Injectable Hydrogel-microsphere Construct With Sequential Degradation For Localy Synergistic Chemotherapy Of Osteosarcoma

Osteosarcoma is a primary malignant bone tumor in childhood and adolescence,with the incidence rate of about 4.0 or 5.0 for the ranges of 0-14 or 0-19 years per year per million persons,which accounts for eighth in the incidence of childhood malignancies.Currently,the combination of preoperative,operative,and postoperative neoadjuvant chemotherapy constitutes the standard treatment program of osteosarcoma.After standardized treatment,the 5-year survival rate for osteosarcoma without metastasis is about 60%-70%,which is limited to only 30% for those with metastasis.Although chemotherapy is still considered as the first choice to treat osteosarcoma,the disappointing pharmacokinetics and serve systemic toxicity of small molecular drugs(SMDs)are the major concerns for patient compliance,which encourage researchers to exploit efficacious system to address these issues.Recently,many studies have focused on the in situ drug delivery systems,like polymer thermo-sensitive hydrogels(Gels).Chemotherapy drugs can be facilely dispersed into the thermo-responsive polymer solution at low temperature,e.g.,4 °C,without the usage of organic solvent,and the drug release depot is automatically formed in situ under the stimuli of body temperature.The local injection is easily manipulated though syringe and could minimize the surgical trauma.Compared with the systemic administration of chemotherapy drugs,the locally and continuously drug release from the Gel in the tumor site can effectively improve the efficacy and reduce the systemic toxicity of drug.Microspheres(MPs)are widely used in controlled drug delivery to treat various diseases.Currently,a lot of polymers have been used to prepare MPs.Among them,the biodegradable synthetic polymer of poly(lactic-co-glycolic acid)(PLGA)has received widespread attention.Previous studies have shown that the PLGA MPs can entrap a variety of SMDs,such as 5-fluorouracil(5-FU),cisplatin(CDDP),dexamethasone(DMS),docetaxel(DTX),doxorubicin(DOX),and paclitaxel(PTX).The loading MPs can gradually release the entrapped drugs with the gradualVI degradation of PLGA backbone to achieve a long-term slow administration.Nowadays,the combination of vascular disrupting agents and cytotoxic drugs has been reported to have improved antitumor efficacy,which can induce the wholly regional apoptosis of tumors.Combretastatin A-4(CA4)can destroy the structure of blood vessel by binding the tubulin of endothelial cells and finally induce tumor necrosis by blocking the transmission of oxygen and other necessary nutrition.In vitro experiments showed that CA4 played a significant role on the blood vessel endothelial cells in the proliferation stage,whereas had little effect in the quiescent stage.As the antitumor effect of CA4 can only act on the internal tumor tissues with rich peripheral vascular proliferation and has no proliferation inhibition effect on tumor cells,it is often the cause of tumor treatment failure and recurrence.Fortunately,the cells at the edge of tumor with rich blood vessels are sensitive to the traditional chemotherapy drugs,and it enables us to explore the alternative strategy to sequentially deliver CA4 and cytotoxic drugs.However,the synergistic therapeutic effects are often unsatisfied by intravenous injection,which mainly result from short circulating half-life in vivo and unselective bio distribution.Additionally,the sequence of administration is also important in the combination of CA4 and cytotoxic drugs.If the peripheral tumor cells were killed by cytotoxic drugs firstly,the tumor cells within the solid tumors will be survived,which will cause the tumor recurrence and metastasis.For this issue,an injectable construct of MP-loaded thermo-sensitive Gel with stepwise degradation was prepared to sequentially deliver CA4,a vascular disrupting drug,and DTX,a cytotoxic agent,for the treatment of osteosarcoma.In this platform,CA4 was released preferentially,which destructed neovascularization and inhibited the formation of new blood vessels,followed by reducing exchange of nutrients between tumor and surrounding tissue to kill the tumor cells inside.Then,the sustained release of DTX led to the destruction of the surface cells of tumor and caused the wholly regional apoptosis of tumor.Specific research program includes the following two parts:1)Research on the preparation and characteristics of docetaxel-based PLGA microspheres with different particle sizesIn this study,the DTX-carrying PLGA microspheres are prepared by water-based and water-in-oil solvent evaporation method with PLAG as the material.This method is simple,feasible,has high encapsulation efficiency and can reduce the loss of drugs.Moreover,the shear rate controlling is used to control the microsphere size.By observing the electron micrographs,it indicates the shear rate controlling can efficiently control the size of microspheres.The microspheres have complete and smooth surface and standard sphere.Moreover,the degradation and release test of drug carrying PLGA microspheres with different particle sizes determine the degradation time of microspheres with different particle sizes,which is conductive to the disease.It also helps to select the microsphere degradation time consistent with the disease treatment time according to needs of different diseases.By studying the explanation and release process of DTX carrying microspheres with different particle sizes,we find the drug carrying microsphere with the particle size of 2.94μm has long and suitable degradation time(49 days)and good release properties.Therefore,it is used as the gradient degrading microsphere/hydrogen complex system for the treatment of osteosarcoma.2)The application of degradable microsphere-carrying thermosensitive in the treatment of osteoscarcoma as drug sustained release carrierThe poly(L-alanine-co-L-phenylalanine)-block-poly(ethyleneglycol)-co-poly(L-alanine-co-L-phenylalanine)(PLAF-b-PEG-b-PLAF)used in this study was synthesized through the ring-opening polymerization(ROP)of L-alanine N-carboxyanhydride(L-Ala NCA)and L-phenylalanine N-carboxyanhydride(L-Phe NCA)using amino-terminated poly-(ethylene glycol)(NH2-PEG-NH2)as a macroinitiator.The physics mixing method is used to mix the drug carrying PLGA microspheres and CA4 into polyamino acid gels to form solution.Relevant representation has been made of the polyamino acid gels,microspheres and composite materials.The polymer solution can transform into gel state at body temperature.After injection into the body,it can make slow gradient release of DTX and CA4 within 42 days,give full play to the synergistic effect of the two drugs,inducing osteosarcoma cell apoptosis.Animal experiments indicate that after the gel formation via the injection of drug-carrying composite material,the composite material microspheres are wrapped inside the gel.With layer-by-layer gel degradation,the microspheres are gradually dispersed.As the polyamino acid gel has faster PLGA microsphere degrading speed and the polyamino acid gel has initial burst,a lot of CA4 is released in the early stage to inhibit tumor angiogenesis and reduce the material exchange between the tumor and surrounding tissue.At the same time,smallmolecule drug DTX enters the tumor tissue via the tissue gap to kill tumor cells.The drug-carrying Gel/CA4-MP/DTX has the best therapeutic effect.More importantly,through systematic histological and immunohistochemical study,this composite material can effectively reduce drug toxicity,with better anti-tumor proliferation effect than free pure medicine in a long time.In summary,the composite material synthesized by this experiment can be injected into the local tumor,maintain the high drug concentration in the local tumor for a long time,show good anti-tumor treatment effect and can reduce the side effects of chemotherapy.Moreover,the material has no toxic side effect.It can be used in local drug synergistic chemotherapy as an injectable biomaterial,which has high application value.