Experimental Study Of Paclitaxel- And GANT61-loaded Peptide-functionalized Recombinant High-density Lipoprotein-targeted Nanoparticles For Inhibition Of Triple-negative Breast Cancer Growth And Lung Metastasis

PART ONE:THE PREPARATION AND CHARACTERIZATION OF TLYP-1-RHDL-PTX/GANT61NANOPARTICLESObject:To prepare the peptide-functionalized targeting tLyP-1-rHDLPTX/GANT61 nanoparticles and explore the size distribution,TEM image and zeta potential value.To detect the drug encapsulation efficiency and the drug release characteristics of nanoparticles.Method:Firstly,cholesterol is conjugated with tLyP-1 penetrating peptide.Next,a thin-film hydration method is used to prepare co-delivery nanoparticles.The transmission electron microscope was used to observe the microscopic morphological structure of each group of nanoparticles,and the laser particle size meter was used to detect the particle size distribution and zeta potential of each group of nanoparticles.High performance liquid chromatography was used to detect the encapsulation efficiency of PTX and GANT61 by nanoparticles.The high performance liquid chromatography was also used to detect the drug release profile of tLyP-1-rHDL-PTX/GANT61 under different pH conditions.Results:Four groups of co-delivery nanoparticles were prepared:LNP-PTX/GANT61,rHDL-PTX/GANT61,tLyP-1-LNP-PTX/GANT61 and tLyP-1-rHDL-PTX/GANT61.Observed by transmission electron microscope,the nanoparticles of each group were uniform spheres.The particle size of the four groups of nanoparticles measured by the laser particle size detector are: 104±27.4nm,124±35.06 nm,119±32.1nm,127.7±29.69nm;the zeta potential are:-7.07±3.66 m V,-17.1±6.3m V,10.9±4.1m V,-5.52±3.52 m V.The encapsulation efficiency of four groups of nanoparticles to PTX measured by high performance liquid chromatography are: 81.6±4.1%,83.2±3.6%,79.2±0.7% and 83.4±2.1%.The encapsulation rates of GANT61 are: 75.9±4.1%,73.1±1.2%,67.3±2.1%,75.2±1.3%.tLyP-1-rHDL-PTX/ GANT61 nanoparticles released 15.6±5.2% and 18.7±4.4% of PTX and GANT61 within 48 hours at pH 7.4.When the nanoparticles were exposed to pH 5.5 acidic conditions for 48 hours,the release of PTX and GANT61 were 53.2±3.7%and 56.2±7.2%,respectively.Conclusion:The preparation process of peptide-functionalized recombinant highdensity lipoprotein co-delivery nanoparticles is simple.The four groups of nanoparticles have uniform diameter distribution,stable surface zeta potential values,and good encapsulation efficiency for both PTX and GANT61.tLyP-1-rHDL-PTX/GANT61 is structurally stable under central conditions,and the release of drugs is significantly increased under acidic conditions.This feature is conducive to the release of drugs in tumor tissues.PART TWO: RESEARCH ON TARGETING PROPERTY OF PEPTIDE-FUNCTIONALIZED RECOMBINANT HIGH-DENSITY LIPOPROTEIN NANOPARTICLESObjective:To observe the targeting efficacy of tLyP-1-rHDL-PTX/GANT61 nanoparticles on human triple-negative breast cancer cells MDA-MB-231 and BT-549,and to observe penetration efficacy in the 3D tumorspheres.Established a mouse breast cancer orthotopic tumor model,to observe the tumor targeting effect of nanoparticles in vivo.Methods:Confocal laser microscope observation the of aggregation of four groups of nanoparticles: LNP-PTX/ GANT61,rHDL-PTX/GANT61,tLyP-1-LNP-PTX/GANT61,tLyP-1-rHDL-PTX/GANT61 on MDA-MB-231 and BT-549.The fluorescence intensity was quantitatively analyzed by flow cytometry.The tumor cell sphere models of MDA-MB-231 and BT-549 were constructed by 3D culture method,and the penetration effect of different groups of nanoparticles on tumor cell spheres was detected through the layer-by-layer scanning of the laser confocal microscope.We establish an orthotopic xenograft tumor model in NCG mice,and use the in vivo imaging system to detect targeting efficiency of different groups of nanoparticles.Nanoparticles were injected via the tail vein,and intravital imaging was performed at 2,6,12,and 24 hours after the injection.After24 hours of image acquisition,the mice were sacrificed.The tumor tissue,heart,liver,spleen,lung,and kidney were extracted for tissue imaging in vitro.Results:Both laser confocal images and flow cytometry showed that LNPPTX/GANT61 were almost not taken up by tumor cells.rHDL-PTX/GANT61 and tLyP-1-LNP-PTX/GANT61 can be taken up by tumor cells.Compared with rHDL-PTX/GANT61,more tLyP-1-LNP-PTX/GANT61 nanoparticles enter the cytoplasm of tumor cells.Dual targeting nanoparticles tLyP-1-rHDL-PTX/ GANT61 has the best targeting efficiency.In the layer-by-layer scanning image of the confocal laser microscope,it can be observed that the LNP-PTX/GANT61 nanoparticles can not enriched on the tumorspheres.The rHDL nanoparticles can be seen to aggregate on the surface of the tumorspheres.The peptide functionalized targeting nanoparticle tLyP-1-rHDL-PTX/GANT61 is not only enriched on the surface of the tumorspheres,but also can penetrate into it.The maximum penetration distance in the MDA-MB-231 tumorsphere of tLyP-1-rHDL-PTX/GANT61 is 41.5um,and the maximum penetration distance in the BT-549 tumor cell sphere is 40.5um.In vivo imaging showed that the four groups of nanoparticles could all accumulate in tumor tissues,and the signal intensity increased over time,reaching the strongest in 24 hours.tLyP-1-rHDL-PTX/GANT61 has the strongest targeting ability in vivo.The imaging results of isolated tumor tissues are consistent with the results of in vivo imaging.Conclusion:tLyP-1-rHDL-PTX/GANT61 has excellent targeting efficacy on MDA-MB-231 and BT-549 cells in in vitro experiments,and can penetrate deep into the tumorspheres.In the tumor-bearing mouse model,tLyP-1-rHDL-PTX/GANT61 nanoparticle can effectively target tumor tissues.PART THREE: IN VITRO EXPERIMENT OF THE CYTOTOXICITY OF PEPTIDE-FUNCTIONALIZED RHDL NANOPARTICLES ON TRIPLE-NEGATIVE BREAST CANCER CELLSObjective:To study the biocompatibility of nanoparticle carriers without drugs,and to detect the anti-proliferation and apoptosis-promoting effects of peptide-functionalized co-delivery nanoparticles on triple-negative breast cancer cells.Methods:The CCK8 assay was used to detect whether the drug-free nanoparticle delivery system(LNP,rHDL,tLyP-1-LNP,tLyP-1-rHDL)have cytotoxic effects on tumor cells.The CCK8 assay detects the cytotoxicity of co-delivery nanoparticles with different targeting efficiencies on MDA-MB-231 and BT-549.The flow cytometry was used to observe the apoptosis of triple-negative breast cancer cells after treat with different groups of nanoparticles.Results:The drug-unencapsulated nanoparticle carrier has no obvious cytotoxic effect on MDA-MB-231 and BT-549 cells.The CCK8 assay and flow cytometry experiments indicate that co-loaded nanoparticles have clear anti-proliferation and pro-apoptosis effects on two triple-negative breast cancer cells,and this effect is enhanced with the enhancement of nanoparticle targeting efficiency,dual targeting co-delivery nanoparticles tLyP-1-rHDL-PTX/GANT61 have the best anti-proliferation and apoptosis-promoting effects on triple-negative breast cancer cells among the drug solution,non-targeted group and single-targeted group co-drug-loaded nanoparticles.Conclusion:The peptide-functionalized recombinant high-density lipoprotein nanoparticle carrier has good biocompatibility and is an ideal choice as a drug carrier.The tLyP-1-rHDL-PTX/GANT61 nanoparticles have better anti-proliferation and apoptosis-promoting effects on triple-negative breast cancer cells than PTX+GANT61 solution,which gives better play to ther effect of combination treatment of GANT61 and PTX.PART ? PEPTIDE-FUNCTIONALIZED DRUG-LOADED NANOPARTICLES INHIBIT TRIPLE-NEGATIVE BREAST CANCER CELL METASTASIS-RELATED PHENOTYPES IN VITROObjective:To analyze the influence of GLI1 expression on the prognosis of breast cancer patients.To verify the inhibitory effect of GANT61 and drug-loaded nanoparticles on SHH signaling pathway in MDA-MB-231 and BT-549 cells.To observe the suppression effects of GANT61 and drug-loaded nanoparticles on the migration,invasion,angiogenesis of TNBC cells.Methods:Through the mining of GEO database,the impact of GLI1 expression on the prognostic indicators of breast cancer patients such as overall survival(OS),disease-free survival(DFS),relapse-free survival(RFS)were evaluated.Western blot experiment was used to detect the effect of GANT61 on the expression of SHH signaling pathway in MDA-MB-231 and BT-549 cells,and was also used to detect drug-loaded nanoparticles with different targeting effects on the expression of GLI1 in triple-negative breast cancer cells.The wound healing assay and transwell assay were used to evaluate the inhibitory effects of GANT61 and each group of drug-loaded nanoparticles on the migration and invasion of triple-negative breast cancer cells.Western Blot was used to detect the change of EMT related protein such as Vimentin,Snail and E-cadherin.The HUVEC cell tube formation experiment was used to evaluate the effects of GANT61 and each group of drug-loaded nanoparticles on the angiogenesis ability of triple-negative breast cancer cells,and the changes of VEGF-A,VEGFR2,s VEGFR2,THBS1 were detected by q RT-PCR.Results:GLI1 is a sensitive indicator of the prognosis of breast cancer patients.Through the analysis of the GSE16446,GSE7390 data sets and the comprehensive database of three-negative breast cancer patients in KM-plotter,it can be found that the OS,RFS and DFS of patients with high expression of GLI1 were significantly inferior to patients with low expression of GLI1.The non-targeted drug-loaded nanoparticles LNP-GANT61 and GANT61 have no difference in GLI1 inhibitory efficacy.The inhibition efficacy of targeted drug-loaded nanoparticles rHDL-GANT61 and tLyP-1-LNP-GANT61 are stronger than that of drug solutions and non-targeted drug-loaded nanoparticles.The inhibitory effect of peptide-functionalized recombinant high-density lipoprotein drug-loaded nanoparticles tLyP-1-rHDL-GANT61 is better than the other experimental groups.In the wound healing assay and the transwell assay,we observed the ability of GANT61 and drug-loaded nanoparticles to inhibit breast cancer cell migration and invasion.The difference in results between the experimental groups is similar to the trend of GLI1 inhibition.Through the Western Blot experiment,we observed the changes of related indicators of epithelial-mesenchymal transition,and the trend of change is the same as before.Through the HUVEC tube formation experiment,we found that GANT61 and each group of drug-loaded nanoparticles can inhibit the angiogenesis ability of MDA-MB-231 and BT-549.Consistent with the previous results,the inhibitory effect of tLyP-1-rHDL-GANT61 nanoparticles is the strongest,which is significantly better than that of each experimental group.Through q RT-PCR experiments,we have observed neovascularization-related molecules VEGF-A,VEGFR2,THSBS1 and s VEGFR2.Conclusion:The high expression of GLI1 in breast cancer patients can lead to a worse prognosis.Inhibition of GLI1 may be a potential target for breast cancer treatment.GANT61 has inhibitory effect on GLI1 in triple-negative breast cancer cells.Peptide functionalized recombinant high-density lipoprotein drug-loaded nanoparticles can enhance the inhibitory effect of GANT61 on GLI1 in breast cancer cells.And through the inhibition of the transcription factor GLI1 to regulate multiple downstream targets,inhibit the migration,invasion and angiogenesis of triple-negative breast cancer cell,showing great potential for inhibiting tumor metastasis.PART 5 EVALUATION OF THE ANTI-TUMOR EFFECT AND BIOLOGICAL SAFETY OF PEPTIDE-FUNCTIONALIZED RECOMBINANT HIGH-DENSITY LIPOPROTEIN CO-DELIVERY NANOPARTICLESObjective:To evaluate the inhibitory efficacy of peptide-functionalized recombinant high-density lipoprotein co-delivery nanoparticles on the primary tumor site and lung metastasis in the highly metastatic triple-negative breast cancer NCG mice model.To investigate the efficacy of peptide functionalized recombinant high-density lipoprotein nanoparticle carrier reducing the toxic and side effects of anti-tumor drugs.Methods:MDA-MB-231/LUC cells were injected into the breast fat pad of severely immunodeficient NCG mice to construct a highly metastatic triple-negative breast cancer xenograft model.The mice were divided into8 groups and were given PBS,PTX solution,GANT61 solution,PTX+GANT61 mixed solution,LNP-PTX/GANT61 nanoparticles,rHDL-PTX/GANT61 nanoparticles tLyP-1-LNP-PTX/GANT61 and tLyP-1-rHDL-PTX/GANT61 nanoparticles via the tail vein,recording the growth curve of the tumor.And at certain time points(day 1,20,and 40 after tumor implantation),a small animal in vivo imaging system was used to detect the LUC luminous intensity of lungs.The mice were sacrificed on the 41 st day after tumor inoculation,the tumors were collected and weighed,and HE staining,Ki-67 immunohistochemistry,TUNEL detection and CD31 immunohistochemistry were performed.Collect mouse lung tissue,count the number of metastatic nodules on the lung surface after fixation with Bouin`s fixative,and perform HE staining.Dividing healthy NCG mice into 8 groups,administer the drug according to the same protocol as above,recording the weight change curve,collecting blood from the mice 15 days later to perform routine blood tests and blood biochemical index tests.Then the mice was sacrificed and HE staining of important organs such as heart,liver,spleen,lung and kidney were performed.Results:The combined treatment of PTX and GANT61 can effectively inhibit the growth of tumor primary tumors.Compared with mice receiving monotherapy,the tumor growth of mice receiving PTX+GANT61combined drug solution was slower.The non-targeted co-delivery nanoparticles LNP-PTX/GANT61 and PTX+GANT61 solution showed no significant difference in the inhibitory effect on the primary tumor.Targeted co-delivery nanoparticles rHDL-PTX/GANT61,tLyP-1-LNP-PTX/GANT61 have enhanced anti-tumor effects,and dual-targeted co-delivery nanoparticles tLyP-1-rHDL-PTX/GANT61 have the strongest suppression efficiency on primary tumors.The average tumor volume of tLyP-1-rHDL-PTX/GANT61 group was 147±11mm~3,which was only 12.1% of the blank control group.The results of Ki-67,TUNEL and CD31 staining show that the dual-targeted co-delivery nanoparticles group has a decrease in cell proliferation index,an increase in apoptotic index,and a decrease in angiogenesis compared with the control group.Pulmonary metastases bioluminescence signal intensity detection,lung surface metastasis nodule count,and lung tissue slice HE staining all indicate that PTX cannot inhibit tumor lung metastasis,GANT61 can reduce the occurrence of tumor lung metastasis,GANT61,PTX+GANT61combination therapy and the non-targeted co-delivery nanoparticles LNP-PTX/GANT61 showed no significant difference in the inhibition of tumor lung metastasis.Targeted co-delivery nanoparticles have a stronger inhibitory effect on tumor lung metastasis,and the inhibitory effect of tLyP-1-rHDL-PTX/GANT61 on lung metastasis is significantly better than that of the other groups.The important organs of healthy NCG mice injected with PTX+GANT61 drug solution showed varying degrees of tissue damage,which was mainly concentrated in the liver.At the same drug-equivalent concentration,the use of co-delivery nanoparticles can reduce tissue damage,and the H&E staining results of each organ in the dual-target co-drug-loaded nanoparticles group also showed no obvious pathological damage.Conclusion:In the highly metastatic triple-negative breast cancer xenograft model,peptide-functionalized recombinant high-density lipoprotein co-delivery nanoparticles tLyP-1-rHDL-PTX/GANT61 can significantly inhibit the proliferation of the primary tumor site and suppress the lung metastasis.In addition,the peptide-functionalized recombinant high-density lipoprotein drug delivery system can significantly reduce the side effects of anti-tumor drugs.tLyP-1-rHDL-PTX/GANT61 is expected to become a reasonable three-negative breast cancer treatment strategy.