| Background:Bone tissue can provide an excellent microenvironment for the growth of cancer cells,and it is the best site for the metastasis of cancer cells.It is easy for bone metastases to form multiple nidus throughout the body.Bone metastases occur in 70%of patients with advanced breast cancer and 85%of patients with advanced prostate cancer.Local surgery or radiotherapy cannot completely remove all cancer nodules.Chemotherapy is an important method for the treatment of bone metastases.However,the growth of cancer cells in bone tissue secretes a variety of cytokines,which induce osteoclast maturation by stimulating bone stromal cells or bone cells to release receptor activator of nuclear factor-κB ligand(RANKL).A large number of mature osteoclasts cause abnormal bone reabsorption at bone metastases sites,leading to severe destruction of bone at bone metastases sites.At the same time,the damaged bone releases various growth factors such as transforming growth factor-β(TGF-β),which induce rapid proliferation and invasion of cancer cells in bone tissue.The rapid proliferation and invasion of cancer cells further deteriorate the bone reabsorption,forming a vicious circle between the proliferation and invasion of cancer cells and bone resorption at the lesion site.This vicious circle severely reduces the sensitivity of bone metastases to chemotherapy drugs.Thus,this vicious circle is also a key factor for drug resistance of bone metastatic cancer cells.Therefore,only by blocking the proliferation of cancer cells and bone resorption in bone tissue at the same time,the drug-resistant bone metastases can be effectively treated.Objective:In this study,zoledronate(ZOL)was used as the bone targeting material and anti-bone resorption drug,and hyaluronic acid(HA)was used as the tumor targeting ligand.The calcium phosphate hybrid nanoparticles were prepared to encapsulate docetaxel(DTX)by using ZOL and the redox-sensitive amphiphilic block polymer hyaluronic acid-polylactic acid(HA-PLA)as the stabilizers.The calcium phosphate hybrid nanoparticles showed the characteristics of bone metastasis tumor targeting and corrosion at the microenvironment of bone metastasis sites.The nanoparticles could not only release ZOL at the bone resorption site and played an inhibitory role in bone resorption,but also enabled ZOL and DTX to efficiently enter drug-resistant bone metastatic cancer cells at the same time,subsequently played a synergistic role in the treatment of drug-resistant bone metastasis through blocking the vicious circle between the growth of drug-resistant bone metastases and bone resorption.As a result,the curative effect of calcium phosphate hybrid nanoparticles on drug-resistant bone metastases was significantly improved.Methods:1.The HA-PLA conjugate was prepared by using HA,PLA and cysteamine.The structure of HA-PLA conjugate was identified by ~1HNMR and IR spectra.2.With HA-PLA as the amphiphilic block polymer,DTX-loaded micelle DTX@HP was prepared by dialysis method.By using particle size and drug loading as indexes,the prescription and preparation process were optimized by changing temperature,stirring time and ratio of drug to material.3.By using DTX@HP,ZOL,calcium chloride and diammonium phosphate as raw materials,the DTX-loaded calcium phosphate hybrid nanoparticles DTX@Capzol/HP were prepared.Taking the particle size and drug loading as indicators,the prescription and preparation process of DTX@Capzol/HP were optimized by changing the concentration of DTX@HP,temperature and stirring time.4.The particle size and zeta potential analyzer,high performance liquid chromatography(HPLC),and transmission electron microscopy(TEM)were used to investigate the particle size,zeta potential,drug loading,morphology,stability,and p H response of DTX@Capzol/HP.5.The drug release characteristics of DTX@Capzol/HP in different release media were investigated by dialysis method.The adsorption properties of DTX@Capzol/HP on the skull tissues of neonatal mice and hydroxyapatite were investigated by using in vivo imaging.6.By using MTT method,the cytotoxicity of DTX@Capzol/HP on human prostate cancer cells(PC-3 cells),DTX-resistant prostate cancer cells(PC-3/DDR cells),human breast cancer cells(MDA-MB-231 cells)and DTX-resistant breast cancer cells(MDA-MB-231/DDR cells)was investigated.The inhibitory effect of DTX@Capzol/HP on the clone formation of PC-3 cells and PC-3/DDR cells was studied by cloning formation experiment.7.The cellular uptake and its mechanism of DTX@Capzol/HP by PC-3 cells and PC-3/DDR cells were determined through laser scanning confocal microscope.8.The effects of DTX@Capzol/HP on the migration and invasion of PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells in bone microenvironment were investigated through migration and invasion experiments.9.Western blot was used to detect the effects of DTX@Capzol/HP on the protein expression of Bcl-2,Bax,Caspase-3,Cleaved caspase-3,FPPS,E-cadherin,N-cadherin,MMP-9,CXCR4 and CD44 in PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells.10.The skull of newborn mice was co-incubated with PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells to establish an in-vitro 3D model of bone metastasis.The effects of DTX@Capzol/HP on DTX-resistant tumor cells and lacunae in in-vitro 3D model of bone metastasis were investigated by field emission scanning electron microscope(SEM).The effect of DTX@Capzol/HP on osteoclasts in in-vitro 3D model of bone metastases was observed by using tartrate resistant acid phosphatase(TRAP)staining.11.PC-3/DDR cells and MDA-MB-231/DDR were injected into the right hind limb bone marrow cavity of nude mice to construct a model of DTX-resistant prostate cancer and DTX-resistant breast cancer bone metastasis.The antitumor activity of DTX@Capzol/HP was investigated by measuring tumor volume in bone metastasis nude mice.The effect of DTX@Capzol/HP on bone mass at the site of bone metastasis was studied by micro-CT.The distribution of DTX@Capzol/HP in bone metastasis nude mice was observed by in vivo imager.The effects of DTX@Capzol/HP on the protein expression of Bcl-2、Bax、Caspase-3、Cleaved caspase-3、FPPS、E-cadherin、N-cadherin、MMP-9、CXCR4、CD44、TGF-β、RANKL and OPG in caner tissue was detected by western blot.The activity of osteoclasts in bone tissue at the site of bone metastases was observed by TRAP staining.Results:1.HA-PLA was identified as the target product by ~1HNMR and IR.2.By using dialytic method,the optimized prescription and preparation process to prepare DTX@HP were as following:the ratio of DTX to HA-PLA was 1:5(g/g),dimethyl sulfoxide was used as solvent and stirring temperature was 20℃.The particle size,zeta potential and drug loading of DTX@HP was 208 nm,-22.2 m V and 4.6%,respectively.DTX@HP exhibited spherical-like particle.3.The optimized prescription and preparation process to prepare DTX@Capzol/HP were as following:the concentration of DTX@HP was 8 mg/m L,stirring temperature was 20℃and stirring time was 20 min.The particle size and zeta potential of DTX@Capzol/HP was144 nm and-7.8 m V,respectively.The DTX loading and ZOL loading in DTX@Capzol/HP was 4.6%and 4.3%,respectively.The particle size of DTX@Capzol/HP increased and density of DTX@Capzol/HP decreased in p H 5.0 medium,indicating that the stability of DTX@Capzol/HP was p H sensitive.4.The in vitro drug release results showed that with the decrease of p H of drug release medium,the release speed of ZOL from DTX@Capzol/HP was accelerated,and the cumulative release amount was significantly increased.The drug release rate of DTX from DTX@Capzol/HP exhibited glutathione concentration dependent manner.This indicated that drug release from DTX@Capzol/HP exhibited p H and redox dual responsive.5.The in vivo imaging results showed that hydroxyapatite and newborn mice skulls exhibited strong adsorption effect on DTX@Capzol/HP,indicating that DTX@Capzol/HP had good bone affinity.6.MTT experimental results showed that compared with free DTX,DTX@Capzol/HP displayed stronger cytotoxicity on PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells.The clone formation experimental results showed that DTX@Capzol/HP could significantly inhibit the clone formation of PC-3 cells and PC-3/DDR cells.7.DTX@Capzol/HP was uptaken by PC-3 cells and PC-3/DDR cells in a time-dependent manner.In weak acidic environment,cellular uptake of DTX@Capzol/HP by PC-3 cells and PC-3/DDR cells was significantly increased.Chlorpromazine and methyl-?-cyclodextrin significantly inhibited the uptake of DTX@Capzol/HP by PC-3/DDR cells,indicating that the uptake of DTX@Capzol/HP by PC-3/DDR cells was mainly through the clathrin and caveolin protein pathway.In addition,the CD44 receptor ligand HA significantly reduced the uptake of DTX@Capzol/HP by PC-3/DDR cells,indicating that DTX@Capzol/HP was uptaken by PC-3/DDR cells through CD44 receptor mediated endocytosis.8.Migration and invasion experimental results showed that DTX@Capzol/HP significantly inhibited the migration and invasion of PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells in bone microenvironment,and the inhibition effect of DTX@Capzol/HP on migration and invasion were significantly stronger than that of free DTX and ZOL.9.Western blot results showed that DTX@Capzol/HP obviously increased the protein expression of Bax,Caspase-3 and Cleaved caspase-3,and reduced the protein expression of Bcl-2 in PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells.This indicated that DTX@Capzol/HP promoted the apoptosis of drug-resistant cancer cells through mitochondrial apoptosis pathway.DTX@Capzol/HP can reduce the expression of FPPS protein.The results indicate that DTX@Capzol/HP can inhibit the activity of farnesyl pyrophosphate synthase(FPPS)and hinder the post-translational modification of proteins,thereby further enhancing DTX@Capzol/HP The role of inducing cancer cell apoptosis.In addition,DTX@Capzol/HP significantly decreased the protein expression of MMP-9,N-Cadherin,CXCR4 and CD44,and significantly increased the protein expression of E-cadherin in PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells.This implied that DTX@Capzol/HP inhibited the migration and invasion of PC-3 cells,PC-3/DDR cells,MDA-MB-231 cells and MDA-MB-231/DDR cells by increasing the adhesion of cancer cells.10.In-vitro 3D model of bone metastases showed that DTX@Capzol/HP significantly reduced the proliferation of drug-resistant cancer cells,compared with free DTX and ZOL.DTX@Capzol/HP played an anti-bone resorption role by reducing the number of osteoclasts in in-vitro 3D models of bone metastases.11.In vivo experimental results in cancer-bearing nude mice showed that DTX@Capzol/HP displayed a good targeting effect on bone metastases.Compared with the same dose of DTX+ZOL and DTX@HP,DTX@Capzol/HP significantly inhibited the growth of bone metastases and increased bone density at the site of bone metastases.Besides,H&E staining results showed that DTX@Capzol/HP exhibited highly specific toxicity on tumor tissues,but displayed no significantly toxicity on normal organs.DTX@Capzol/HP obviously reduced the number of osteoclasts in bone tissue at the site of bone metastasis.DTX@Capzol/HP significantly reduced the proteins expression of TGF-β,RANKL,Bcl-2,FPPS,MMP-9,N-cadherin,CXCR4 and CD44,and increased the proteins expression of OPG,Bax,caspase-3,Cleaved caspase-3 and E-cadherin in tumor tissue.The above data indicated that DTX@Capzol/HP reduced the activation of osteoclasts by decreasing RANKL and increasing OPG expression,subsequently attenuated bone resorption.DTX@Capzol/HP inhibited the growth of DTX-resistant bone metastases by inhibiting TGF-β、FPPS and Bcl-2 protein expression and decreasing Bax and Cleaved caspase-3protein expression.DTX@Capzol/HP inhibited the invasion of DTX-resistant bone metastases by increasing the adhesion between cancer cells.Conclusions:DTX@Capzol/HP exhibited p H sensitivity and targeting of bone metastases.DTX@Capzol/HP effectively delayed the growth of DTX-resistant bone metastases through inhibiting TGF-β、FPPS and Bcl-2 protein expression and decreasing Bax and Cleaved caspase-3 protein expression.DTX@Capzol/HP inhibited the invasion of DTX-resistant cancer cells via increasing the adhesion between cancer cells.DTX@Capzol/HP attenuated bone resorption by decreasing RANKL expression and increasing OPG expression.DTX@Capzol/HP blocked the vicious cycle between proliferation and bone resorption of DTX-resistant bone metastases via dual pathway through the synergy of DTX and ZOL,consequently improved the therapeutic effect on DTX-resistant bone metastases in vivo. |
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