Preparation Of Docetaxel-loaded Bone Targeting Dendrimer Nanoparticle And Its Anti-tumor Activity In Bone Metastases Of Lung Cancer

In recent years,the incidence and mortality of lung cancer rank first in our country.Bone is one of the prone metastatic sites of lung cancer,and 30%~40%of patients with advanced lung cancer will develop into bone metastases.The most of bone metastases of lung cancer are osteolytic destruction.In the bone metastases sites,a variety of cytokines secreted by tumor cells can up-regulate the expression of receptor activator for nuclear factor-?B ligand(RANKL)and inhibit the secretion of osteoprotegerin(OPG)in osteoblast or osteocyte,resulting in the increase of bone resorption.While in the process of bone resorption,bone matrix releases various growth factors,which stimulate the proliferation of tumor cells in turn.Finally,a"vicious circle"between bone resorption and tumor growth is formed.Bone metastases destroy bone mass and subsequently lead to a series of skeletal-related events such as hypercalcemia,severe pain,pathologic fractures,spinal cord compression and myelodysplasia.These complications severely lower the quality of life of patient.Besides,due to the poor permeability and low blood flow of bone tissue,the conventional chemotherapy shows low efficacy.Docetaxel(DTX),a sort of common anti-tumor agents in clinic,exhibits a broad anti-tumor spectrum and high anti-tumor activity.However,the low water solubility and poor bone targeting ability limit its clinical application in bone metastases of lung cancer.Nano-delivery system shows significant effect on improving drug solubility and targeting ability.Therefore,it is of great importance to develop a bone targeting DTX delivery system with anti-bone resorption effect for the treatment of bone metastases of lung cancer.Objective:In order to enhance the distribution of DTX in bone metastases sites and inhibit bone resorption and bone metastases,a pH-responsive DTX@ALN-PAMAM and a pH and redoxdual-responsiveDTX@ALN-HA-PAMAMwerepreparedbyusing polyamidoamine dendrimer(PAMAM G5.0)as drug carrier,alendronate sodium(ALN)as bone targeting ligand and hyaluronic acid(HA)as tumor cell targeting ligand.Methods:(1)ALN-PAMAM and ALN-HA-PAMAM were prepared by using PAMAM,ALN and HA.Their structures were identified by ~1H NMR and IR.(2)The pH-responsive DTX@ALN-PAMAM and pH/redox dual-responsive DTX@ALN-HA-PAMAM were prepared by solvent evaporation method.The particle size and drug loading were used as indexes to optimize the prescription composition and preparation process by changing the ratio of DTX to material,solvent and stirring time.(3)The particle size,zeta potential,drug loading,morphology and stability of drug-loaded nanoparticles were investigated by using zeta potential and nano-particle size analyzer,high performance liquid chromatography(HPLC)and transmission electron microscopy(TEM).(4)The drug release properties of drug-loaded nanoparticles in different release mediums were investigated by dialysis method.The zeta potential of nanoparticles in different mediums was measured by zeta potential and nano-particle size analyzer.(5)The adsorption and desorption of nanoparticles on hydroxyapatite were estimated by using HPLC.(6)The cytotoxicity of drug-loaded nanoparticles against A549 cells and MDA-MB-231 cells was evaluated by MTT assay.The uptake of nanoparticles in A549cells and RAW 264.7 cells was investigated by using HPLC and confocal laser scanning microscope(CLSM).(7)In vitro 3D bone metastases model of lung cancer was established by co-incubating of A549 cells with cranium of neonatal mouse.The effects of drug-loaded nanoparticle on tumor cells and bone lacuna number were observed by using field emission scanning electron microscopy(SEM).The effect of drug-loaded nanoparticles on osteoclasts was estimated by using tartrate resistant acid phosphatase(TRAP)staining.(8)The bone metastases model of lung cancer in nude mice was established by injecting A549 cells to medullary cavity of right hindlimb.The in vivo antitumor activity of drug-loaded nanoparticles was determined by measuring tumor volume and pain response after injecting by tail vein.The toxicity of drug-loaded nanoparticles was evaluated by body weight changes of tumor-bearing nude mice and H&E staining.The bio-distribution of nanoparticle in tumor-bearing nude mice was observed by using in vivo imaging.The effect of drug-loaded nanoparticle on bone mass in bone metastases sites was evaluated by using micro-CT.Result:(1)By using ~1H NMR and IR,the synthesized ALN-PAMAM and ALN-HA-PAMAM were identified as the target compounds.(2)The drug-loaded nanoparticles were prepared by solvent evaporation method.The optimized prescription composition and preparation process were conducted as follows:the ratio of DTX to material was 1:5(g/g),the solvent was the mixture of methanol and acetone(v/v=1:1)and the stirring time was 12 h.The particle size of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM was(99±13)nm and(164±3)nm respectively.The zeta potential was+(4.35±0.28)mV and-(2.28±0.27)mV respectively.The drug loading was(5.2±0.4)%and(4.3±0.3)%,respectively.The TEM results showed that the drug-loaded nanoparticles were spherical in appearance.The content of ALN in DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM was 0.49%and 0.54%,respectively.(3)The in vitro DTX release rate of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM increased significantly with the decrease of pH.As the GSH concentration increased,more amount of DTX released from DTX@ALN-HA-PAMAM.The above data indicated that DTX@ALN-PAMAM exhibited pH-responsive drug release characteristics and DTX@ALN-HA-PAMAM exhibited pH/redox dual-responsive drug release characteristics.When the pH value dropped from 7.4 to 5.0,the zeta potential of DTX@ALN-PAMAMincreasedgradually,andthezetapotentialof DTX@ALN-HA-PAMAM changed from negative to positive.This indicated that DTX@ALN-HA-PAMAM exhibited charge-reversal property.(4)DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM showed strong adsorption on hydroxyapatite.DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM also exhibited good desorption from hydroxyapatite in pH 6.5.This indicated that DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM exhibited good bone targeting ability and they could desorb from bone matrix in the weak acidic microenvironment in bone resorption sites.(5)Compared with free DTX,the cytotoxicity of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM against A549 cells and MDA-MB-231 cells was significantly enhanced.(6)The uptake of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM in A549cells increased significantly in pH 6.5.The uptake of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM in A549 cells and RAW264.7 cells increased in time-dependent manner.The modification of ALN and HA significantly reduced the uptake of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM by RAW264.7 cells.In addition,exogenous HA reduced the uptake of DTX@ALN-HA-PAMAM in A549 cells.Sucrose(clathrin pathway inhibitor)significantly inhibited the uptake of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM in A549 cells.This indicated that the uptake of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM in A549 cells was mainly through clathrin-mediated endocytosis.(7)DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM effectively inhibited the proliferation of osteoclasts and tumor cells,and reduced the number of bone lacunae in in vitro 3D bone metastases model of lung cancer.This indicated that DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM exhibited anti-bone resorption and anti-tumor activity in in vitro 3D model.(8)DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM could markedly inhibit the growth of bone metastases and alleviate the pain of tumor-bearing nude mice.In vivo imaging results showed that DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM selectively accumulated in bone metastases sites.The bone mineral density in bone metastases sites of DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM group was significantly higher than that in normal saline group and free DTX group.This indicated that DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM significantly inhibited bone resorption.H&EstainingindicatedthatDTX@ALN-PAMAMand DTX@ALN-HA-PAMAM exhibited obvious toxicity on tumor tissue,but they did not exhibit apparent toxicity on normal organs.Conclusion:DTX@ALN-PAMAM showed pH-responsive characteristics.DTX@ALN-HA-PAMAM exhibited pH/redox dual-responsive characteristics.DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM selectively accumulated in bone metastases sites.DTX@ALN-PAMAM and DTX@ALN-HA-PAMAM obviously inhibited bone resorption and tumor growth.Consequently,they significantly improved the therapeutic effect of DTX on bone metastases of lung cancer.Compared with DTX@ALN-HA-PAMAM,DTX@ALN-PAMAM exhibited better therapeutic effect on bone metastases of lung cancer.