Anti-tumor Basic Research Of Cisplatin-PHA Composite Materials With Photothermal Effects

BackgroundAt present,the treatment of bone tumor in our country has transited from simple surgery to comprehensive treatment.However,surgery is generally difficult to completely remove tumor cells and can result in different degrees of bone defects.In order to kill residual tumor cells,traditional chemotherapeutics such as doxorubicin,ifosfamide and platinum are widely used.However,these drugs lack specificity to tumors,and the dose of drugs reaching the tumor sites is small.Meanwhile,it will produce serious side effects and bring a lot of pain to patients.In recent years,with the development of near-infrared laser technology,local hyperthermia of tumors at near-infrared laser level has aroused people’s interest.It is easy to operate and has few complications.Moreover,it can not only kill local tumor cells effectively,but also enhance the sensitivity of tumor tissues to chemotherapy.Therefore,hyperthermia as an adjuvant therapy technology for tumors has good application prospects.At present,some material systems capable of responding to near-infrared light,such as gold nanorods and carbon nanorods,have been developed,but they have defects such as difficulty in obtaining and absorption of near-infrared light.After long-term exploration,it is found that there is a substance in many organs of the human body--polydopamine,which is the main component of eumelanin,which can respond to near-infrared light and has high photothermal conversion efficiency.At the same time,its structure is rich in active functional groups,and can load anti-tumor drug cisplatin,sothatcisplatinisreleasedlocallyintumortissues,and photothermal-chemotherapy is combined to achieve better tumor treatment effects.In addition,autogenous bones,allogeneic bones and xenogeneic bones are often used to fill the bone defects after tumor resection.However,there are some drawbacks in their clinical application,such as the limited amount of autogenous bones,the increase of surgical trauma and the sequelae of bone extraction sites;the rejection reaction of allogeneic bones and xenogeneic bones and the possibility of carrying deadly viruses.Therefore,this study intends to use the photothermal effects of polydopamine to load the anti-tumor drug cisplatin,and at the same time modify the nano-hydroxyapatite to obtain a composite material to achieve tumor treatment and bone defect repair.ContentsIn the study,nano-hydroxyapatite(n-HA),polydopamine(PDA),cisplatin(DDP),oxidized sodium alginate(OSA)and chitosan(CS)were used as main components to prepare a novel composite material with near-infrared light response at room temperature.Firstly,dopamine(DA)self-polymerized under weak alkaline conditions to form PDA,and then PDA loaded the chemotherapeutic drug DDP,and simultaneously functionalized the modification of n-HA,thereby finally forming the PHA-DDP complex.Subsequently,they were introduced into the Schiff base reaction system between OSA and CS.Furthermore,the photothermal effects under near-infrared light irradiation,the release properties of drugs,and the degradation characteristics were investigated.Secondly,tumor cells(4T1 cells)were cultured on the surface of composite material to study the cytotoxicity,and the tumor therapy effects were investigated by establishing a mouse tumor model.Then,it was co-cultured with bone marrow mesenchymal stem cells(BMSCs)to study the proliferation and differentiation behavior of BMSCs,and the osteogenesis ability of the material was investigated by establishing a model of lateral femoral condyle defect in rabbits.Methods1.The composite materials were prepared by Schiff base reaction and the materialswere optimized.These composite materials were divided into three groups:OSA-CS-Borax,OSA-CS-PHA,and OSA-CS-PHA-DDP.2.The formation time of composite materials were determined via the rheologicalprinciples.3.The microstructure of the composites was observed via SEM scanning.4.In vitro simulated body fluid environment,the photothermal effects of materials(irradiation wavelength:808nm),drug release(OD value measured by UVspectrophotometer)and degradation were detected.5.4T1 cells(1×10~4/well)were cultured in vitro,and the toxicity on the surface of thecomposites was detected by immunofluorescence and microplate reader.Groups:controlgroups(OSA-CS-Borax,OSA-CS-PHA),experimentalgroups(OSA-CS-PHA-DDP,OSA-CS-PHA-DDP+NIR).6.BMSCs(1×10~5/well)were cultured in vitro,and the proliferation and differentiationactivities on the surface of the composites were evaluated by immunofluorescence,microplate reader and BCA protein assay.Groups:control group(OSA-CS-Borax),experimental groups(OSA-CS-PHA,OSA-CS-PHA-DDP).7.The BALB/c mouse right-wing ectopic tumor model was established by cellsuspension injection(1×10~6/mouse)to monitor the tumor growth.H&E staining wasperformed on the tumor tissue and internal organs after treatment.Fifty BALB/cmice were randomly divided into the blank group,control groups(OSA-CS-Borax,OSA-CS-PHA),andexperimentalgroups(OSA-CS-PHA-DDP,OSA-CS-PHA-DDP+NIR).There were 10 mice in each group.8.The model of lateral femoral condyle defect in rabbits was established.Aftertreatment,the defects were observed by 64-slice spiral CT scanning and H&Esection staining.Forty rabbits were randomly divided into the blank group,controlgroup(OSA-CS-Borax),andexperimentalgroups(OSA-CS-PHA,OSA-CS-PHA-DDP).There were 10 rabbits in each group.ResultsBased on the above research contents,we obtained the following results:1.The results of rheological test,SEM observation and degradation rate test showed that the introduction of DDP not only accelerated the reaction speed of the composite material,made the material more compact,but also reduced the rate of degradation,thereby making it more advantageous for clinical application.2.The photothermal effects under near-infrared irradiation showed that the introduction of PDA endowed the composite material with excellent photothermal effects,and it could absorb near-infrared light to rapidly raise temperature,thus achieving local hyperthermia.3.The in vitro release test of chemotherapeutic drug DDP showed that the introduction of PDA significantly slowed down the release of DDP,which could not only avoid its burst release toxicity,but also continue to kill tumors.Moreover,near-infrared irradiation also promoted the drug release.4.Through the MTT assay,alkaline phosphatase(ALP)detection and laser confocal scanning experiments,we learned that the photothermal effects of PDA could synergize with DDP to promote the apoptosis of 4T1 cells and achieved the purpose of killing tumors.At the same time,the introduction of PDA could promote the adhesion,proliferation and differentiation of BMSCs,and its immobilized DDP did not significantly affect the proliferation of BMSCs.5.The results of in vivo animal experiments showed that near-infrared light and DDP exhibited synergistic effects on local treatment of tumors.Furthermore,n-HA endowed the composite material with good bone repair performance,and the introduction of DDP did not significantly affect the osteogenesis of the material.Conclusion1.The OSA-CS-PHA-DDP composite has excellent injectability.By optimizing the concentration of various substances,the reaction rate of is moderate and can be used for clinical operation.The prepared composite material can be heated rapidly under near-infrared light irradiation,sustain the release of DDP,and has good degradation performance,thus it has great clinical application value.2.The OSA-CS-PHA-DDP composite can release DDP to kill 4T1 cells and exert synergistic therapeutic effects under near-infrared light.In addition,it can promote the proliferation and osteogenic differentiation of BMSCs and achieve good bone repair effects.