Study On Self-targeting,Dual ROS/pH-responsive Intelligently Controlled Release Nanosystem Based On Apoferritin Nanocage

Objective: In order to increase the accumulation of drugs in the tumor site and improve the therapeutic efficiency,a dual-responsive ROS/p H system with self-targeting characteristics was constructed.The physicochemical properties,targeting,antitumor activity in vivo and in vitro,and pharmacokinetic behavior were systematically studied.The effect of intelligently controlled release and the self-targeting effect of L-apoferritin were evaluated.Methods:(1)Preparation and characteristics DOX was encapsulated in the cavity of AFt by using its reversibly dissociation-reassemble characters upon p H changes.Then,we introduced photosensitizer Rose Bengal(RB)onto AFt surface through amido bond under the catalysis of EDC and NHS.The structure of nanocomposites was successfully characterized through UV-vis and FT-IR.The size and zeta of nanocomposites were determined by dynamic light scattering(DLS).The shape of nanocomposites was observed by transmission electron microscopy(TEM).(2)The study of intelligently controlled release properties DPBF method was used to investigate the contents of ROS produced by the nanocomposites.Native-PAGE was used to investigate the effects of p H and ROS on the protein structure.Besides,the effects of p H and ROS on the release behavior in vitro were also investigated.(3)The studies of in vitro antitumor activity and targeting 4T-1 breast cancer cells as a model,the specific binding of SCARA 5 receptor to AFt was investigated by immunofluorescence.Fluorescence microscopy and flow cytometry were used to investigate the cellular uptake of the nanocomposites.SRB method investigated the nanocomposites on the 4T-1 cell activity;In addition,the effect of the therapeutic system on apoptosis was further examined.(4)The studies on pharmacokinetics and targeting in vivo SD rats were used for animal models,the changes of drug concentration in plasma with time were measured by HPLC method,and the pharmacokinetic parameters were calculated by PK Solver analysis software and the drug time curve was drawn.Using IR783 as a fluorescent probe,the drug distributions in tissues of IR783 and IR783@AFt-RB were observed by in vivo fluorescence imaging system.(5)The study on in vivo pharmacodynamics Using 4T-1 tumor-bearing mice as animal models,The antitumor effect and side effects of the drug delivery system were investigated through changes of weight,tumor volume and HE staining.Results: The results of UV-vis,FT-IR and TEM showed that DOX was successfully loaded into the AFt protein cage and the photosensitizer RB was linked to the protein shell through amide bond.The nanocomposites showed spherical structure and good dispersion.Native-PAGE showed that there was obvious protein fragmentation under the acidic conditions or the presence of ROS,and the protein damage was more serious with the decrease of p H value or the increase of ROS content.In vitro release results also indicated that the release rate under acidic conditions and in the presence of ROS was significantly higher than that under normal physiological conditions or single response conditions.Cytotoxicity test results showed that AFt had excellent biocompatibility,and the controlled release system showed strong cytotoxic effect under 532 nm laser irradiation.Cell uptake experiments showed that the nanocomposites can target 4T-1 breast cancer cells.DOX can release from AFt under the laser irradiation and enter into the nucleus to exert its cytotoxicity.The results of apoptosis experiments further demonstrated that the apoptotic rate of the drug delivery system was significantly higher than that of DOX,and the late apoptosis rate was significantly increased after laser irradiation.In addition,we carried out a series of evaluation on the anti-tumor effect and targeting of the controlled release system in vivo.Pharmacokinetic results showed that the retention time of the controlled release system was 4 times as high as the retention time of DOX in the body,which obviously enhanced the antitumor effect.In vivo imaging results showed that the accumulation of the nanocomposites increased significantly at the tumor site.The nanocomposites have good targeting ability.In vivo pharmacodynamic results showed that this formulation has a potent tumor-suppressive effect and almost no side effects on normal tissues.Conclusions: The DOX@AFt-RB intelligently controlled-release system can be rapidly targeted and uptaked into the tumor site through the AFt’s L-chain receptorSCARA 5,and can rapidly release at tumor site under 532 nm laser irradiation,which showed an strong tumor inhibition effect.