Fabrication And Anti-tumor Study Of Baicalin-Loaded Multifunctional Nanoparticles

Scutellaria baicalensis Georgi is a Labiatae,Scutellaria,and an annual or perennial herbaceous medicinal plant with anti-inflammatory,anti-infection,anti-tumor,immune regulation and other a variety of pharmacological activities.In recent years,S.baicalensis Georgi has been widely used in regulating immunity,antiviral and anti-tumor.Baicalin(C21H18O11)is one of the main flavonoids in the roots,stems,and leaves of S.baicalensis Georgi.In recent years,it has been reported that low-dose baicalin can regulate the body's immunity and attack tumor cells by activating adaptive immune system.Therefore,the development of baicalin as drug for tumor chemotherapy and immunity has a broad application prospect.However,the disadvantages including the weak stability in solution,poor absorption,and low bioavailability of baicalin limit its clinical application.In order to increase the clinical application of medicinal plant resources,could only change the way of drug application.At present,nano-drug delivery system is an effective strategy to solve these issues.The biomimetic modification and targeted design could enlong circulation in vivo,and targeted delivery of nano-drug.The nano-drug delivery system could also reduce the drugs' toxic,side effects,and the dosage,which improve the effective application of drug.In hence,our aim is to fabricate a multiple function polymer nanoparticle containing baicalin and immunostimulants for anti-tumor.The multiple function baicalin-loaded nanoparticles could exert the dual effects of tumor-killing chemotherapy and immunotherapy,and improve the antitumor application efficiency of baicalin.Furthermore,targeted modification could deliver chemotherapy and immune drugs to tumor site,which further promoted tumor microenvironment and released cytotoxicity of baicalin for a combined effective of anti-tumor.The nano-delivery-system offered a new way to improve the utilization of medicinal plant.The combined antitumor effect provided new ideas for the study of new formulation for anti-tumor of combination chemotherapy and immunotherapy.And these studies will lay a theoretical foundation for the development of efficient and safe tumor therapeutic agents.In detail,this study includes the following aspects:1.We developed a baicalin-loaded Poly(D,L-lactic-Co-glycolic acid)nanoparticle(PLGA-B)with small particle size in our study.The results showed that the baicalin-loaded PLGA nanoparticles(PLGA-B)were spherical in shape with a particle size around 120 nm,which showed narrow size distribution as an excellent polydispersity index of 0.103(PDI).The cell experiments revealed that baicalin and baicalin-loaded PLGA nanoparticles(PLGA-B)could activate dendritic cells(DCs)with higher expression of the surface marker molecules and the costimulatory molecules than control in vitro.Moreover,both baicalin and baicalin-loaded PLGA nanoparticles triggered apoptosis in melanoma cells via arrest of cell cycle at G2/M phase.All the detailed evidence showed that baicalin-loaded PLGA nanoparticles might have the dual effects of activating immune cells and inducing tumor cells apoptosis.2.In order to increase the immune stimulate ability of PLGA-B,in this study,we further developed baicalin-loaded poly(D,L-lactide-co-glycolide)(PLGA)nanoparticles containing an antigenic peptide(Hgp 10025-33,Hgp)and immunostimulant(CpG).The nanoparticles were further coated with a galactose-inserted erythrocyte membrane,which actively targeted the tumor site.The tumor treatment effectiveness of the nanoparticles was evaluated.The biomimetic nanoparticles showed enhanced cell uptake in vitro and targeted tumor effects in vivo.In addition,compared with baicalin-loaded PLGA-NPs(B@NPs),the biomimetic nanoparticles,actively targeted the tumor site and significantly polarized the TAMs such that they changed from the M2 type to the M1 type both in vitro and in vivo.Subsequently,the infiltration of CD4+T and CD8+ T cells into tumor sites after induced by the biomimetic nanoparticles was greatly increased,which suggested a significant enhancement of the immune activation effect and T cells response.In addition,the activation of the T cells and induction of the CTL responses effectively suppressed melanoma tumor growth in vivo;the tumor inhibition rate was reached 65.7%.These results indicated that the biomimetic nanoparticles of co-delivery baicalin,tumor antigen(Hgp 10025-33,Hgp)and CpG targeting tumor sites,and exerting the significant effect on the tumor microenvironment for achieving an antitumor effect.3.In order to further explore the nano-complexes loaded with baicalin,CpG and melanoma antigen peptide(Hgp25-33)on tumor microenvironment,the nanoparticles were further conjugated to M2pep and ?-pep peptides(B/H@NPs@CpG-?mp),which actively target M2 tumor associated macrophage(TAMs).Targeted M2-TAMs loaded-baicalin nano-complexes(B/H@NPs@CpG-?mp)were spherical in shape with a particle size 123.6 run,which had excellent PBS storage stability and cells uptake.The nano-complex showed enhanced targeting to M2-TAMs,where uptake in the acidic lysosomal environment triggers the disintegration of polydopamine from the nanoparticle surface.These results in a slow release of the baicalin and immune-modulator payload,release for 7 days,and the release rates are all greater than 80%.The extended release of CpG has the desired effect of transforming the TAMs and activating release stress cytokines.The combined application of targeted M2-TAMs loaded-baicalin nano-complexes with macrophages has shown exellent anti-tumor effects in vitro,the highest anti-tumor ratio is 69.72%.These results laid the foundation for anti-tumor experiments in vivo.4.In order to explore whether the targeting M2-TAMs baicalin-loaded nano-complexes(B/H@NPs@CpG-?mp)effectively target M2-TAMs in tumor microenvironment,stimulate the phenotype reversal of M2-TAMs,and induce the subsequent anti-tumor chemotherapy and immunotherapy microenvironment,the anti-tumor mechanism of B/H@NPs@CpG-?mp in vivo were further studied in our paper.The results showed nano-complex enhanced targeting to M2-TAMs,where uptake in the acidic lysosomal environment triggered the disintegration of polydopamine from the nanoparticle surface.This results in a slow release of the cytotoxin and immune-modulator payload.The extended release of baicalin and CpG showed the desired effect of transforming the TAMs,from M2-TAMs to M1-TAMs,which increased the expression of CD86 and decreased CD206 to 38.42%and 6.03%respectively.Activated TAMs further increased the release of IL-12,IL-2 and IFN-? and carried baicalin to tumor cells.The TAMs also presented antigen to T cells,further stimulated the antitumor response.In addition,baicalin release exercises tumoricidal action on local melanoma cells.It also promoted Th1,CTL and NK cell infiltration in tumor microenvironment(increased to 21.2%,24.93%and 25.38%respectively in the frozen section of tumor),reduced tumor angiogenesis,and suppressed tumor metastasis.The B/H@NPs@CpG-?mp showed the strongest antitumor effect,and the tumor inhibition rate reached 73.5%.These results indicated that the targeted M2-TAMs modified baicalin-loaded nona-complexes had changed the tumor microenvironment from tumor promoting microenvironment to anti-tumor microenvironment.The nano-complex facilitates baicalin delivery to tumor cells and achieves a dual-synergistic effect by recruiting immune cells to do some killing while delivering chemo-active agents that achieved contributory cell killingIn conclusion,these results indicated that the multifunctional nanoparticles effectively reversed the TAM phenotype from M2 to M1,which further improved the tumor immune microenvironment and promoted tumor immunotherapy.These results suggested that the new designation and fabrication of baicalin-loaded nano-complexes delivery system by us has played an important role in promoting anti-tumor chemotherapy and immunotherapy,improved the efficiency of the clinical application of baicalin,promoting the value of the medicinal plant resources of S.baicalensis Georgi,also provided a new idea for the future development of a safe and effective anti-tumor delivery system.