decreased,c SLNs had more a more toxic effect on red cells and platelets and adsorbed more serum protein in vitro.2.All c SLNs had no influence on the concentration of red cells and white cells in vivo.But as surface charge increased and PEG density decreased,the concentration of platelets significantly reduced,whereas the ratio of both activated platelets and platelets binding to c SLNs increased.3.As surface charge increased and PEG density decreased,the number of thrombus induced by c SLNs increased.And 5-doses administration induced more thrombus than one dose.4.All c SLNs showed no change in prothrombin time(PT),while c SLNs with high surface charge and low PEG density reduced the activated partial thromboplastin time(APTT).5.Heparin pretreatment inhibited the aggregation and activation of platelet and the thrombus formation induced by c SLNs with high surface charge and low PEG density(c SLN3-3).6.After heparin pretreatment,the half-life in blood circulation of c SLNs with high surface charge and low PEG density(c SLN3-3)was prolonged from(1.43 ± 0.03)min to(16.34 ± 0.32)min;the tumor accumulation was obviously enhanced;the growth of tumor was significantly inhibited.Conclusion:Our study demonstrated that c SLNs with high surface charge and low PEG density could induce the aggregation and activation of platelets,and induce the thrombus formation by activating the intrinsic coagulation pathway.Furthermore,inhibitor of intrinsic coagulation pathway could inhibit the thrombotic toxicity,thus prolong the half-life in blood circulation,improve the drug targeted delivery and finally enhance the therapeutic effect.