Study Of Reversibly Modified RNaseA And Its Nanocomposites For Cancer Cell-specific Response

Ribonuclease A?RNaseA?is a cytotoxic protein that cleaves RNA inside cells and leads cell apoptosis.An H₂O₂-responsive molecule 4-nitrophenyl?4-?4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl?-benzyl?carbonate?NBC?can modify RNaseA and inhibit its activity.After treating with hydrogen peroxide,the borate molecule NBC can undergo rearrangement and result in the release of modified NBC on RNaseA,leading to restoring of the enzyme activity.We then prepared liposomal nanocomposites based on different positively charged lipids to transport RNaseA prodrugs as well as substrate RNA strands simultaneously into cells.Due to the high expression of H₂O₂ in cancer cells,the H₂O₂ can activate the caged RNaseA inside cells and restore its activity.On one hand,the activated RNaseA can cleave the mRNA in cancer cells and lead to cell apoptosis.On the other hand,the activated RNaseA can cleave the RNA substrates with fluorophore modification and result in the fluorescence turn-on,which can be used to monitor the activation kinetics of the caged RNaseA prodrug.In the kinetic experiments,we clearly observed t he activation of RNaseA in cells.?1?We synthesized H₂O₂-responsive molecule NBC.The activity of RNaseA can be caged after NBC modification.After treating caged RNaseA with H ₂O₂,the activity of the enzyme can be efficiently restored.We used MALDI-TOF and other methods to characterize the modified RNaseA before and after treatment with hydrogen peroxide.?2?Using four different lipid formulations?DPPC,DPPC+CHOL,EC16-1,and EC16-80?,we prepared liposomal nanocomposites containing RNaseA-NBC and RNA substrates.The size,surface charge,and morphology of the formed nanocomposites were characterized using DLS and AFM.The results show that the synthesized nanoparticles are round and uniform with the size of ca 100 nm.We further measured the loading rates of the four liposomal nanocomposites.The results show that three nanocomposites have good loading rates except for the liposomal nanocomposites prepared by only DPPC.Moreover,we studied the H₂O₂ response of the formed nanocomposites in vitro and found that the liposomal nanocomposites based on DPPC and DPPC+CHOL showed the best fluorescence recovery.Based on the characterization results as well as the response of H₂O₂ of the liposomal nanocomposites,we chose the DPPC+CHOL formulation for following cell experiments.?3?We selected three different cell lines in the cell experiments,MCF-7,HeLa,and HEK-293T.The first two are cancer cells and the latter is normal cell.To test the ability of liposomal nanocomposites entering cells and find an optimal incubation time,we used Rhodamine 6G to label liposomals and test the fluorescence intensity in defferent time points using flow cytometry.After determining the optimal incubation time,we studied the kinetics of intracellular H₂O₂-response of caged RNaseA.By using flow cytometry and confocal microscopy,we found that in MCF cells the fluorescence of RNA substrate increased fast with the fluorescence enhancement of¹0-12 times.In HeLa cells which are also cancer cells,the turn-on of fluorescence is slower and the turn-on ratio is³-4 times.For the normal cell HEK-293T,almost no fluorescence turn-on was observed.Both flow cytometry and confocal microscope imaging confirmed these results,which can be attributed to the different concentrations of H₂O₂ in different cells.In addition,we studied the cytotoxicity of the nanocomposite and found that it showed highest cytotoxicity to MCF-7 and some to HeLa cells,but almost no cytotoxicity to HEK-293T cells.These results indicated that the liposomal nanocomposites can response to cancer cells with high H ₂O₂concentration specificallyIn summary,we demonstrated the reversible modification of RNaseA using a H₂O₂ responsive molecule NBC and prepared nanocomposites which containing RNaseA pro-drug and RNA substrates using different lipids.We further explored cancer-cell specific activation of caged RNaseA nanocomposites in different cell lines and studied the activation kinetics and cytotoxicity.