Photosensitive ROS Liposomes Encapsulated With Dimerization Small Molecule Drugs Remotely Activate CAR-T Cell Immune Responses

Chimeric Antigen Receptor T-cell（CAR-T）therapy is the typical representative of adoptive immunotherapy and has developed rapidly in recent years.In short,it is to isolate healthy T cells from patients,transfection methods such as electrical transfer,lentivirus,adenovirus and other transfection methods to introduce the CAR gene constructed by genetic engineering technology into the cells,culture and expression in vitro,and finally transfection back into patients to achieve targeted killing of tumor cells.The main components of CAR gene include Sc Fv,extracellular hinge,transmembrane and intracellular signal domain.Successful expression of CAR confers the ability to recognize tumor cells and rapidly activate and kill tumor cells while bypassing MHC constraints.At present,there are many products on the market.However,CAR-T therapy is also associated with serious adverse events,including off-target effects and cytokine storms.Several strategies have been proposed to address the adverse effects of CAR-T therapy.For example,the introduction of suicide genes into CAR-T cells can induce apoptosis of CAR-T cells when cytokines are oversecreted.Considering that the suicide gene would interrupt the expensive treatment process,researchers proposed the idea of constructing on-switch CAR-T cells,that is,the traditional CAR gene is constructed in two segments and expressed separately in the cell,for example,FKBP and FRB protein domains are designed and expressed On the peptide.In the presence of a dimerized small molecule drug such as rapamycin or its derivative AP21967,two segments of CAR bind to perfect the T cell activation signaling pathway to produce immune effects.By regulating the release and distribution of dimerized small molecule drugs,On-switch CAR-T can be"On/off"and unnecessary side effects can be reduced.Liposome is a classical nano drug carrier,which has been widely used in clinical practice because of its advantages of enhancing drug solubility and excellent biocompatibility.Especially because of the existence of high permeability and strong retention effect（EPR）,liposomes have a place in the field of tumor therapy.ROS responsive liposomes are stimulus-responsive carriers prepared based on ROS high level expression in tumor microenvironment,in order to deliver drugs for precise release at tumor site.The researchers further combined photodynamic therapy with ROS-responsive liposomes to develop photosensitive ROS-responsive liposomes to avoid the uncertainty of ROS expression in tumor microenvironment.Photosensitive ROS responsive liposomes can load photosensitizer and drug together into liposomes.When reaching the target site,external light is given to the photosensitizer to generate ROS（singlet oxygen ¹O₂）to destroy the stability of liposomes and promote the release of encapsulated drugs.In this study,On-switch CAR-T cells targeting mesenchymal were constructed,and photosensitive ROS liposomes co-loaded with octa-butylphthalocyanine palladium Pd PC（OBU）₈ and dimerization small molecule drug rapamycin or AP21967 were prepared based on our previous study on photosensitive liposomes.It was introduced into On-switch CAR-T cells.On-switch CAR-T cells carry photosensitive ROS liposomes to the tumor site.External near-infrared irradiation and high ROS at the tumor site can release dimerization small molecule drugs in photosensitive ROS liposomes,and remotely activate the immune response of On-switch CAR-T cells at the tumor site.Precise control of On-switch CAR-T cell activity levels to avoid side effects caused by CAR-T cells.In this study,we investigated the in vitro and in vivo antitumor effects of the constructed novel On-switch CAR-T cells on ovarian cancer with high expression of mesotheliins.We first constructed traditional mesothelin-targeting CAR-T to verify the in vitro effects of CAR-T on ovarian cancer.The constructed secondary generation CAR gene was transformed into DH5α,and the target plasmid was extracted and encapsulated with lentivirus.CAR-T were then transfected with lentivirus and activated by magnetic beads.The CAR gene expression rate was over 70%by flow cytometry,and the CAR protein expression was verified by Western blotting with a clear protein band.Results:（1）CAR-T showed higher cytotoxicity to OVCAR-3,which were not transfected with T cells.（2）the cytotoxicity of CAR-T to OVCAR-3 increased with increasing reaction time,and the cytotoxicity of CAR-T to OVCAR-3 was complete after 24h.（3）The cytotoxicity of CAR-T against OVCAR-3 cells increased with the increase of target ratio.The levels of TNF-αand IL-2 in the CAR-T group were higher than those in the non-transfected T group by ELISA,indicating that CAR-T had higher immune activity.Secondly,On-switch CAR-T targeting mesothelin were constructed,and red fluorescence was observed after CAR protein chain expression under inverted microscope.Flow cytometry was used to detect the CAR gene expression rate above 90%.Western blot was used to verify the CAR protein expression,and a clear protein band was visible.The killing effect of On-Switch CAR-T on ovarian cancer cells was verified by LDH release assay.Under the dimerization of Rapamycin and AP21967,the killing effect of On-switch CAR-T on ovarian cancer cells increased with the increase of small molecule concentration.The secretion levels of TNF-αand IL-2 in On-switch CAR-T were higher than those in non-transfected T cells by ELISA,indicating that On-switch CAR T had higher immune activity.Thirdly,a method for the content determination of rapamycin,AP21967 and Pd PC（OBU）₈ was established.Rapamycin and AP21967 were determined by reversed-phase high performance liquid chromatography,and Pd PC（OBU）₈by UV-visible spectrophotometry.Examine specificity,linear range,precision,and accuracy.The linear regression equation of Rapamycin was A=0.6240C-0.1738,R2=0.9990.The linear range was 0.2～40μg·ml^-1.The linear regression equation of AP21967 was A=0.2016C-0.0119,R2=0.9968.The linear range was 0.25～10μM.The linear regression equation of Pd PC（OBU）₈was A=0.0665C+0.0022,R2=0.9999.The linear range was 2～12μg·m L^-1.Photosensitive ROS responsive liposomes containing Rapamycin or AP21967 were prepared using DSPC,18:2（Cis）PC（DLPC）,cholesterol,m DSPE-PEG₂₀₀₀ as membrane materials.Pd PC（OBU）₈ was selected as the photosensitizer.DLPC was unsaturated phospholipid,in which the carbon-carbon double bond structure could be oxidized by ROS to promote the release of small molecule drugs.The effects of DLPC,cholesterol,DSPE-m PEG₂₀₀₀,Pd PC（OBU）₈ dosage on liposome indexes were investigated by single factor method.The results showed that when the dosage mole ratio of DSPC,DLPC,cholesterol and DSPE-m PEG₂₀₀₀ was 60:10:25:5,and the dosage mass ratio of Pd PC（OBU）₈ was 1:100,the liposome prescription was the best.The particle size of liposome was 141.7±1.5nm,the PDI value was 0.036±0.001,and the Zeta potential was-12.8±0.6m V.The EE was（98.43±0.47）%,the LE was（1.1±0.01）%.The cumulative drug release rate reached more than 60%within 6 hours.Under low temperature environment,the particle size of liposomes was stable at 110-120nm within 7 days,and the PDI remained below 0.2,indicating that liposomes were relatively stable.Within 48h,liposomes were stable in 20%ethanol solution and serum-containing medium.Finally,On-switch CAR-T cells carrying photosensitive ROS responsive liposomes were prepared to investigate their antitumor effects in vitro.It was proved that liposomes and near infrared light had no toxicity to On-switch CAR-T cells and OVCAR-3 cells.After infrared irradiation,On-switch CAR-T cells carrying photosensitive ROS responsive liposomes had specific killing effect on mesothelin-high expression ovarian cancer cells OVCAR-3.The effects of two photosensitive ROS liposomes On on-Switch CAR T cell immune function at the same level of drug concentration（200n M）were compared,and the results showed no difference,which laid a foundation for subsequent in vivo experiments.