Dual Functional Nanoprobe Based On Alpha-helical Peptide Self-assembly For The Role Of Tumor Diagnosis And Treatment

Research background:Cancer is a disease characterized by abnormal cell proliferation,which as a leading cause of human death,it has attracted significant attention from clinical researchers worldwide.Nanomedicine has recently made impressive progress in the field of cancer treatment.However,the biocompatibility and targeted penetration of these nanomaterials remain a limitation,which leads to serious side effects and significantly narrows the scope of their applications.The self-assembly of nanoparticle structures can be induced through the formation of hydrophobic,ionic,or hydrogen bonds within and between different chains under certain conditions.Non-immunogenic self-assembled peptide from natural amino acids with good biocompatibility,and their further modified perhaps can solve the problem of limited tissue penetration.The human vimentin intermediate filaments(IFs)are the basic component of the cytoskeleton in cells,which realizes the cross-linking with the visible components such as microfilaments and microtubules in the form of homologous?-helical dimer.This study can promote the formation of polypeptide diversification three-dimensional spatial structure by using its variable expansion and contraction properties.An extremely conserved?-helical motif containing 36 amino acids was selected for the main sequence region of the self-assembled peptides,due to its ability to form the polymer shape via their parallel coiled-coil“rod”domains.7-amino actinomycin D(7-AAD)acts as a homologous structural analogue for the antitumor drug of actinomycin D(ACTD),which with a 2-cyclic peptide structure kept.In the connection chain of the seventh hydrogen atom of 7-AAD is replaced with the amidogen to emit the fluorescence by an intramolecular charge transfer effect,and it also have the ability to bind to DNA.The stokes shift of 7-AAD is approximately 100 nm,which can help effectively suppress the self-quenching process and reduce the non-specific background fluorescence.And making 7-AAD potentially suitable for diagnostic imaging and therapeutic value.Research Objectives:In this study,based on the respective properties of the polypeptide self-assembly and 7-AAD,a tumor targeting and p H-responsive bifunctional nanoprobe has been aimed to design and synthesize.Research methods:1.Design and molecular docking of RGD-IFP/7-AAD self-assembled peptide nanoprobeThe luminescent characteristics of 7-AAD were detected by fluorescence spectrometer,the binding effect between 7-AAD and DNA was analyzed by agarose gel electrophoresis and ultraviolet-visible spectra.The hydrophobic and electric charge properties of RGD-IFP peptide were analyzed by Novopro,and their tertiary structure were constructed by PDB database and SWISS-MODEL,and the molecular docking calculation and analysis of RGD-IFP/7-AAD were performed by MOE software.2.Preparation and characterization of RGD-IFP/7-AAD nanoprobe7-AAD was encapsulated by RGD-IFP into the self-assembled ordered nanoparticles(RGD-IFP/7-AAD)with a convenient one-step method.The self-assembled RGD-IFP/7-AAD nanoparticles were characterized by Transmission electron microscope(TEM),Dynamic light scattering(DLS),fluorescence spectrum,Zeta potential,X-ray photoelectron spectroscopy(XPS),circular dichroism spectrum(CD)and Fourier transform infrared spectroscopy(FTIR).Under the assembly and release behavior of nanoparticles,the fluorescence spectra of 7-AAD and dialysis method were used to determine the contents of 7-AAD,and the encapsulation rate,drug loading rate and release rate of nanoparticles were calculated.TEM was used to characterize the acid-sensitive disintegrated morphology of nanoparticles,and DLS and Zeta potential were used to detect the stability of nanoparticles in vitro.3.Application in vitro tumor cells of RGD-IFP/7-AAD nanoprobeRGD-IFP/7-AAD,IFP/7-AAD and free 7-AAD were incubated with A549 lung cancer cells and 293T cells respectively,and Lyso-Tracker fluorescent probe was used to trace lysosome,the uptake and positioning of nanoparticles in cells were observed by laser confocal microscope.The intensity of cell uptake,targeting property of tumor cells,the cell cycles were detected and analyzed by flow cytometry,CCK-8 experiments and Ed U detection were used to analyze the cell activity and proliferation.4.Fluorescence imaging and antitumor efficacy of RGD-IFP/7-AAD nanoprobe in tumor-bearing miceThe tumor-bearing mice model was established by human non-small cell lung cancer cells,and RGD-IFP/7-AAD,IFP/7-AAD and free 7-AAD were administered by caudal vein.The vivo fluorescence imaging of nanoprobes were observed by the animal living imaging system,and the intravital distribution of the nanoprobe and?_V?₃ integrin was observed by the immunofluorescence of frozen tissue sections.Treatment groups with continuous administration for several days,body weight and tumor volume were measured before each administration.At the end of experiments,isolated organs and tumor tissues were stained with immunohistochemistry and hematoxylin-eosin(H&E)staining,and the collected bloods were detected by biochemistry and routine blood analysis.Research results:1.The designed RGD-IFP self-assembled peptide sequence was consisted of three parts:the main sequence is an?-helical peptide composed of 36 amino acids in the vimentin intermediate filaments,the N-terminal fused with tumor targeting sequence(RGD)and drug carrying sequence of negative charge(SEE,IP=3.79),the final fused amino acid sequence is as follows:RGD–SEEKVELQELNDRFANYIDKVRFLEQQNKILLAELEQL.The hydrophobic cationic7-AAD drug of red fluorescence(excitation/emission wavelength:546/647 nm)has been shown to have the properties of combining DNA through experiment.The theoretical docking calculation model of 7-AAD and RGD-IFP peptide dimer showed that the lowest free energy and docking score was decided by 7-AAD binding with the middle of RGD-IFP peptide dimer as-12.16 kcal/mol and-8.37 respectively,and this conformation is the most stable one.They could dock into a stable ordered three-dimensional conformation by noncovalent interaction such as electrostatic interaction,hydrophobic effect,?-?stacking and so on.2.RGD-IFP/7-AAD nanoparticles were successfully prepared,and the morphology of TEM and DLS showed that the spherical particles were dispersed uniformly with 92.5±26.7 nm,their Zeta potential was 23.20±2.35 m V,while the successfully self-assembled RGD-IFP/7-AAD nanoparticle had obvious fluorescence reducing phenomenon.The XPS spectra showed that the self-assembled nanoparticles were mainly composed of C,N and O,and the composition was relatively simple.The circular dichroism spectra showed that the conformations of the peptide of self-assembled RGD-IFP/7-AAD was changed.And the vibrational frequencies of RGD-IFP/7-AAD intermolecular functional groups were characterized by FTIR,which showed obvious coordination.The derivative peptides of RGD-IFP contributed to the encapsulation and dissolution of the hydrophobic cationic drug 7-AAD,and the encapsulation and drug loading rates were up to 76.26±6.7%and 43.26±2.2%respectively.The particle size and Zeta potential of RGD-IFP/7-AAD nanoparticles remained stable for a long time in PBS buffer and 10%FBS serum diluent under the neutral p H7.4 in vitro.RGD-IFP/7-AAD nanoprobes showed the obvious acid-sensitive properties in vitro study.When p H was less than 6.5,the irregular disintegration and obvious 7-AAD drug release were showed in nanoparticles.3.RGD-IFP/7-AAD nanoprobes can be used to target A549 tumor cells for high expression of?_V?₃ integrin via RGD,and display the strongest red fluorescence imaging in intracellular nuclear location.The co-localization of RGD-IFP/7-AAD nanoprobe and lysosome was observed after intracellular uptake,suggesting that RGD-IFP/7-AAD could be absorbed by lysosomes and released 7-AAD in acidic environment and lysosome,so that 7-AAD could bind to intracellular DNA.Low-dose of 0.01?g/m L RGD-IFP/7-AAD nanoprobes had cell viability inhibition on tumor cells after 24 h continuously effect,and the Ed U test for 3 h showed obvious inhibition of cell proliferation.It is possible that 7-AAD released from RGD-IFP/7-AAD nanoprobes were inserted into DNA,and the tumor cell cycle was significantly blocked in the G0/G1 phase.4.The living imaging results of tumor-bearing mice showed that RGD-IFP/7-AAD nanoprobes have the high ability to target and retain fluorescence imaging at tumor sites for 4h and 24 h.And the targeted distribution of RGD-IFP/7-AAD nanoprobe and?_V?₃ integrin were shown in tissue sections of isolated organs.After continuous periodic administration,the RGD-IFP/7-AAD nanoprobe shown the obvious antitumor therapeutic effect.The immunohistochemical staining showed that the proliferation of Ki67 was significantly decreased(p<0.05)and the expression of TUNEL was significantly up-regulated(p<0.05)in RGD-IFP/7-AAD group.The results of H&E pathology sections,blood biochemistry and blood tests showed that RGD-IFP/7-AAD nanoprobes had low toxic side effects and well biocompatibility.Research conclusions:The designed and synthesized RGD-IFP/7-AAD self-assembled nanoprobes had well cell imaging effect and tumor cell growth inhibition ability,and had been successfully applied in nude mouse model of human non-small cell lung cancer for tumor targeted fluorescence imaging and treatment.Therefore,this study provides the theoretical and practical basis for the design and application of the integrated dual-functional nanoprobe with diagnosis and treatment.