Locally Controlled Release Of Chemotherapeutics And Bisphosphate By Polypeptide Thermogel For Synergistic Inhibition Of Osteosarcoma Progression

Background:Osteosarcoma(OS)is the most common primary malignant bone tumor which is usually seen in children and teenagers.The treatment method and efficacy of OS haven't made much progress since neoadjuvant chemotherapy was introduced in the 1970 s.In the past five decades,the five-year survival rate of OS has always been around 60% without much improvement.When metastasis or recurrence is found in patients,the five-year survival rate can reduce to lower than 20%.Although many researches have been conducted to improve the therapeutic effect by altering treatment methods and regulating the immune response,there hasn't been a more effective or less toxic strategy.The high rate of local recurrence and lung metastases often lead to decreased survival rate.Nowadays,conventional clinical treatments of OS include neoadjuvant chemotherapy combined with surgical removal of tumor and postoperative chemotherapy.However,these traditional chemotherapy can bring about severe side effects,such as allergy,gastrointestinal toxicity,viscera toxicity and myelosuppression et al.,causing unfavorable prognosis of OS patients.Therefore,it is urgent to develop a new treatment method that possesses minimal adverse effect and exerts maximal anti-tumor effect.The in situ drug delivery system could locally release drugs at tumor tissues,owing to which the therapeutic effect can be significantly enhanced and the impact toward normal organs is also reduced.Injectable biodegradable hydrogel(Gel)has found wide applications in biomedical field and has provided new platforms for the treatment of OS with low toxicity but high effectiveness.Due to the fine biocompatibility and degradability,biodegradable hydrogel spares the trouble of operation for removal and avoids obvious inflammation.At the same time,it has high drug loading capacity,controllable drug release rate and responsiveness toward tumor microenvironment,which can significantly improve the prognosis of patients and reduce the possibility of recurrence and metastasis.Purpose:In this paper,we intend to achieve a biodegradable drug-loading injectable hydrogel through convenient but highly efficient synthesis and preparation methods using polypeptide as basic material.By co-loading methotrexate(MTX),a chemotherapeutic drug and alendronate(ALN),an osteoclast inhibitor into hydrogel,we hope to obtain enhanced therapeutic effect,reduced bone damage,effective suppression over lung metastasis and less side effects.On the basis of this result,in order to further improve the therapeutic efficacy,we substituted MTX with oxaliplatin(OXA),which could induce immungentic cell death(ICD)of tumor cells.The combination of OXA and ALN could further improve the efficacy of OS treatment and the mechanisims of immune-response induced anti-tumor effect was expoled.It is expected that the injectable biodegradable polypeptide hydrogel drug delivery system prepared in this paper could effectively deliver chemotherapeutic drugs to enhance the therapeutic effect of OS and elevate the biosafety of co-loaded drugs,which would provide theoretical basis for in situ treatment of OS.Materials and Methods:The methoxy poly(ethylene glycol)-poly(L-valine)(m PEG45-PLV19)copolymer was synthesized via the ring-opening polymerization(ROP)of L-Valine N-carboxyanhydrides(L-Val NCA),which was initiated by the amino-terminated m PEG(m PEG-NH2).The chemical structure of m PEG45–PLV19 was analyzed by 1H NMR and fourier transform-infrared spectroscopy(FT-IR).The structural characterization of polypeptide m PEG45–PLV19 thermo-sensitive hydrogel was examined by phase diagram,rheology and scanning electron microscope.In order to develop the primary OS model of mice,mice oriented K7M2 OS cells were transplanted into 4-week old female BALB/c mice's marrow cavity of right proximal tibia.The phosphate buffered solution(PBS),two free drugs,single drug loaded hydrogel and two drugs co-loaded hydrogel were injected near tumors,with PBS used as control.During the treatment period of 16 days,the variation trend of tumor volume and body weight of tumor-bearing mice was recorded.At the end of the treatment,model animals were sacrificed,and tumor tissue and main organs(heart,liver,spleen,kidney and lung)were isolated.Hematoxylin and eosin(H&E)staining was used as histopathological examination to evaluate the necrosis and metastasis of tumor tissues after different treatments.The proportional change of CD8+ T cells in tumor tissues and lymph nodes were determined by flow cytometry.The ICD of tumor cells and the regulation of immune response against tumor after each treatment were also analyzed.Results:In this paper,we successfully prepared thermo-sensitive poly(valine)hydrogel.It showed great mechanical property and stable gelation ability after a series of chemical characterization and gel-related examination.In comparison with other treatment groups,the hydrogels co-loaded with MTX and ALN showed the strongest in situ anti-tumor effect and also effectively inhibited lung metastasis with minimal side effects.Subsequently,in order to further improve therapeutic efficacy,we replaced MTX with OXA,which could induce ICD of tumor cells.The combination of OXA with ALN synergistically inhibited the progression of OS.In addition to killing tumor cells,OXA also triggered the production of tumor antigens and activated CD8+ T cells.The combination of anti-tumor effect and immune-stimulating effect could synergistically enhance the therapeutic effect in tumor treatment and suppress lung metastasis.Conclusion:In this paper,the copolymer m PEG45–PLV19 was synthesized via the ROP of L-Val NCA which was initiated by m PEG45-NH2.m PEG45–PLV19 based injectable thermo-sensitive gel could realize effective co-loading of MTX,a chemotherapeutic drug and ALN,an osteoclast inhibitor.After injected near tumor tissue,the drug-loading hydrogel realized sustained and controlled drug release and achieved excellent inhibitory effect on tumor growth and lung metastasis.Besides,the bio-safety of drugs was also greatly improved.Furthermore,with the significant therapeutic effect of MTX and ALN co-loading hydrogel being proven,we verified that OXA and ALN co-loaded hydrogel could enhance anti-tumor effect through elevating the accumulation of cytotoxic T cells and triggering immune-response against tumor by expolring the mechanisms of tumor cell ICD.These two kinds of injectable drug delivering hydrogel systems developed in this paper may help improve the drug delivering strategies for OS chemotherapy,showing great prospect in clinical application.