Design Of Nano-drug Delivery Systems For Celastrol And Study On Synergistic Anti-tumor Effect Of Phototherapy-chemotherapy

BackgroundsHepatocellular carcinoma(HCC)is one of the most serious malignant tumors in the world,with high incidence rate,high mortality,difficult to cure,poor prognosis and serious harm to human health.Chemotherapy,as a traditional tumor treatment method,is widely used in inhibiting tumor growth and prolonging the survival time of patients.However,single chemotherapy has many disadvantages,such as large toxic side effects and easy to induce multidrug resistance of tumor,which affects the treatment effect.Local drug delivery and nano-drug delivery systems are the development trend of drug therapy.Multi-mode collaborative anti-tumor therapy is also conducive to reducing drug toxicity and enhancing the efficacy.Celastrol(Celastrol,CEL)is one of the most important antitumor active monomers in traditional Chinese medicine Tripterygium wilfordii.Pharmacological studies have shown that CEL has broad-spectrum and efficient anti-tumor activity,has a good inhibitory effect on a variety of tumor cells,and can reverse the multidrug resistance of the body.However,its poor water solubility,low dissolution,low bioavailability and certain hepatorenal toxicity affect its clinical transformation.Nano-drug loading system,local drug delivery and other technical means help to improve the bioavailability of CEL,enhance the efficacy and reduce toxicity.ObjectiveIn this study,CEL is proposed to be used as a model drug,and the polymer polydopamine(PDA)is used as a photothermal agent.In combination with nanotechnology,two types of nano drug delivery systems with photothermal chemotherapy synergistic effect are designed.They are used to overcome the shortcomings of CEL,make up for the shortcomings of single mode,and play the role of enhancing efficiency and reducing toxicity.Methods(1)Preparation and characterization.Firstly,a carrier-free nano-drug delivery system was prepared.Poloxamer 188 was used as stabilizer.CEL self-assembled nanoparticles(CEL-NS)were prepared by precipitation method.The surface of polyamine(PDA)was modified by oxidative polymerization,and the amount of modification was investigated by single factor to obtain photothermal modified drug-loaded nanoparticles(PDA@CEL-NS).The particle size,morphology,crystal characteristics and infrared spectrum were characterized by nanoparticle size analyzer,scanning electron microscope,X-ray diffraction and infrared spectrometer.The storage stability and the stability of different physiological media were investigated.Drug release in vitro and photothermal temperature rise in vitro were used to evaluate its release performance and photothermal performance in vitro.Then,a nano-drug delivery system based on metal-organic framework materials(MOFs)was prepared.Firstly,the preparation processes of UIO-66,ZIF-8 and MIL-101(Fe)were optimized and compared by single factor investigation and orthogonal experimental design.The most suitable one was selected from three common nano-MOFs as drug carrier.CEL was loaded by adsorption method and PDA was modified by oxidation polymerization method,and the drug loading process and PDA modification ratio were investigated by single factor.Photothermal modified drug-loaded MOFs with high drug loading and good photothermal properties were prepared(PDA@MOF s-CEL).The physicochemical properties such as particle size,morphology;crystal form,thermogravimetry,drug loading,in vitro release and in vitro photothermal properties were characterized.In addition,temperature sensitive gel based on poloxamer 407,poloxamer 188 and HPMC were prepared.The metal organic framework/temperature sensitive gel composite system(PDA@MOFs-CEL-GEL)was prepared by dispersing nanoparticles in gel.And the gelling temperature,viscosity change and gel dissolution were investigated.(2)In vitro cytological evaluation.HepG2 cells were used as cell model.CCK-8 method was used to evaluate the biological safety,cytotoxicity to tumor cells and the efficacy of photo-thermal combined chemotherapy cells of blank nanoparticles.DAPI staining was used to observe the effect of nanoparticles on nuclear morphology.The cell scratch repair experiment was used to explore the effect of nanoparticles on cell migration,and the effect of nanoparticles on cell apoptosis was measured by flow cytometry.(3)In vivo efficacy evaluation.H22 hepatoma bearing mouse model was established.The retention of thermosensitive gel injected subcutaneously was observed directly.The photothermal heating effect and retention of nanoparticles in tumor-bearing mice were evaluated by infrared thermography.The biological distribution and retention time of nanoparticles in tumor-bearing mice was monitored by near-infrared fluorescence in vivo imaging.Through intratumoral injection,the anti-tumor effect of photothermo-chemotherapy was evaluated by monitoring tumor volume and tumor weight.GPT/GOT test kit was preliminarily used to evaluate the hepatotoxicity of nanoparticles.After the efficacy test,the main organs of mice were taken for HE staining,and the systemic toxicity of nanoparticles was evaluated by histopathology.Results(1)In vitro characterization results showed that the preparative PDA@CEL-NS are aqueous dispersion solution of spherical nanoparticles with a particle size of 189.67±2.08 nm,uniform distribution,and a drug loading of up to 60.33±1.09%,PDA@CEL-NS have good stability during the investigation,and the release in vitro shows that PDA@CEL-NS can significantly enhance drug dissolution,and the cumulative release rate can reach 60%.The study of photothermal temperature rise in vitro shows that it has a concentration-dependent temperature rise and good photothermal stability.In vitro cytological evaluation showed that,PDA@CEL-NS can inhibit tumor cell proliferation in a concentration-dependent manner,which IC50(calculated by CEL)is 2.238μg/mL,the efficacy is better than that of free drugs(IC50 value is 2.609 μg/mL).After 808 nm laser treatment,the tumor cell inhibition effect of PDA@CEL-NS was significantly enhanced.The DAPI staining experiment confirmed that nanoparticles can affect the morphology of nucleus and have apoptosis-inducing properties.The cell scratch injury experiment showed that nanoparticles can inhibit cell migration.The apoptosis experiment showed that inducing apoptosis is one of its cytotoxic mechanisms.In vivo research confirmed that PDA@CEL-NS has good photothermal warming effect in mice.Its heating efficiency decreases with time,indicating that nanoparticles are gradually metabolized.The drug distribution in vivo shows that the tumor retention effect of nanoparticles is better than that of the original drug.The drug is mainly distributed in tumor tissue in vivo,but less in other major organs,reflecting the advantages of nanoparticles and intratumoral drug delivery.The results of anti-tumor efficacy showed that,the tumor inhibition rate of PDA@CEL-NS-laser group can reach 92%,which is 1.57 times higher than PDA@CEL-NS and 2.73 times higher than that of CEL.It shows the good effect of enhancing the curative effect of nanometer preparation and the superiority of the synergistic anti-tumor effect of photo-thermal-chemotherapy dual mode.Finally,the determination of serum hepatotoxicity biochemical indicators and histopathological examination of mice proved that the nano preparation had basically no toxic and side effects on the main organs of mice,and had a certain liver protection effect compared with the original drug.(2)Three common MOFs nano-carriers UIO-66,ZIF-8 and MIL-101(Fe)were prepared.Through the comparison of particle size and preparation difficulty,ZIF-8 was selected as the nanometer carrier to prepare the CEL nanometer drug delivery system.PDA@ZIF-8-CEL nanoparticles with high drug loading and good photothermal properties were prepared by surface modification with PDA.The prepared PDA@ZIF-8-CEL nanoparticles have suitable particle size(305.8 ±8.4 nm),spherical shape,regular shape,good crystallinity,and drug loading of 23.47±0.26%.In vitro release study shows that it has good sustained release effect.PDA@ZIF-8-CEL has good photothermal heating performance in vitro and high photothermal stability after being treated with 808 nm laser.The thermosensitive gel based on poloxamer and HPMC was prepared,and the gelling temperature was 29℃.The PDA@ZIF-8-CEL thermosensitive gel(PDA@ZIF-8-CEL-GEL)composite system can be obtained by dispersing the nano particles in the blank gel,and the gelling temperature is basically unchanged.The cytotoxicity of PDA@ZIF-8-CEL nanoparticles was evaluated in vitro.At the experimental dose,ZIF-8 and PDA@ZIF-8 have good biological safety,and the IC50 of PDA@ZIF-8-CEL on HepG2 cells is 2.078 μg/mL,significantly better than free drugs.PDA@ZIF-8-CEL plus near-infrared laser treatment can further enhance the anti-tumor effect,reflecting a good photo-thermal synergistic effect.In addition,DAPI staining test,cell scratch test and cell apoptosis test confirmed that PDA@ZIF-8-CEL can induce nuclear rupture and deformation and inhibit cell migration by inducing cell apoptosis.In vivo studies in animals show that after intratumoral injection of thermosensitive gel,it has a good effect of intratumoral retention,which can be verified by visual observation,photothermal temperature rise in tumor,and drug distribution in vivo.Nanoparticles are mainly distributed in tumor tissues,less in other major organs.They have a long retention time in tumor tissue and the temperature can still rise to more than 50℃after laser treatment on the fifth day after injection.The efficacy test confirmed that the tumor inhibition effect of PDA@ZIF-8-CEL-GEL plus laser group was significantly better than that of other groups.After the treatment,two mice ’tumors completely disappeared,and the tumor inhibition rate of this group was as high as 97%,showing that the temperature sensitive gel composite system has a good effect of promoting the synergistic effect of photothermal chemotherapy.Take the serum of mice after the efficacy test and measure the biochemical indexes related to hepatotoxicity.The results show that nanoparticles can significantly reduce the content of glutamic-pyruvic transaminase in serum,indicating that nanoparticles can improve the liver injury compared with the original drug,and have a "detoxification"effect.Histopathological studies also confirmed that the nano drug delivery system has basically no toxic and side effects on the main organs of mice.ConclusionBased on CEL,the active monomer of traditional Chinese medicine,and combined with nanotechnology,this paper constructed two kinds of nano-drug delivery systems with and without carriers,which are used for the dual-mode synergistic anti-tumor effect of photothermo-chemotherapy.Firstly,the self-assembled nanodrug delivery system of polydopamine modified CEL was constructed,which can enhance drug dissolution,overcome the shortcomings of single mode,and reflect the complementarity of photothermal therapy and chemotherapy dual mode anti-tumor;Secondly,based on the consideration of long-term retention in tumor,a metal organic framework/thermosensitive gel composite nano drug delivery system was designed.The organic combination of nanoparticles and temperature sensitive gel realizes local long-term retention,promotes the synergistic effect of photothermal chemotherapy,and plays a role in enhancing efficiency and reducing toxicity.Through the use of nanotechnology to combine the dual modes of photo-thermal therapy and chemotherapy for anti-tumor,it provides new ideas for tumor treatment and provides a certain foundation for the anti-tumor application of tripterygium CEL.