Study On The Antitumor Effect Of T7-mediated Dual Sensitive PAMAM/DOX Delivery System Combined With Immunogenic Cell Death

Objective:In this paper,based on the G5.0 PAMAM,the polymer carrier T7-PEG-SS-PAMAM-CD(T7-PSSPCD)with active targeting and pH-redox sensitivity was designed and synthesized.And then doxorubicin(DOX)was efficiently loaded into T7-PSSPCD,to construct a functional drug delivery system T7-PSSPCD/DOX.At the same time,the cancer cell apoptosis was induced via DOX in vitro,and immunogenic death(ICD)apoptotic cells were creened out as anti-tumor vaccine(ICD-VAC),which cooperated with T7-PSSPCD/DOX to kill tumor.A series of in vivo and in vitro experiments were carried out to investigate the co-antitumor effect and related mechanisms.Method:Based on the Generation 5 PAMAM(G5.0 PAMAM)as the basic carrier,carboxymethyl-β-cyclodextrin(COOH-β-CD)was connected to the surface of PAMAM through dehydration condensation reaction to generate PAMAM-CD(PCD).Then,one end(-HS)of the bi-functional PEG(HS-PEG-COOH)was connected to PAMAM via cross-linking agent SPDP(3-(2-pyridine dimercaptol)n-hydroxysuccinimide propionate)to form reduction-responsive disulfide bond,and PEG-SS-PAMAM-CD(PSSPCD)was obtained.The the other end(-COOH)of HS-PEG-COOH was coupled with active targeting peptide T7 to synthesize polymer T7-PEG-SS-PAMAM-CD(T7-PSSPCD).The intermediates and final products were characterized by 1H-NMR and DLS.Subsequently,DOX was loaded by hydrophobic interaction to construct drug-loaded nanoparticles T7-PSSPCD/DOX,and its particle size,potential,appearance,drug loading,encapsulation efficiency and in vitro release were evaluated.Using 4T1 as a model cell,the cytotoxicity and cellular uptake were investigated by MTT method and flow cytometry.The escape ability of nanoparticles in the lysosome was investigated by confocal microscope.Mouse model of 4T1 breast cancer was established,then the in vivo distribution of T7-PSSPCD and anti-tumor activity of T7-PSSPCD/DOX were investigated by small animal in vivo imaging technology,and the pathological changes of tumors and major organs were evaluated by H&E staining.Secondly,the immunogenic cell death(ICD)of 4T1 was induced in vitro by DOX,and ICD tumor cells were screened and evaluated using flow cytometry,immunofluorescence and ELISA.DOX-induced ICD tumor cells were used as anti-tumor vaccine(ICD-VAC),and the in vivo immune response and anti-tumor effect of ICD-VAC were investigated through tumor growth and the immune microenvironment changes.Finally,the co-antitumor effect of ICD-VAC vaccine and T7-PSSPCD/DOX was evaluated in vivo,and the combined anti-tumor mechanism was discussed.Results:(1)A series of polymers such as PCD,PSSPCD and T7-PSSPCD were successfully synthesized,and the structures were verified by 1H-NMR.The results of dynamic light scattering(DLS)showed that the particle size and potential of T7-PSSPCD were 37.06±4.09 nm and 1.44±0.36 mV,respectively,and the particle size was uniform,and the stability was good.(2)The particle size and potential of the T7-PSSPCD/DOX nanoparticle were 52.07±3.55nm and 0.12±0.19mV,respectively.The TEM image showed that the shape of the nanoparticles is uniform and spherical.The drug loading of T7-PSSPCD/DOX was 15.08±1.21%,which was significantly increased compared with PP/DOX(9.12±0.23%),indicating that modification of β-CD could indeed increase the drug loading of the carrier.In vitro stability experiments showed that the nanoparticle have good stability.In vitro release experiments showed that the nanoparticles had obviously pH and reduction-sensitive drug release characteristics.(3)The result of the MTT experiment showed that the cell survival rates of the blank carriers at various concentrations were all above 80%,indicating good biological safety.The cytotoxicity of the DOX loading carrier was concentration-dependent,and the modification of T7 peptide could further increase the nanoparticle’s cytotoxicity against tumor cells.Cell uptake experiments showed that,compared with the non-targeted group,the modification of T7 peptide could significantly increase the uptake of nanoparticles by 4T1 cells and enhance tumor targeting.The investigation of the cell uptake mechanism suggested that T7-PSSPCD/DOX entered into cells mainly through clathrin-mediated endocytosis to exert anti-tumor effects.(4)In vivo fluorescence imaging experiments showed that Cy7-PSSPCD-T7 stayed longer in the body compared to free Cy7.Free Cy7 was quickly excreted through the kidneys,while Cy7-PSSPCD-T7 was distributed in the kidneys,liver and tumors.Compared with the Cy7-PSSPCD group,the Cy7-PSSPCD-T7 group had more accumulation in the tumor site after 36h and had obvious targeting ability.Using 4T1 tumor-bearing mice as tumor animal models,the anti-tumor effect of T7-PSSPCD/DOX was investigated,and the result showed that the nanoparticles could significantly inhibit tumor growth.The results of mouse body weight investigation and H&E tissue staining showed that T7-PSSPCD/DOX could significantly reduce the side effects of DOX.(5)The apoptosis assay was used to detect the apoptosis of 4T1 cells induced by DOX.The results showed that the cytotoxicity of DOX was highly related to apoptosis,which led to immunogenic cell death(ICD).The ICD of 4T1 cells was analysed by immunofluorescence and flow cytometry,and the results showed an increased release of damage-associated molecular patterns(DAMPs),including calreticulin(CRT),high mobility group box 1(HMGB1)and ATP,compared with control group.We screened the 4T1 apoptotic cells(ICD-VAC)with immunogenic death and co-cultured with dendritic cells(DC2.4)in vitro.The experimental results showed that ICD-VAC couldstimulate the mature of dendritic cells(DC2.4),increasing the expression of CD80 and CD86 and promoting the secretion of cytokines such as IL-6 and IL-1β.The distribution of the ICD-VAC in the relevant immune organs in the body was investigated by near-infrared fluorescence imaging.The results showed that ICD-VAC enters the lymph nodes and spleen after subcutaneous injection,and then exerted immune function.The immune response in vivo showed that ICD-VAC could obviously activate the immune system.In vivo tumor suppression experiments further showed that the ICD-VAC vaccine significantly inhibited tumor growth by activating the immune system.(6)The in vivo anti-tumor experiments showed that the combination group of T7-PSSPCD/DOX and ICD-VAC displayed the best tumor suppressing effect,compared to single treatment.During the treatment,the life characteristics of the mice were normal,which showed that each treatment group is safe in vivo.Flow cytometry and ELISA methods were used to evaluatethe activation of the immune system and the secretion of serum cytokines in lymphatics and spleen.The results showed that the combined treatment of T7-PSSPCD/DOX and ICD-VAC vaccine could activate a stronger anti-tumor response in vivo,thereby producing the better anti-tumor effect,which would open up new ideas for anti-tumor research.Conclusion:The T7-PSSPCD polymer was successfully synthesized.The carrier could efficiently load DOX,actively target tumors,and achieve rapid and effective release of DOX under the stimulation of the microenvironment within tumor cells.Besides,immunogenic death of cancer cells was induced by DOX as a vaccine(ICD-VAC),which could promote the maturation of DC cells and the antigen presentation,and activated T cells.Therefore,the combination therapies of T7-PSSPCD/DOX and ICD-VAC was used for antitumor study,and displayed good tumor inhibition effect,which may provide new strategies for improving the limitations of cancer chemotherapy and immunotherapy,and enhancing the anti-tumor effect.