| Docetaxel(DTX)is a second-generation taxane antineoplastic agent that has a definite therapeutic effect as a primary or second-line clinical treatment for primary lung cancer.However,the clinically high-risk lung cancer bone metastasis has poor efficacy and large side effects.The main reason is that docetaxel has poor targeting to bone metastatic lung cancer tumor cells and does not reach a sufficient therapeutic dose.In order to solve this bottleneck problem,this study first used tumor vascular-targeted docosahexaenoic acid(DHA)-conjugated bovine serum albumin(BSA)as a carrier(DHA-BSA)to entrap drugs to enhance the tumor targeting of drugs.Secondly,considering the toxicity of DHA-BSA-encapsulated docetaxel sustained release,the docetaxel prodrug(DTX-S-DTX)was designed by using redox double?sensitive monosulfide bond as chemical linker.Controlled release administration of docetaxel tumor tissue is achieved due to the presence of a high redox microenvironment in the tumor tissue.Finally,the prodrug and the carrier were prepared into nanospheres(DTX-S-DTX-DHA-BSA-NPs)by emulsification.This study constructed a tumor-targeted delivery system for the treatment of lung cancer metastases to bone with a docetaxel prodrug in response to tumor microenvironment,and evaluated the system.The results are as follows:1.Synthesis of DTX-S-DTX prodrug:The DTX-S-DTX prodrug was designed by using monosulfide bond as the redox sensitive chemical bonding arm.The synthesis of DTX-S-DTX was confirmed by the use of mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy and the combination with the activity intensity of hydroxyl esterification reaction in DTX molecular structure.2.Preparation and Optimization of DTX-S-DTX-DHA-BSA-NPs:the preparation of DTX-S-DTX-DHA-BSA-NPs nanoparticles were prepared and the preparation eonditions were optimized by single factor experiment.The results were shown:coupling protein(DHA-BSA)concentration was 5 mg/mL,the volume ratio of the organic phase to the aqueous phase was 1:16,the homogenate speed was 6000 rpm,the homogenization time was 6 min,homogenization pressure was 800 bar,and the homogenization times were 7 times.Under this optimization condition,the particle size of DTX-S-DTX-DHA-BSA-NPs prepared was 98.8±11.7 nm,potential-20.90±1.24 mV drug loading 7.86%±1.4%,entrapment efficiency 84.6%±4.3%,the DHA content in the sample was 4.04%±1.2%,n=3.3.Characterization of DTX-S-DTX-DHA-BSA-NPs:?Scanning electron microscope(SEM)was used to observe the morphology of DTX-S-DTX-DHA-BSA-NPs.The results show that the DTX-S-DTX exhibits a non-uniform long-form distribution and the particle size reaches the micron level;the DTX-S-DTX-DHA-BSA-NPs exhibit an approximate spherical shape with a particle size of about 10nm.?The results of X-ray diffraction(XRD)showed that the peak intensity of the original drug was the largest,and a large number of diffraction peaks appeared at 10.71°,12.15°,13.57°,and 17.14°,19.29°,and there are two diffraction peaks in the physical mixture at 19.27°and 31.66°;DTX-S-DTX-DHA-BSA-NPs showed a diffraction peak at 19.28°,and the peak intensity was significantly smaller which indicates that DTX-S-DTX-DHA-BSA-NPs changes its crystal structure due to the smaller particle size,and the crystallinity decreases.DTX-S-DTX exists in the nanoparticles with the amorphous form make DTX-S-DTX-DHA-BSA-NPs have a better water solubility,which increases the bioavailability of the drug.?Using thermal differential analysis(DSC)to detect the properties of nanoparticles,the DTX-S-DTX drug showed a strong heat absorption peak at 216.07?,indicating that it showed crystal morphology,while the physical mixture of DTX-S-DTX and DHA-BSA has a very weak heat absorption peak at 216.07?,indicating that a small amount of DTX-S-DTX crystal is adsorbed by the surface of DHA-BSA,the DTX-S-DTX-DHA-BSA-NPs,its heat absorption peak disappeared at 216.07?,indicating that DTX-S-DTX in DTX-S-DTX-DHA-BSA-NPs existed in an amorphous state.4.DTX-S-DTX-DHA-BSA-NPs redissolving stability testing:The lyophilized DTX-S-DTX-DHA-BSA-NPs were dissolved in deionized water and their particle size and potential were measured at 0 h,1 h,2 h,4 h,8 h,12 h and 24 h.The result were 97.4±15.3 nm and-20.90±1.63mV,101.0±14.8nm and-21.37±1.83mV,104.2±15.9nmand-21.56±1.36mV,110.3±15.9nm and-20.76±0.92 mV,122.0±17.1nm and-19.33±1.88 mV,129.1±16.6un and-19.92±1.43mV,146.0±18.1 nm and-19.86±1.47 mV,respectively,n=3,the particle size and potential value did not change much within 8 h,indicating that its stability was better.5.DTX-S-DTX-DHA-BSA-NPs in vitro release experiments:To investigate the condition of DTX-S-DTX-DHA-BSA-NPs nanoparticles release DTX with the addition of hydrogen peroxide(H2O2)or dithiothreitol(DTT)a mixed solvent containing 30%ethanol in PBS buffer(pH=7.4)was used as the release medium.The results showed that 10.4%± 2.4%DTX was released from the nanoparticles in the release medium without the addition of hydrogen peroxide(H2O2)or dithiothreitol(DTT).While 88.1%± 8.7%,91.3%±8.1%,34.7%±5.2%,and 41.6±3.8%of DTX were released from DTX-S-DTX-DHA-BSA-NPs in the release media containing 5mM H2O2,10 mM H2O2 and 5 mM DTT,10 mM DTT within 12 h respectively,n=3.This indicates that the designed DTX-S-DTX prodrug can release DTX rapidly in a redox microenvironment.6.Acute toxicity experiment of DTX-S-DTX and DTX-S-DTX-DHA-BSA-NPs:Using C57BL/6 mice as models,DTX-S-DTX and DTX-S-DTX-DHA-BSA-NPs were administered at doses of 5?moL/kg,10?moL/kg,15?moL/kg,20?moL/kg and 25 ?moL/kg,respectively,and observed.The survival status of the mice and the weight of the mice were recorded.The results showed that the maximum drug resistance dose of DTX-S-DTX was 5 ?moL/kg,and the maximum drug resistance dose of DTX-S-DTX-DHA-BSA-NPs was 10?moL/kg.7.In vivo anti-tumor activity of DTX-S-DTX-DHA-BSA-NPs:C57BL/6 mice were used to establish a bone metastasis model of lung cancer,and the effect of DTX-S-DTX-DHA-BSA-NPs on bone metastasis of lung cancer was studied.In the study of anti-lung cancer bone metastasis activity,the average survival time of the control group was 19.86±2.96 d,and the average survival time of the 5 ?moL/kg DTX group was 25.43±1.38 d.The average survival time of the 2.5 ?moL/kg DTX-S-DTX group was 26.71±1.34 d(P<0.05),2.5?moL/kg DTX-S-DTX-DHA-BSA-NPs group had an average survival of 29.29±2.37 days(P<0.01),5?moL/kg DTX-S-DTX-DHA-BSA-NPs group was 33.00±2.55 d(P<0.01).In the study of anti-lung cancer bone metastasis activity,the average body weight of the control group was 26.08±1.67g,the average body weight of the 5 ?moL/kg DTX group was 24.45±1.89g,and the average body weight of the 2.5?moL/kg DTX-S-DTX group was 24.30±1.88 g.The mean body weight of the 2.5 ?moL/kg DTX-S-DTX-DHA-BSA-NPs group was 26.39±2.30g,and the average body weight of the 5 ?moL/kg DTX-S-DTX-DHA-BSA-NPs group was 26.98±2.43 g.The above data indicate that DTX-S-DTX-DHA-BSA-NPs has stronger inhibitory effect on bone metastasis of lung cancer than DTX and DTX-S-DTX,which improves the average survival time of mice and greatly reduces the cytotoxicity of drugs.The DTX-S-DTX-DHA-BSA-NPs prepared in this reseach have good water solubility,redissolving stability,tumor targeting,controlled release in tumor tissue and low toxic side effects,and thus have strong anti-lung cancer bone metastasis.It has the potential to be further developed as a drug for anti-lung cancer bone metastasis.This study will provide new ideas for the design of targeted delivery system for lung cancer bone metastasis,and lay a solid theoretical and practical foundation for clinical breakthroughs in the treatment of bone metastasis of lung cancer. |
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