Application Of Tumor-Targeting Peptide-Decorated Nanogel With Doxorubicin For The Treatment Of Osteosarcoma

Background:Osteosarcoma is the most frequent primary malignant bone tumor in children and young adults,which is composed of mesenchymal cells producing osteoid and immature bone.At present,the management of osteosarcoma comprises neo-adjuvant chemotherapy,surgical resection and adjuvant chemotherapy.This yields a 5-year event free survival of approximately 70%for patients with localized osteosarcoma while patients with metastatic or recurrent disease fare poorly.Unfortunately,the outcome has not changed in the past 30years.This plateau in survival rates highlights the need for a novel approach towards research.Current problems in treating cancer include low specifity,rapid drug clearance and biodegration,and limited targeting.On this topics,we aim to design redox-responsive nanogel as well as targeting modifications for improving the controlled release of the drug which is able to enhance the efficacy and reduce the toxicity.Objective:In this study,we want to design and develop a novel nanomedicine which is able to respond to the intratumoral overexpressed GSH and increase the targeting ability of the loaded drugs.The development of nanogel-based drug delivery systems lies on handling of the relationship among loading capacity,stability and delivery efficiency.To enable the active tumor-specific function,a tumor-targeting peptide is incorporated.The construction of nanomedicine by encapsulating the doxorubicin is one of ideal routes to overcome the drawbacks of nonspecific distribution,poor bioavalibility,rapid blood clearance,and poor solubility in physiological environments.Thus,we want to assess these properties in our experiment.Method:1 The mPEG-P(Phe-co-Cys)was synthesized through the simultaneous ROP of L-Phe NCA and L-Cys NCA with amino-functionalized PEG(mPEG-NH2)as macroinitiators.Second,t-BOC-NH-PEG-NH2 was used as a macroinitiators to synthesize the MI-PEG-P(Phe-co-Cys)following the same proposal with.Then the characterizations and biocompatibility of copolymers and nanogels were assessed.(2)DOX was embedded into nanogels through a nanoprecipitation technique.Drug loading content and drug loading efficiency were detected after the encapsulation of DOX.The in vitro drug release was measured with a dialysis method.The intracellular DOX release and antitumor effect was detected via confocal laser scanning microscopy(CLSM)and flow cytometry(FCM)detections and MTT assays toward human osteosarcoma cells,respectively.(3)We choose five-week-old male BALB/c nude mice for xenografted models of human osteosarcoma and randomly divide them into four groups.The tumor volume and body weight were real-time documented for the evaluation of chemotherapy efficacy and security.Immunofluorescence was then performed to determine the form of proliferation and apoptosis.Moreover,histopathological examination were performed to observe the systemic toxicity and the tumor necrosis rate.The tissue biodistribution of DOX and pharmacokinetics were measured to calculate drug metabolism.Results:1 Both the mPEG-P(Phe-co-Cys)and STP-PEG-P(Phe-co-Cys)could selfassemble into nanogels in aqueous solution with the particle diameters of61.6±34.8nm and 61.0±33.6nm,respectively.The cell viability(%)at 72 h was above 90%,indicating the good biocompatibility.Subsequently,DOX was loaded into nanogels obtaining the DLC at 8.95±0.07 wt.% in NG/DOX and 9.82±0.14 wt.% in STP-NG/DOX,respectively.Both the DOX-loaded nanogels exhibited controlled realease in PBS at p H 7.4,but the release contents of DOX from NG/DOX and STP-NG/DOX were accelerated up to 74.3% and 84.4% in the presence of GSH.(2)The cytotoxicity of DOX,NG/DOX and STP-NG/DOX toward 143 B cells in vitro was measured by a MTT assay.The IC50 of NG/DOX group and STP-NG/DOX group was significantly lower than that of F-DOX group(*P<0.05).There was no significant difference in IC50 between NG/DOX group and STP-NG/DOX group(P>0.05).10.0m M GSH could significantly acelerate the drug release from STP-NG/DOX and NG/DOX,which was consistent with the results of CLSM and FCM.(3)The pharmacokinetic results showed that the average half-lives(T1/2)of NG/DOX and STP-NG/DOX were9.77 and 12.12 hours,which were 1.34 and 1.66 times of F-DOX respectively.(4)The three treatent groups showed the growth of tumors was suppressed.After euthanization,the tumor volume of STP-NG/DOX was significant smaller than the other three groups(**P<0.01).H&E staining showed a large number of nuclear pyknosis or fragmentation in the tumor tissues,and extensive necrosis areas were observed in the control group(3.17±1.94%),F-DOX group(16.35±5.15%),NG/DOX group(40.26±2.62%)and STP-NG/DOX group(75.26±4.28%).Immunohistochemical results showed that the weakest Ki-67 fluorescence signal was observed in STP-NG/DOX group.The order of Caspase-3 content in each group was STP-NG/DOX group > NG/DOX group >F-DOX group > PBS group.(5)On the 30 th day after the establishment of tumor-bearing model,compared with the other three groups,the weight loss of STP-NG/DOX group was the least(*P < 0.05);compared with PBS group,the weight loss of F-DOX group was the most,the difference was significant(** P <0.01).H&E staining was used to observe the damage of main organs.The heart,liver,kidney and lung of F-DOX group showed more serious damage,while STP-NG/DOX group showed the least damage.Conclusions:This work demonstrates a paradigm of dual intratumoral redox-responsive/ tumortargeting nanogel for tumor-specific and high-efficacy cancer therapy in osteosarcoma.Also,it can reduce the systemic toxicity.