Smart Delivery Of Nucleic Acids Based On A Surface Charge Tunable Fluoresecent Protein H₃₉GFP

Gene delivery is a process in which exogenous genetic materials are transported into somatic cells of patients for the purpose of modulating gene expression of desired proteins.It is applied in many areas,such as structural and functional analysis of genes;expression and regulation of gene;gene therapy and transgenic animal research etc.Though many delivery systems have been developed to efficiently deliver vehicles for nucleic acids into cells,concerns have been raised regarding their immune responses,toxicity,instability and unintelligency.In terms of practical applications,it is necessary to make the vehicle capable of encapsulating the nucleic acid on command in response to external stimuli,such as pH and metal ions.Recently,a new kind of ScGFPs,containing 39 histidine residues on the surface(H39GFP),was developed by further resurfacing of ScGFP.Histidine residues on the surface of H39GFP possess several unique properties,such as pH-mediated charge change and the ability of coordination with metal ions.Hence,we predicted that the surface charge of H39GFP could be controlled by pH and Ni2+ ions.The addition of H+ or Ni2+ions not only make H39GFP positively charged and deliver nucleic acids to cell membrane.Similar to other ScGFPs,positively charged H39GFP can delivery nucleic acids into mammalian cells.Consequently,H39GFP can be regulated as a stimuli-responsive vector for gene delivery.1)It is known that the tumor microenvironment is weakly acidic(pH 5.7-7.0)compared to normal physiology(pH 7.0-7.4).According to histidine pKa 6.10,the surface charge of H39GFP could be 26 positive charge at pH 6.0 in theory.As we expected,zeta potential measurements showed that in buffer solution at pH 7.4,H39GFP was electrically neutral with zeta potential of 1.08±0.33 mV,while at pH 6.0 or the addition of Ni2+,they were positively charged with zeta potential of 4.56±0.45 mV and 6.69±1.16 mV.And the H39GFP/nucleic acids are formed the nano-complex under the condition of H39GFP positively charge which was high 35-70 nm by atomic force microscopy.Thus,these prove that H39GFP can self-assemble with nucleic acids under different stimuli.2)According to the surface change of H39GFP,we employ the microenvirmental change as activator-like to complete the construction of OR and AND gates to realize logic circuits.We use the change of pH and the existence of nickel ion as the inputs and the internalization of H39GFP or DNA in cells as the outputs to construct the OR logic gates.According to the images of laser scanning confocal microscopy and the results of flow cytometer,the outputs totally correspond to "OR" logic gates.Additionally,we present the AND gates by defining the existence of DNA and the existence of nickel ion as the inputs and the cell’s internalization of DNA as the output.Based on the above findings,we used H39GFP as a smart vector to deliver theβ-actin-targeting siRNA,which mediate efficient siRNA-induced 48%suppression of β-actin expression by western blot.These prove that we can keep the function of nucleic acids in cells.3)Based on H39GFP as the vector,we also construct the stimuli-triggered drug delivery systems to promote the therapeutic efficacy of anticancer drugs.Here,we utilize adenosine-5’-triphosphate(ATP)as a trigger for the controlled release of anticancer drugs.We use flow cytometer and MTT to demonstrate that H39GFP/ATP-Duplex/Dox nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment.