| Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults and accounts for 60% of primary bone cancers diagnosed in the first two decades of life. Doxorubicin (DOX), cisplatin, methotrexate, Ifosfamide, and curcumin(CUR) are the commonly used chemotherapeutics to treat OS. But, in the treatment of cancer, single drug chemotherapy is unacceptable because of drug side effects and multidrug resistance thus affecting the prognosis of patients. Therefore we focus on combination therapy of different drugs which have different mechanisms and side effects to play a synergistic anti-tumor effect, with the lowest effective dose, to achieve the best therapeutic effect.Nanoparticle drug delivery system is able to increase the drug solubility, prolong the blood circulation time; improve drug pharmacokinetic and pharmacodynamic characteristics, promote the intracellular drug delivery and mediated the tumor targeting of the drugs they carried; increase the cellular uptake of delivery systems, and improve bioavailability of the drugs; significantly reduce the toxic and side effects of the drug, etc. Owing to the advantages nanoparticle is considered to be the optimum formulation of anticancer drugs.Doxorubicin is one of the anthracycline drugs which is known for its antineoplastic against a number of tumors, such as osteosarcoma, liver cancer, gastric cancer, lymphoma. Experiments have proven that DOX had a curative effect on tumors combined with other anti-tumor drugs. However, DOX has cardiac toxicity, influence liver and kidney function, and can induse bone marrow suppression and other toxic side effects.Curcumin, the constituent of Curcuma longa, is a kind of polyphenol yellow pigment which contain the structure of Dione in its molecules. CUR has a variety of pharmacological effects, like induce the apoptosis of human osteosarcoma, prostate cancer, breast cancer, colon cancer and other cancer cells. CUR is a natural pigment from the plant, whose toxic side effect is small, and have no damage to the human body, that can be used as a potential high efficiency and low toxicity anti-cancer drugs. But, the solubility of CUR is low, leading to a low bioavailability, and difficult to reach the effective blood drug concentration which limits its application. In addition, the short half-life, the fast metabolism and clearance rate also restricts its clinical application.Purpose:In our study, in order to overcome the multi drug resistance and serious adverse reaction of DOX, increase the solubility and prolong the half-life of CUR we focused on the combination therapy of DOX and CUR. We have explored DOX plus CUR co-encapsulated LPNs (DOX+CUR LPNs) which could be an innovative strategy for the synergistic therapy of OS.Methods:1. Preparation and Characterization of DOX plus CUR co-encapsulated LPNs (DOX+CUR LPNs)(1) Preparation of LPNs:DOX was stirred with TEA, DMSO to obtain the base. DOX, CUR, PLGA were dissolved in DCM to form the oil phase; DLPC, Cholesterol, PEG2k-DSPE were dissolved in water to form the aqueous phase. The oil phase was then poured into the water phase, and the drug loaded nanoparticles were preparaed by improved solvent extraction method.(2) The shape and surface morphology of LPNs were examined by transmission electronic microscopy. The mean particle size, polydispersity index, and zeta potential were analyzed by photon correlation spectroscopy. The DEE and DLC of DOX or CUR were measured by UV-vis method, and the selected wavelength for DOX and CUR was 485 nm and 430 nm respectively.(3) Stability of LPNs. The LPNs were re-suspended in 0.9% physiological saline, and the samples were incubated at 4? under agitation. At scheduled times (1?2?3? 4?5?10?15?20?25?30?35d) the samples were diluted to measure the mean particle size. Stability study in the serum was determined in phosphate buffers solution containing 10% fetal bovine serum at 37?. At scheduled times(0?2?4?8?12h)the samples were diluted to measure the mean particle size.(4) The in vitro drug release study of LPNs were measured by the dialysis method. The LPNs were placed in the dialysis bag separately. Then, the bag was incubated with 50 mL release medium at 37?. The medium was collected at predetermined time points(0?2?4?6?12?24?48?72?96 h) and replaced with 50 mL of fresh medium. The concentrations of released DOX or CUR were determined by the UV-vis method in the above section.2. Anticancer evaluation of DOX+CUR LPNs in vitro and in vivo(1) The in vitro cytotoxicity was evaluated by MTT assay. KHOS cells were seeded in a 96-well plate at a density of 5*103 cells/well and allowed to adhere for 24 h. The drugs were added to each well at a concentration of 0.5?1.0?5.0?10?50 ?M. After 24 h aliquots of MTT solution were added into each well, and after a 3 h of incubation the DMSO were added to dissolve the internalized purple formazan crystals. The plates were then assayed at 570 nm. The inhibitory activity of the drug was calculated according to the absorbance value IC50.(2) The BALB/c mice were used to study the tissure sidtribution. The mice were inoculating subcutaneously with 1*106 KHOS cells. The drugs were given into the mice by tail vein injection separately after a week after inoculating. At predetermined time(15 min?1h?2h?8h?24h?48h?120h after injection) intervals, mice were sacrificed and the tumor, heart, liver, spleen, lung, and kidney of mice were collected. The tissures were initially weighted and homogenized to determine the amount of DOX or CUR in each tissue. The concentration of released DOX or CUR were determined by the UV-vis method.(3) The tumor beared BALB/c mice were used to evaluate the in vivo anticancer effect. BALB/c mice were inoculating subcutaneously with 1*106 KHOS cells. The tumors were allowed to develop on the posterolateral side of the mice for one week prior to treatment. Mice were randomly divided into 8 groups (DOX+CUR LPNs, DOX LPNs, CUR LPNs, LPNs, DOX+CUR solution, DOX solution, CUR solution, 0.9% physiological saline). The drugs were administered intravenously every 3 days until 3 weeks. The volumes of the solid tumor were measured with a digital caliper every 3 days. Tumor weights were measured and the tumor growth inhibition ratio was calculated.Results:1. Preparation and Characterization of DOX plus CUR co-encapsulated LPNs (DOX+CUR LPNs)(1) DOX+CUR LPNs, DOX LPNs, CUR LPNs, LPNs were prepared by a modified solvent extraction/evaporation method. The LPNs had spheroidal shapes and there are grey coats on the darker spherical shaped particles. The size of LPNs were about 100 nm which had no significant variation(P>0.05) loaded with one or two drugs. The PDI of LPNs were 0.17-0.21 and the zeta potential of DOX+CUR LPNs was -33.4 mV, no obvious variations were observed in different samples(P>0.05).(2) The DEE of DOX loading in DOX+CUR LPNs and DOX LPNs was around 85%, the DEE of CUR was 80%, and no significant change was observed in different particles. The DLC of DOX loading in DOX+CUR LPNs was 9.1, the DLC of CUR was 8.3, which was a little lower than LPNs loading one drug.(3) No significant change in particle size and DEE was observed up to 35 days in the physiological saline condition, indicating that all the samples were stable in one month in the physiological saline condition. Also, No significant change was observed in all samples in the FBS. Thus the LPNs were considered very stable after incubation with serum and suggest that this formulation will not aggregate or disassemble after intravenous administration.(4)In vitro drug release were measured by the dialysis method. The drugs release from the LPNs had a fast release of drugs during the first 12 h, followed by a sustained release in the subsequent time until 48 h. However, the releases of drugs from solutions were completed in the first 2 h. The in vitro release profiles show that drugs loaded LPNs has the capacity to release DOX and CUR at a sustained rate over 48 h.2. Anticancer evaluation of DOX+CUR LPNs in vitro and in vivo(1) The in vitro anti OS efficacy of different samples were evaluated by MTT assay. The results illustrated than over the studied drug concentrations, the cytotoxicity of the dual drugs loaded LPNs were higher than single drug loaded LPNs (P< 0.05); cytotoxicity of the drug loaded LPNs were higher than free drug solutions (P< 0.05). The cytotoxicity of thel drugs loaded LPNs were higher than drug solutions.(2) The IC50 values were also measured to evaluated the inhibition of cell proliferation. The IC50 of DOX+CUR LPNs was the lowest (0.6?M). The IC50 values of drug loaded LPNs were many folds dose advantage over the free drug solutions.(3) The in vivo tissue distribution were measured using mice. The results showed that the drug solution mainly distributes in heart and kidney. DOX and CUR distribution of DOX+CUR LPNs was higher in the tumor tissue compared with the other tissues. The higher concentration in the tumor tissue remained relatively stable at all time points until 48 h after injection, indicate the sustained-release behavior of the DOX+CUR LPNs.(4) The in vivo antitumor efficiency was evaluated in the KHOS cells bearing OS mice model. As illustrated most obvious tumor regressions were clearly observed in the DOX+CUR LPNs group, the tumor growth was prominently delayed, which attained about 182 mm3, whereas in saline-treated group, tumor volume grew rapidly to 973 mm3 on 21-day post treatment. The TGI of OS mice treated with the DOX+CUR LPNs was 81.3%, which significantly higher than that treated with other samples. The obviously emaciation could be observed in the free drug solutions groups, while the LPNs groups did not cause significant difference body weight lost. During the treatment, reduction in food intake, energy sag and inactive in moving were also observed in the free drug solution groups but not in other groups.Conclusions:DOX+CUR LPNs was prepared and used for the delivery of DOX and CUR to the OS cells to inhibit the cell viability. Furthermore, the DOX+CUR LPNs effectively improves anticancer efficiency for OS bearing mice without causing obvious toxicity in vivo. These results demonstrate that the dual drugs co-encapsulated LPNs may have potential to be used as promising drug delivery system for the treatment of OS. |
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