| Research background and purposeGlioma is a most common,primary malignancy with high invasiveness and metastasis rate,poor prognoses and high mortality that is extremely harmful to humans.The reason for these results may be non-controlled cell proliferation,invasive growth,angiogenesis and antagonism of apoptosis.In recent years,the treatment of gliomas is still based on"surgery,chemotherapy and radiotherapy".Only few patients have improved survival rates after surgery because of its high malignancy and invasive growth.And with the blood-brain barrier(BBB),most anticancer drugs can’t enter the brain.Even if entering,these drugs have poor efficacy due to toxic side effects or drug resistance.Resulting in a limited variety of drugs that can be used to treat for gliomas.Therefore,how to improve the therapy of glioma has become an urgently problem to be solved.In addition,special growth site of glioma restrict early diagnosis and affect the therapeutic effect.At present,the diagnosis of gliomas still relies on imaging techniques.However,any single-modality molecular imaging technique has not been able to fully display the structure,function,and molecular information of tumors.The various modals have their insurmountable limitations,so the use of multi-modal molecular imaging technology to achieve mutual complementarity and mutual verification aims to fully reflect different aspects of information of gliomas.And becomes a focus of contemporary imaging research.In addition,active target imaging and therapy of gliomas has attracted more and more attention for the integration of diagnosis and treatment in nanomedicine.holo-transferrin(holo-Tf)is an endogenous biological protein in human body that specifically binds to the transferrin receptor(TfR)(highly expressed in glioma cells)and can transport iron ions at the same time.As a dominant carrier for oncological drugs,biologically active substances,and gene therapy,holo-Tf has a certain function across BBB and can be applied to the targeted treatment of gliomas.Indocyanine green(ICG)is the only near-infrared fluorescent(NIRF)contrast agent approved by the US Food and Drug Administration(FDA)for clinical use.Clinically,ICG is widely used for the auxiliary diagnosis of liver function,cardiac output,retinal vasculature in clinical.ICG is used as a fluorescent probe to emit NIRF at a wavelength of 820 nm,and as a photoacoustic(PA)contrast agent for PA imaging.At the same time,ICG is used as a photosensitizer that can absorb light energy and convert it into heat energy or generate singlet oxygen to recult in the dead of tumour cells.Based on the above research background,the study proposes that holo-Tf can be utilized to load with ICG for dual-modality imaging and photothermal therapy(PTT)of tumor.ICG was used as a NIRF/PA imaging contrast agent to synthesize holo-Tf-ICG nanoprobes in a protein-small molecule-linked manner.And investigated NIRF/PA imaging ability and synergy with PTT killing effect of holo-Tf-ICG on glioma in both cell-level in vitro and animal experiments in vivo.The purpose is to provide a valuable theoretical and experimental basis for the integration of holo-Tf-ICG in the diagnosis and treatment of gliomas and improve the anti-tumor efficacy.Research methods and results1)Self-assembly by one-step is used to combine the NIRF probe ICG with the holo-Tf and optimized the synthesis conditions.The interaction mechanism between ICG and holo-Tf is analyzed by computer molecular simulation,and its structure and spectral behavior are characterized.The main conclusions are as follows:①The particle size,potential measurement,drug loading and encapsulation efficiency of holo-Tf-ICG with dose ratios of 5%,10%,20%,30%,40%and 50%respectively were calculated and 30%was optimum dose ratio and and the drug loading reached 8.61%.②The interaction mechanism between holo-Tf and ICG was studied by molecular docking.holo-Tf-ICG was formed by the existence of hydrophobic interactions and hydrogen bond pairs from the result of computer molecular simulation.③The structural characterization results shown that the synthesized holo-Tf-ICG nanoparticles were uniform in size,stable in properties,and had no obvious change in secondary structure.The synthesis method was green and mild,and didn’t interfere with the inherent geometry of holo-Tf.④The spectral characterization shown that the maximum ultraviolet absorption of the holo-Tf-ICG nanoassemblies was at 780 nm,and the excitation energy at this wavelength produced a maximum emission peak around 810 nm.The fluorescence intensity of holo-Tf-ICG was reduced by 73%compared with free ICG,indicating that holo-Tf efficiently loaded ICG and caused fluorescence quenching due to excessive local concentrations.2)U87 cells was cultured with free ICG and holo-Tf-ICG nanoparticles.When the concentration of ICG reached 30 μg/mL,cell viability was more than 90%detected by CCK-8 assay,indicating that the synthesized holo-Tf-ICG is not nontoxic.The expression of TfR was detected on the surface of glioma cells U87 and bEnd.3 of brain microvascular endothelial cells by immunofluorescence assay,and the expression level of U87 cells was higher,suggesting that U87 cells can bind efficiently holo-Tf-ICG nanoparticles.The uptake of holo-Tf-ICG nanoparticles for U87 cells and bEnd.3 cells was investigated by laser confocal fluorescence microscopy.The study found that the holo-Tf-ICG-treated cells had higher fluorescence intensity than the free ICG,and the U87 cells exhibited stronger red fluorescence than the bEnd.3 cells.Flow cytometry analysis showed that the fluorescence intensity of the holo-Tf-ICG-treated group was 2.4-fold higher than that of the free ICG treated group in U87 cells,and slightly higher in bEnd.3 cells,but weaker than the U87 cells,indicating that holo-Tf-ICG has an excellent ability to actively target tumor cells,selectively enhance cellular uptake of ICG.Laser confocal microscopy results based on the 3D tumor-based sphere model of U87 cells shown that holo-Tf-ICG nanoparticles can enhance the penetration of ICG into tumor tissue.Infrared thermography in vitro exhibited that holo-Tf-ICG nanoparticles had better temperature-raising effect than free ICG,which was favorable for later PTT.Under the laser irradiation of 808 nm(0.8 W/cm2),the killing effect of the holo-Tf-ICG nanoparticle-treated group on the cells was time-dependent and concentration-dependent,when the ICG concentration was 20 μg/mL and the irradiation time was 10 minutes,cell killing rate was more than 95%,but the non-laser irradiation group and free ICG plus light group were less than 20%.The Calcein AM/PI staining further visually demonstrated that holo-Tf enhanced cellular uptake of ICG in U87 cells and exerted an effective PTT.3)The holo-Tf-ICG nanoparticles group displayed higher tumor accumulation by NIRF imaging in vivo.As an effective tumor targeting ligand,holo-Tf not only can increase the selectivity and permeability of the drug to tumor cells,but also it is possible to cross the BBB and bind to glioma cells.The PA imaging demonstrated that the holo-Tf-ICG nanoassemblies can be used as a NIRF and PA contrast agent,and can cross BBB by effective tumor active targeting and high permeability,enhancing the aggregation of ICG to tumor.The holo-Tf-ICG combined with laser treatment significantly inhibited tumor growth and can be used as an effective photothermal therapeutic agent for cancer.It is reflected that the research strategy has certain system safety by monitoring mouse tumor weight and H&E staining of major tissues. |
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