| Hepatocellular carcinoma(HCC)poses a severe threat to human health.Chemotherapy is a well-established paradigm to treat HCC clinically.Sorafenib(SF)was the first drug approved by the FDA for advanced HCC therapy,being recommended later for HCC therapy by several clinical guidelines.However,SF exhibited certain immunosuppressive effects.It could promote the high expression of the immune checkpoints on the T cell surface by inducing the hypoxia environment of tumor site,causing tumor immune escape and reducing the clinical therapeutic effect.Therefore,based on the chemoimmunotherapy strategy,immune checkpoints inhibitor was proposed to combine with SF in this project,to reactivate the immune system while kill the tumor cell and then enhance the therapeutic effect of HCC through different pharmacological mechanism.T-cell immunoglobulin and mucin domain 3 monoclonal antibody(Tim-3 mAb)was the hot topic in the field of immune checkpoints inhibitor.Tim-3 mAb could block the T cell activation suppression of SF,reactivating the T cell to play its role to kill the tumor cells.The combination of SF and Tim-3 mAb could kill the tumor cells by different mechanisms,possessing superior prospects of HCC therapy.However,SF has some barriers for clinical application,such as poor solubility,low oral bioavailability and some side effects(e.g.gastrointestinal bleeding),which greatly restricts its therapeutic efficacy against HCC.Tim-3 mAb is protein drug,which is not suitable for oral administration,and it also has immune-related side effects.Both drugs have non-specific biodistribution,which may cause serious damage to normal tissue when killing tumor cells.To improve the therapeutic effect and synergize the two therapeutic mechanisms,proper drug delivery strategy should be induced to co-delivery the drugs to tumor site.Mesoporous silica nanoparticles(MSNs)possesses multi-functions of negative target,co-loaded and controlled drug release,providing a good strategy to solve the above problems.Based on the design strategy,two type of stimuli-responsive drug release system based on MSNs were designed in this project:(?)the pH-responsive SF loaded MSNs with "on-off" switch;(?)MMP2-responsive SF and Tim-3 mAb co-loaded differ-target co-delivery MSNs.To improve the therapeutic effects,HCC was treated through the two levels(chemotherapy and chemoimmunotherapy)in this study.For the first level,SF was chosen as the HCC immunotherapy drug;pH sensitive material Poly(L-histidine)(PLH)was chosen as the "Gatekeeper" of MSNs;(Poly(ethylene glycol)(PEG)was chosen as the hydrophilic coating.Then,the pH-responsive SF loaded MSNs with "on-off" switch(SF/MSNs-PLH-PEG)was constructed for chemotherapy,to enhance the therapeutic effect of HCC.For the second level,SF was chosen as the HCC chemotherapy drug;Tim-3 mAb was chosen as immunotherapy drug and "Gatekeeper";Tim-3 mAb was covalently linked to MSNs through Matrix metalloproteinase 2(MMP2)sensitive peptide.Then,the MMP2-responsive SF and Tim-3 mAb co-loaded differ-target co-delivery MSNs(SF/MSNs-pep-Tim-3 mAb)was constructed for chemoimmunotherapy,to achieve the differ-target co-delivery and further enhance the therapeutic effect of HCC.The main contents and results in this study are listed as follows:1.Determination of SF contentDetermination method of SF was established by HPLC.The results showed that the linearity of SF in the concentration of 1?30 pg/mL was good(R2=0.9999).The precision of within-day and between-days could meet the requirements(RSD<2%),The method recovery could meet the requirements(98%?102%?RSD<2%).2.The construction and evaluation of the pH-responsive SF loaded MSNs with "on-off" switchMSNs were synthesized through sol-gel process.Drug was loaded by solvent evaporation method.PLH and PEG were covalently grafted to MSNs in sequence.Transmission electron microscope,Zeta Potential,Fourier Transform Infrared Spectroscopy,N2 Adsorption,Thermogravimetric Analysis and Small Angle X-ray Scattering were adopted to characterize the construction of the system.The results showed that SF/MSNs-PLH-PEG was constructed successfully.The average diameter of MSNs-PLH-PEG was about 160 nm.The zeta potential of SF/MSNs-PLH-PEG was-(13.8±1.7)mV.The drug loading content of SF was(22.5±2.5)%.In vitro release of SF in different pH was monitored by the dialysis bag diffusion technique.SF released from SF/MSNs-PLH-PEG in pH 7.4 was significantly higher than that in pH 5.0(p<0.01),indicating that the carrier possesses pH-controlled release property.Besides,with the pH changes of release media,the release rate of SF was also changed,meaning the carrier have "on-off"switches.The in vitro cytotoxicity was evaluated using MTT assay.SF/MSNs-PLH-PEG exhibited good anti-proliferation effect.In vivo anti-tumor effects were studied on H22 tumor-bearing mice.The tumor weight of SF/MSNs-PLH-PEG group mice was lower than that in SF/MSNs group(p<0.05),indicating the good anti-tumor effect in vivo.In the in vitro haemolysis assay,the haemolysis percentage of SF/MSNs-PLH-PEG was less than 5%,revealed that SF/MSNs-PLH-PEG possesses good hemocompatibility.The histological changes were assessed by H&E staining,no obvious damage was observed,indicating that MSNs-PLH-PEG exhibited good biocompatibility and safety at the test dose.All the results above demonstrated that SF/MSNs-PLH-PEG possesses pH-controlled"on-off"switch,good cytotoxicity,superior anti-tumor effect and favorable safety.3.The construction and evaluation of MMP2-responsive SF and Tim-3 mAb co-loaded differ-target co-delivery MSNsTim-3 mAb was covalently linked to SF/MSNs through MMP2 sensitive peptide to prepare the SF/MSNs-pep-Tim-3 mAb.Transmission electron microscope,Fourier Transform Infrared Spectroscopy,N2 Adsorption,Thermogravimetric Analysis and Small Angle X-ray Scattering were adopted to characterize the construction of the system.The results showed that SF/MSNs-pep-Tim-3 mAb was constructed successfully.SF/MSNs-pep-Tim-3 mAb were well-dispersed nanoparticles,with an average diameter of about 150 nm,and the zeta potential was-(22.3±1.9)mV.The loading content of SF and Tim-3 mAb were(13.5±3.1)%and(10.1 ±0.1)%respectively.In vitro release of SF and Tim-3 mAb was monitored by the dialysis bag diffusion technique and centrifugation method.Both SF and Tim-3 mAb released from SF/MSNs-pep-Tim-3 mAb in release media containing enzyme were significantly higher than that without enzyme(p<0.01),indicating that the carrier possesses MMP2 sensitive release property.The in vitro cytotoxicity was evaluated using MTT assay.SF/MSNs-pep-Tim-3 mAb exhibited similar anti-proliferation effect on HepG2 and H22 as free SF did.In vivo anti-tumor effects were studied on H22 tumor-bearing Balb/c mice.The tumor weight of SF/MSNs-pep-Tim-3 mAb group mice was lower than that in SF and Tim-3 mAb mixed solution group(p<0.05),indicating the good anti-tumor effectin vivo.In cell co-culture experiment,the HepG2 cellular uptake ratio of Dil was significantly higher than that on Jurkat cell(p<0.01)and uptake ratio of Tim-3 mAb was significantly lower than that on Jurkat cell(p<0.01),indicating that the carrier possess differ-target delivery ability.Ki-67 staining,H&E staining and Tunel staining was used to further evaluate the anti-proliferation and apoptosis-promoting ability.The results showed that SF/MSNs-pep-Tim-3 mAb exhibited superior anti-proliferation and apoptosis-promoting effects.In ELISA assay,SF/MSNs-pep-Tim-3 mAb promoted the expression of the immunostimulator IFN-y and IL-12,possessing the ability to neutralize or reverse the immunosuppression of SF.In the in vitro haemolysis assay,the haemolysis percentage of SF/MSNs-pep-Tim-3 mAb was less than 5%,revealed that SF/MSNs-pep-Tim-3 mAb possesses good hemocompatibility.The histological changes were assessed by H&E staining,no obvious damage was observed,indicating that MSNs-PLH-PEG exhibited good biocompatibility and safety at the test dose.All the results above demonstrated that SF/MSNs-pep-Tim-3 mAb possesses MMP2 sensitive release property,superior anti-proliferation and apoptosis-promoting effects,relieve immunosuppression of SF and favorable safety.In summary,based on the chemoimmunotherapy strategy,stimuli-responsive drug release system was designed in this study to co-loaded the chemotherapy drug and immunotherapy drug.This system combined passive targeting,stimuli responsive release and differ-target co-delivery functions,providing a kind of new idea and new strategy for HCC chemoimmunotherapy. |
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