Preparation Of Targeted Nanoparticle For Platelet Inhibition And Its Antitumor Mechanisms

Malignant cancer is the number one killer for people's health,and metastasis is responsible for most of the cancer-related death.Surgery and chemoradiotherapy is the main clinical treatment of cancer.However,once diagnosed,most patients are already in advanced stages of cancer,leading to less possibility with surgery.Due to the ineffective tumor drug accumulation and specificity,commonly used chemotherapy can result in severe side effects.As a new developed science,nanomedicine has offered a new dawn for cancer therapy.By fine-tune design of nanoparticles,this strategy can not only prolong the half-life of chemotherapeutics but also realize controlled drug release within tumor microenvironment.By increasing the drug accumulation in tumor,EPR effect(enhanced permeability and retention effect)can decrease systemic toxicity.Several nanoparticulated drugs have been approved in cninical usage,including Genexol?-PM,Abraxane?,Transdrug?,Doxil/Caelix? Dauno Xome?,Myocet? and so on,and also some in different clininical stage.However,in contrast to preclinical animal models,researches did not find obvious EPR effect in tumor-bearing patients.Thus,exploiting new approaches to enhance the tumor drug accumulation becomes more important.In conclusion,there are two challenges for cancer therapy,one is metastasis and another one is efficient drug delivery.Platelets are anuclear fragments of cytoplasm that are derived from the megakaryocytes and its main physiological functions are coagulation and hemostasis.Recently,many clinical observations and preclinical experiments indicate platelets play a vital role during the development of cancer and its metastasis.1.By secreting many kinds of cytokines,platelets can promote the proliferation and angiogenesis of tumor cells.2.By secreting TGF?,platelets can induce tumor cells to acquire invasive VI properties.3.In the circulation,platelets have been shown to bind to circulating tumor cells to protect them from immune system attack and high-speed blood flow as they travel to distant sites.4.By secreting chemotactic factors,platelets can assist metastatic cells to become established at distant sites.5.By secreting inflammatory protective factors or by physical adsorption,platelets can maintain the integrity of tumor blood vessles.In summary,based on those important roles platelets play in tumor progress,platelets inhibiton will be a new strategy to treat cancer.Many clinical data indicate long-term use of asprin can greatly decrease the risk of cancer morbidity.There are also many preclinical evidences indicate the useage of platelets inhibitor can efficiently reduce the metastasis,while systemic drug administration brings the risk of bleeding complications,which limits its clinical application.We first designed a platelet inhibitor loaded and tumor microenvironment targeted liposomal nanoparticle named CREKA-Lipo-T.By targeting the fibrinfibronectin complexes within the microthrombi on tumor blood vessles,CREKALipo-T can slowly release platelets inhibitor Ticagrelor within tumor microenvironment and inhibit platelets function and further inhibit the metastasis of tumor cells.We prepared the CREKA-Lipo-T using an improved thin-film dispersion method.In vitro cell experiments,we investigated whether platelets can induce the EMT of cancer cells and whether CREKA-Lipo-T can prevent this process.The results indicated CREKA-Lipo-T can block EMT by inhibiting platelets function.We also investigated whether CREKA-Lipo-T could prevent the platelet-induced invasion of tumor cells.As shown in in vivo anti-metastasis results,CREKA-Lipo-T can significantly inhibit lung metastasis of 4T1 tumor cells and almost no bleeding complication was observed.However,free drug treated group leads to serious lung bleeding.In summary,by targeting delivery of platelets inhibitor to tumor using microenvironment targeted nanoplateform,we realized efficient metastasis inhibition.Based on the important role platelets play in maintaining the integrity of tumor blood vessels,we next designed a lipid-polymer hybrid nanocarrier simultaneously loaded with chemotherapeutic agent Dox and platelet depletion antibody R300.We also designed a matrix metalloproteinase 2(MMP2)-cleavable peptides to the outer lipid layer.When the nanoparticles come into tumor tissue,the lipid layer was cleaved and the R300 was site-specifically released.Tumor-associated platelets depletion would increase the perfusion of nanoparticles.Meanwhile,the released positive charged inner core would be efficienly uptaken by tumor cells and killed them.We first prepared the inner Dox-loaded core by double emusion method and optimized the drug loading efficiency.Then we prepared MMP2-responsive peptide chelated lipid layer by filming-rehydration method to construct microenvironment-responsive lipid-polymer hibird nanoparticle PLP-D-R.In vitro enzyme-responsive experiments indicated in the presence of MMP2,PLP-D-R could efficiently release platelets depletion antibody and Dox,with their biological functions stablized.The measurement of cell viability also indicated PLP-D-R possessed MMP2-dependent cytotoxicity.When the MMP2 inhibitor was added,the cytotoxicity of PLP-D-R decreased.In vivo tumour targeting and pharmacokinetics of PLP–D–R indicated nanoparticated Dox and R300 could extend their half-life.Compared with PLP-D or LP-D-R,PLP-D-R showed an enhanced tumor accumulation.Immunohistochemical staining of platelets indicated PLP-D-R chould eliminate intratumoral platelets while no effect with circulation platelets.By Immunostaining,SEM,high resolution TEM scanning,we demonstrated PLP-D-R could enlarge the branch of adjacent endothelial cells and further increase the permeation of red cells and nanoparticles.Two tumor models' in vivo anti-tumor activity evaluation indicated PLP-D-R has a MMP2-dependent anti-tumor activity.Compared with other control-treated groups,PLP-D-R showed an improved anti-tumor activity in both tumor models,simultaneously without bleeding complication.In summary,we realized a safe delivery of platelets depletion antibody by microenvironment targeted lipid-polymer hybrid nanocarrier and also realized the enhanced drug accumulation in tumor.This strategy offered a new way to overcome the low efficiency of EPR effect in clinical practice.