Preparation And Anti-tumor Activity Of Bone-targeted Micelles

Background:Bone cancer is often happened in bone tissue or its affiliates, and it can be divided into primary and secondary bone cancer. Study found that at least 50% of advanced cancer patients will occurr bone metastases. The following cancers are proved to be easy to metastasize to the bone: breast cancer(the rate of bone metastases is 65%-75%), prostate cancer(the rate of bone metastases is 65%-75%), thyroid cancer(the rate of bone metastases is 60%), bladder cancer(the rate of bone metastases is 40%), lung cancer(the rate of bone metastases is 30%-40%), kidney cancer(the rate of bone metastases is 20%-25%) and malignant melanoma(the rate of bone metastases is 4%-45%). When the tumor cells metastasize to the bone tissue, bone is damaged. The destruction of bone produce bone related events including pathologic fractures, hypercalcemia, spinal cord and nerve root compression, decreased bone marrow function, etc. These complications severely affect the treatment of cancer and increase the pain, and seriously affect the quality of life of patients.The main component of bone tissue is hydroxyapatite [(Ca10PO4)6(OH)2, HA]. Because of the poor permeability, specific physiological and biochemical processes of bone tissue, it is difficult for drug to effectively accumulate in bone metastases tumor. In order to achieve effective therapeutic concentrations in bone tissue, higher dose and long-term medication are inevitable. This not only reduces the therapeutic index, but also causes more serious adverse reactions. Pierce in 1986 for the first time proposed the concept of "bone-targeted", the compound deposited in the bone and bounded with calcium. Thus, the compound that has a specific affinity with hydroxyapatite can be used as a bone targeting molecule. Bisphosphonates(BPs) has a strong affinity with HA. Moreover, once BPs is administrated, it is rapidly cleared from the blood circulation and adsorbed onto the bone mineral. Alendronate(ALN) belongs to the third generation BPs, its structure is simple and can be chemically modified. ALN has a strong binding affinity with the bone site and has been widely used as a bone targeting ligand.Studies have shown that the response to environmental stimuli plays a key role for the controlled release of drugs. Hydrazone bond is a p H-sensitive bond, it is stable in the blood circulation(p H =7.4), but it can be broken in the tumor cell lysosomes(p H <6). The block copolymer conjugated by hydrazone bond can form a p H-sensitive drug delivery system. Disulfide bond is a kind of chemical bond, which is sensitive to glutathione(GSH). The concentration of GSH varied enormously inside and outside of the cells. The intracellular GSH concentration is 2-10 m M, and the extracellular GSH concentration is approximately 2-20 μM. Thus, disulfide-linked drug delivery systems were used to selectively release the drug in the tumor cells.Polyethylene glycol(PEG) is a water-soluble, good biocompatibility and no immunogenicity polymer. PEG is widely used in drug delivery systems. The active targeting drug delivery systems can be obtained by connecting a targeting ligand with PEG. Dextran is a class of natural origin types of polymer material. Compared with many synthetic polymers, dextran has low level of toxicity. Based on the above advantages, the dextran is chosen as the redox-sensitive micellar bridging arm.Objective:In this study, alendronate(ALN) is chosen as the bone targeting ligand, doxorubicin(DOX) as a model drug, PEG-based p H-sensitive micelles and DEX-based redox-sensitive micelles were prepared. The bone targeted property and antitumor activity of drug loaded micelle were investigated to explore a new method to treat bone metastases.Methods:1. ALN is chosen as the bone targeting ligand, DOX as a model drug, PEG-based p H-sensitive micelles DOX-hyd-PEG-ALN, and DEX-based redox-sensitive micelles OA-DEX(5K)-ALN, MA-DEX(5K)-ALN, ODA-DEX(5K)-ALN, DSPE-DEX(5K)-ALN, DSPE-DEX(20K)-ALN, DSPE-DEX(70K)-ALN and DSPE- DEX(150K)-ALN were respectively prepared. 2. The preparation procedure of drug-loaded micelles was optimized by changing the amount of water, temperature and ratio between DOX and micellar material. 3. The micelle was characterized by measuring drug loading, encapsulation efficiency, CMC value, particle size, zeta potential, stability and the micellar morphology. 4. The drug release properties of micelles were observed in different media solution. 5. The adsorption of micelle with hydroxyapatite(HA) was studied. 6. The cytotoxicity of drug-loaded micelles was determined by the MTT assay. The cellular uptake of the drug-loaded micelles was observed by using fluorescence microscopy and flow cytometry. The distribution of DOX in tumor cells was observed by using confocal laser scan microscopy after micelles were cultured with the cells. The apoptosis was tested by Caspase-3 kits. The flow cytometry was used to observe the effect of the drug-loaded micelles on tumor cell cycle. 7. The bone metastases of lung cancer model was prepared by injection of A549 cells(1×107 cells/animal) via intra-tibia. The effects of bone metastases on bone were observed by Micro-CT. The distribution of the DOX in tumor-bearing nude mice was measured by the in vivo imaging and fluorescence microscopy after micelle was intravenously administered. The therapeutic effect of micelles on nude mice was determined by measuring tumor volume, body weight and survival rate.Results:Part one: The preparation and anti-tumor activity of DOX@DOX-hyd-PEG-ALN.1. 1H NMR and IR confirmed DOX-hyd-PEG-ALN was the desired polymeric material.2. The dialysis method was used to prepare DOX@DOX-hyd-PEG-ALN. The diameter, drug loading, encapsulation efficiency and CMC value of DOX@DOX-hyd-PEG-ALN was 114±27 nm, 24.3%, 64.3% and 2.3 mg/L, respectively. TEM data showed that DOX@DOX-hyd-PEG-ALN had spherical structure. XPS analysis showed ALN presented outside of the micelle surface. DOX@DOX-hyd-PEG-ALN showed p H dependent drug release characteristics. DOX@DOX-hyd-PEG-ALN was stable in the blood condition, and released drugs quickly and thoroughly in acidic environments. DOX@DOX-hyd-PEG-ALN showed high binding affinity with HA. 3. Compared with free DOX, DOX@DOX-hyd-PEG-ALN showed no significant toxicity on A549 cells after 24 h of incubation. When the incubation time increased to 48 h, the DOX@DOX-hyd-PEG-ALN showed higher toxicity than free DOX on A549 cells. The cellular uptake of the micelles showed a time-dependent manner. Caspase-3 data showed DOX@DOX-hyd-PEG-ALN induced apoptosis in A549 cells in a time and concentration-dependent manner. Cell cycle analysis showed that DOX and DOX@DOX-hyd-PEG-ALN were able to induce A549 cells arrest into G2 phase and S phase in time-dependent manner. 4. Bone metastasis model was confirmed by Micro-CT. HE staining showed that the nucleolus of the tumor cells were clear and large. The tumor cells were also irregular in formation. 5. Living imaging and fluorescence microscopy showed that DOX@DOX-hyd-PEG-ALN exhibited better bone-targeted property and enhanced the permeability of DOX in the tumor tissue as compared with free DOX. At the same time, DOX@DOX-hyd-PEG-ALN decreased the distribution of the DOX in heart. In vivo anti-tumor experiment results showed that compared with free DOX, DOX@DOX-hyd-PEG-ALN significantly prolonged the lifetime of the tumor-bearing mice, inhibited the tumor growth. Micro-CT results showed that compared with the free DOX, DOX@DOX-hyd-PEG-ALN could effectively weaken the effect of tumor cells on the bone density. HE staining indicated that DOX@DOX-hyd-PEG-ALN micelles had no significant toxic effects on the normal organs, but showed obvious toxicity in the tumortissue.Part two: The preparation and anti-tumor activity of DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN.1. 1H NMR and IR confirmed the OA-DEX(5K)-ALN, MA-DEX(5K)-ALN, ODA-DEX(5K)-ALN, DSPE-DEX(5K)-ALN, DSPE-DEX(20K)-ALN, DSPE-DEX(70K)-ALN and DSPE-DEX(150K)-ALN were the desired polymeric material. 2. The dialysis method was used to prepare the drug-loaded micelles. The size of DOX@OA-DEX(5K)-ALN, DOX@MA-DEX(5K)-ALN, DOX@ODA-DEX(5K)-ALN, DOX@DSPE-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN was about 60 nm. The size of DOX@DSPE-DEX(70K)-ALN and DOX@DSPE-DEX(150K)-ALN was about 90 nm. The zeta potential of micelles was between-15 m V and-25 m V, the drug loading was more than 17%, and the encapsulation efficiency was more than 70%. TEM data showed that the micelles had spherical structure. XPS analysis showed ALN presented the surface of the micelle. The OA-DEX(5K)-ALN, MA-DEX(5K)-ALN, DSPE-DEX(5K)-ALN and DSPE-DEX(20K)-ALN was stability in five days. However, with the increasing of incubation time, the particle size of DSPE-DEX(70k)-ALN, DSPE-DEX(150k)-ALN and ODA-DEX(5K)-ALN became large. Drug release of the micelles showed GSH concentration-dependent characteristics. The micelles modified by the ALN showed high binding affinity with HA. 3. DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN showed the strongest cytotoxicity on A549 cells. The cellular uptake of the DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN showed time-dependent characteristics. DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN induced apoptosis of A549 cells and MDA-MB-231/ADR cells in time and concentration-dependent manner. Compared with free DOX, DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN induced more cancer cells arrest into G2 phase and S phase. 4. Living imaging and fluorescence microscopy showed that DOX@ MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN exhibited better bone-targeted property and enhanced the permeability of DOX in tumor tissue as compared with free DOX. At the same time, DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN decreased the distribution of the DOX in heart. In vivo anti-tumor experiment results showed that compared with free DOX, DOX@MA-DEX(5K)-ALN micelles significantly prolonged the lifetime of the mice and inhibited the tumor growth. Compared with the free DOX, DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN could effectively weaken effect of tumor cells on the bone density. HE staining results indicated that MA-DEX(5K)-ALN micelles had no obvious toxic effects on the normal organs, but showed significant toxicity in the tumor tissue.Conclusion:The drug loading and encapsulation efficiency of DOX@DOX-hyd-PEG-ALN was high. DOX@DOX-hyd-PEG-ALN was effectively uptaked by the tumor cells, and showed higher cytotoxicity as compared with free DOX. DOX@DOX-hyd-PEG-ALN could effectively accumulate in bone metastases site, and enhanced the antitumor activity of DOX. Thus, DOX@DOX-hyd-PEG-ALN prolonged the survival time of tumor-bearing nude mice, and reduced the systemic toxicity of DOX.The size of DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN was less than 100 nm. DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN could be effectively uptaked by tumor cells, and showed a stronger cytotoxicity as compared to the free DOX. DOX@MA-DEX(5K)-ALN and DOX@DSPE-DEX(20K)-ALN could effectively accumulate in bone metastases site, and enhanced the antitumor activity of DOX. Thus, DOX@MA-DEX(5K)-ALN prolonged the survival time of tumor-bearing nude mice, and reduced the systemic toxicity of DOX. As compared with DOX@DOX-hyd-PEG-ALN, DOX@MA-DEX(5K)-ALN delivered more DOX to the tumor site, and showed better anti-tumor activity.