| Background and objective:Tumor is a new creature result from a variety of carcinogenic factors that lead to abnormal proliferation of cells in the body.Nowadays,treatments of tumor mainly including surgery,radiotherapy and chemotherapy.The radiotherapy and chemotherapy can kill tumor cells,however,it also do damage to normal cells.It also bring a series of side effects such as poor immunity and radiation dermatitis and so on.In recent years,the anti-angiogenic therapy targeting to tumor vascular endothelial cells,because of its lowly resistance and slightly side effects has gradually developed into "The fourth treatment of tumor".In 1971,Folkman proposed a significant doctrine that the growth and metastasis of tumor depend on angiogenesis,destructing angiogenesis is an effective strategy to inhibit tumor growth.Angiogenesis of tumor plays a key role in the growth and metastasis of primary malignant solid tumor.Therefore,the anti-angiogenic therapy targeting to the angiogenesis of tumor has become a hot spot in recent years.The complexity of angiogenesis depends on the complexity of the biological behavior of tumor cells.A tumor or other different tumors at different growth stages have different angiogenic microenvironments.Therefore,every phase of angiogenesis can be used as a target of anti-angiogenic therapy.However,the current anti-angiogenic therapy always target to only a phase,and therefore it do not have sustained effect at different phases of tumor.Given the current state of antiangiogenic therapy of tumor,this study proposed to design multi-targeted nanoparticles for tumor antiangiogenesis which can target to both tumor vascular endothelial cells and tumor cells.Vascular endothelial growth factor(VEGF)is one of the most important targets in antiangiogenic therapy of tumor,which plays a key role during the process of tumor angiogenesis.Some studies found that heparin can inhibit the proliferation of vascular endothelial cells and the formation of angiogenesis.Heparin also have an effect on the fibrin aggregation structure and the sensitivity of plasminogen activator.Therefore,heparin can be used as an antagonist of VEGF.It is well-known that integrin ?v?3 as another important target also plays a key role during the process of tumor angiogenesis.Some studies found that RGD peptides can be competitive with the integration of the tumor surface receptor and thus to prevent the adhesion between tumor cells and stromal.It also have an influence on the formation of tumor angiogenesis,and thus to induce the growth and migration of tumor cells.Therefore,RGD peptides can be used as an inhibitor of ?v?3.Some studies found that folate receptor(FR)was obviously overexpress on the surface of many cancer cells(such as breast cancer,ovarian cancer),whereas it was not or little express on the surface of normal cells.Binding of folate receptor and folate is highly specific.Because of the relatively small molecular weight,easily modification and lowly immunogenicity of folic acid,it can be used as a potential target for a variety of ligand and antibody-directed cancer therapeutics.Biotin is an cofactor of enzyme which can participate in the body's carbohydrate metabolism of lipids,proteins and nucleic acids.Biotin can easily to combine with the antibody,which have a multi-level zoom effect between Biotin and avidin.Therefore,it can be widely used in the quantitative detection of antigen and antibody.So the role of Biotin in the biomedical fieldis is very important and can not be ignored.At present,the administration of antiangiogenic drugs mainly through intravenous injection.However,antiangiogenic drugs are mostly common form which are susceptible to degradation in the blood because of various enzymes.So the effective concentration of the drug decreases,leading to the decline of its bioavailability and the effect of therapy.Some studies found that biological macromolecules as self-assembled nanocarriers have good biocompatibility,easy modification of surface characteristics,high permeability and retention(EPR effect)in tumor tissue and so on.It can prolong the cycle time of drug in the body,evade the reticular of macrophage phagocytic cells,increase the utilization of drugs.At present,the self-assembled nanodrugs have been widely used in the field of cancer treatment.Above all,this issue first to use heparin as a carrier,coupled with biotin,RGD peptides chemically to build an antiangiogenic drug Heparin-biotin-RGD(HBR).In vitro,experiments show that it has a good inhibit effect on the proliferation of tumor cell and the microtubule formation of HUVEC.However,it does not self-assemble to form nanoparticles,and thus seriously limite its application in vivo.Our research studies found that conjugation to the heparin backbone promoted the formation of self-assembled nanoparticles.Therefore,based on the structure of antiangiogenic drug heparin-biotin-RGD(HBR),we introduced folate for the formation of multi-target antiangiogenic drug heparin-biotin-folic acid-RGD(HBFR)and screened optimal formulation of nanoparticles.We choose human umbilical vein endothelial cells,ovarian cancer cells,lung cancer cells as experimental subjects to investigate biological activity of nanoparticles in vitro and in vivo.It might provide research base for the development of safe and effective antiangiogenic drugs applied in cancer.Materials and methods:1.Preparation and characterization of drugsSuccinylated-heparin was dissolved indry DMSO and maintained by gentle heating.Heparin,Biotin,folate,RGD,EDC and NHS were successively added to the mixture,and the reaction was allowed to proceed at room temperature.Fluorescence dye Oregon green was also added if needed.After overnight stiring,DMSO was then removed by a dialysis memberane for 48 h to get HB,HR,HBR,HBFR or Oregon green488-Tabled HB,HR,HBR,HBFR.Characterization of nanoparticles drugs were measured by dynamic light scattering(DLS)and transmission electron microscopy(TEM).The absorbance of folic acid of nanoparticles drugs at a concentration of 366 nm standard curve was measured by UV spectrophotometry.The content of folic acid of HBFR was then calculated according to the standard curve.The content of Biotin of HBFR was measured by Quant*TagTM Biotin Kit.The content of RGD of HBFR was measured by CQBCA protein assay kit.2.Qualitative analysis of cellular uptake of nanoparticlesTo investigate the cellular uptake of nanoparticles,we choose ovarian cancer cell A2780,lung cancer cell A549 and HUVEC to carry it out.Cells were inoculated into 6 well plate,after incubation for 12 h,different ratio of nanoparticles were added into the well with the same amount of Oregon green4 88-labled.After incubation for 4h,cells were incubated with Hochest 33342 nuclear stain and directly introduced to a FACSort flow cytometer equipped with a 488 nm argonion laser.3.Cell Viability AssayA2780 cells,A549 cells and HUVEC were incubated with HB,HR,HBR and HBFR(NP1 and NP2)with different drug concentrationsfor 48 h.MTT assay was used to measure the relative cell viability in different groups.4.Transwell assayIn order to evaluate the ability of nanoparticles in inhibiting cells migration,we conducted the transwell assay.A2780 cells,A549 cells and HUVEC were added into transwell chamber with LMWH,NP1 and NP2with different drug concentration for 48 h.Then we calculated the relative cell migrate rate in different groups.5.Tube formation assayTo demonstrate the effective tube disruption ability of HBR and HBFR(NP1 and NP2),we used the tube formation assay at an equivalent concentration with different groups.HUVEC and nanoparticles were added into the 96 well plate,after incubation for 7-14 hours,we observed the tube formation respectively under the inverted optical microscope.Calculation of tube formation inhibition rate(computational formula:inhibition ratio(%)=(Tube length of control-Tube length of sample)/Tube length of control x 100%).6.Matrigel plug assayThe matrigel plug assay was conducted to assess the antiangiogenesis potency of nanoparticles in vivo.We mixed the low growth factor matrigel and b-FGF,and then added to three groups with PBS,low molecular weight heparin and nanoparticles respectively whose total volume were 700 ?L.The mixture were then injected into both abdominal sides of nude mice,3 mices of each group.After 7 days,mices were sacrificed.We removed the matrigel plug to conduct immunohistochemistry analysis and observe the vascular intensity of each group under the optical microscope.7.In vivo primary tumor experimentTo evaluate the effect of nanoparticles in inhibiting the tumor cell growth in vivo,we used nude mices as experimental subject and divided into 3 groups including blank control group,low molecular weight heparin group and nanoparticles group.A2780 cells were implanted subcutaneously into 4-5 weeks old nude mice.Tumor size and animal weight were monitored every other day.The tumor volume wascalculated as follows:v=1/2×a×b2(v represents the size of tumor,a represents the longest diameter of tumor,b represents a tumor on the shortest diameter).The nanoparticle drug,LMWH and PBS were administrated through tail vein every other day at drug dose of 90 U/mL respectively.After two weeks,mices were sacrificed.We removed the tumor tissue and took immunohistochemical assay.8.Immunohistochemistry evaluation of angiogenesisIn order to further investigate the inhibit effect of nanoparticles of tumor angiogenesis,we used thematrigel plug tissues and subcutaneous tumor tissues to conduct immunohistochemical staining.The process includes dewaxing,antigen repairing,blocking the endogenous peroxidase,serum blocking,antibody incubation,DAB staining,transparent,mounting and so on.Staining of vascular of each group were observed under the optical microscope.9.Statistical analysisStatistical analysis was performed using the SPSS 13.0 software package.All data were performed in triplicate and presented as a mean value with its standard deviation indicated(meanąSD.)Difference of cell viability in different drug delivery system groups were analyzed with one-way ANOVA.Cytotoxicity of HB?HR and HBR,HBR?NP1 and NP2were analyzed with One-Way ANOVA.Inhibition of cell migration effect of HBR?NP1 and NP2 were analyzed with One-Way ANOVA.Inhibition of tube formation ability of HBR?NP1 and NP2 were analyzed with One-Way ANOVA.HBR?NP1 in vivo angiogenesis ability was analyzed with two-sample t-test.P<0.05 was considered statistically significant.Results:1.Preparation andidentify physicochemical properties of drugsHeparin-Biotin-RGD(HBR)was conjugated by chemical method,the dynamic light scattering instrument displayed that although HBR has a larger particle size in aqueous solutions and the polydispersity index(PDI)is very big.So they haveno self-assembled behavior in aqueous solutions.Based on the structure of HBR,we introduced folate to construct two kinds of multitarget nanoparticle with different proportions:NP1 and NP2.It wasshown in water the size of NP1 and NP2 was around 25 nm and 30 nm respectively and their zeta potential was around-25 mV and-28 mV in water measured by Dynamic light scattering.To further observe the morphology of two NPs,we clearly found that two NPs have an approximately spherical shape under transmission electron microscope.Polydispersity index(PDI)of two NPs was 0.201 and 0.257 respectively.The content of folic acid was around 8.7%and 6.3%respectively measured by UV spectrum.The content of Biotin was around 1.6%and 1.3%respectively measured by Quant*TagTM Biotin Kit.The content of RGD was around 11.2%and 9.38%respectively measured by CQBCA Protein Quantification Kit.2.Quantitative analysis of cellular uptake of nanoparticles by Flow cytometryResults of flow cytometry showed that the fluorescence intensity of nanoparticles in the A2780 cells?A549 cells and HUVEC was significantly higher than the control group.The fluorescence intensity of NP1 was slightly higher than that of NP2.It illustrated that these three kinds of cell have good uptake ability of nanopartices.3.Determination of cytotoxic effect of nanometermeter drugs using MTTThe results of cell viability for A2780 and HUVEC cell lines were:HR<HBR<HB(P<0.05).With the increase of the concentration of nanoparticles,cell viability decreased gradually.The results displayed that although HBR has no self-assembled behavior in aqueous solutions,it still has well activity of inhibition of tumor cells and endothelial cells.Based on the structure of HBR,we introduced folate to construct two kinds of multitarget nanoparticle with different proportions:NP1 and NP2.MTT results demonstrated that the cell viability of A2780 cells.A549 cells and HUVEC decreased significantly treated by HBR?NP1and NP2 for 48 h.Telling that nanoparticles can effective inhibit proliferation of tumor cell and endothelial cell.4.Transwell assayCompared with the LM WH,the number of transmembrane cells of nanoparticles(NP1 and NP2)clearly decreased under the optical microscope.The inhibit effect of NP1 was better than NP2.Suggesting that the inhibit effect of LMWH was not better than the nanoparticles on cell migration.5.Tube formation assayNanoparticles have the ability to inhibit tube formation of HUVEC under the optical microscope.Compared with LMWH,the number of tube of nanoparticles(NP1 and NP2)in HUVEC was significantly reduced and the morphology structure of tube was obviously incomplete.It demonstrated that the inhibit effect of nanoparticles significantly better than LMWH on the formation of tube in HUVEC and NP1 exhibit the best inhibit effect.6.Matrigel plug assayAfter the matrigel plug removed from the nude mice,we found that:compared with the control group and the LMWH group,angiogenesis of nanoparticle on the surface of plug was significantly reduced,indicating that nanoparticle have an effect on the inhibition of angiogenesis in vivo.To further clarify the ability of antiangiogenesis of nanoparticle,we fixed the plug,paraffin-embedded sections and conduct immunohistochemistry staining.The result found that the expression of CD34 of nanoparticle significantly decreased compared with LMWH,indicating that nanoparticle can effective inhibit the angiogenesis in vivo.7.In vivo primary tumor experimentTo evaluate the effect of nanoparticles in inhibiting the tumor cell growth in vivo,we used nude mices as experimental subject.A2780 cells were implanted subcutaneously into 4-5 weeks old nude mice.Tumor size and animal weight were monitored every other day.After two weeks,mices were sacrificed.We removed the tumor tissue and took immunohistochemical assay.The result found that the tumor size of nanoparticle group decreased significantly compared with the blank control group and LMWH group.8.Immunohistochemistry evaluation of angiogenesisAfter the Matrigel plugs and tumor tissue removed from the nude mice,we embedded it in paraffin,sliced and selected CD34 as vascular marker to conduct immunohistochemical staining analysis.The results showed that the expression of CD34 of control and LMWH significantly higher than nanoparticle.It was means that nanoparticle can effective inhibit tumor angiogenesis in vivo compared with LMWH.Conclusion:1.First,we applied heparin as carrier to conjugate with Biotin,RGD peptide by chemical method and then we obstained antiangiogenic drug Heparin-Biotin-RGD(HBR).Experiments in vitro showed that HBR can inhibit the proliferation of ovarian cancer cell A2780,human umbilical vein endothelial cells HUVEC and it also can inhibit the tube formation of human umbilical vein endothelial cells HUVEC.Dynamic light scattering instrument displayed that HBR has a larger particle size in aqueous solutions and the polydispersity index(PDI)is very big.Indicating that they have no self-assembled behavior in aqueous solutions.2.Based on the structure of HBR,we introduced folate to construct multitarget nanoparticles Heparin-Biotin-folate-RGD(HBFR)and screened the optimized nano-formulation.It was shown that the size of HBFR was around 25 nm in aqueous solutions and its zeta potential was around-25 mV in aqueous solutions measured by Dynamic light scattering.Poly dispersity index(PDI)of HBFR was around 0.201.3.Experiments in vitro showed that multitarget nanoparticles(HBFR)can be intake by ovarian cancer cell A2780,lung cancer cells A549 and human umbilical vein endothelial cells HUVEC.It also can inhibit proliferation and migration of ovarian cancer cell A2780,lung cancer cells A549 and human umbilical vein endothelial cells HUVEC.The tube formation of human umbilical vein endothelial cells HUVEC can be destroyed by multitarget nanoparticles(HBFR).4.Experiments in vivo displayed that multitarget nanoparticles(HBFR)have better antiangiogenic ability compared with LMWH via matrigel plug assay.In vivo primary tumor experiment showed that multitarget nanoparticles(HBFR)have good inhibit effect of tumor proliferation in the animal model of ovarian cancer cell A2780. |
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