Monitoring The Trans-membrane Process Of Targeted Peptide-modified Nano-drugs And Studying The Dynamic Mechanism

Biopharmaceuticals have transformed the treatment of disease and are increasingly used in clinical medicine.It contains an altered active pharmaceutical ingredient and is characterized by enhanced efficacy.In the pharmaceutical industry,drugs based on small organic molecules have been the most widely used therapeutic agents,and targeting peptides have become important ligands for the construction of biosmart nano-drug delivery systems.Due to their physical,chemical,and biological properties,they can actively target receptors overexpressed in tumor cells,bind and penetrate different barriers existing in cells and tissues,and these functional peptides can be used as effective drugs and carrier ligands to overcome extracellular and intracellular barriers.The targeted interaction between peptides and receptors is the key to affecting the efficacy of targeted nanodrugs.Different types of peptides as ligands have different interactions with their corresponding receptors,thus reflecting the different targeting efficiencies of peptides.Transmembrane transport is the first step for nanodrugs to enter the cell,understanding its transmembrane dynamic process is crucial to the rational design and screening of targeted nanodrug ligands.However,due to the limitation of technology,the dynamic transmembrane process of peptide-targeted nanodrug and virus entry into cells is still unclear.Herein,this paper comprehensively applies the mechanical measurement technology based on atomic force microscopy(AFM),single-molecule force spectroscopy(SMFS),and force tracing technology,combined with fluorescence imaging technology to study the interaction of peptides,targeted nanobodies,viruses and cell membranes under near-physiological conditions dynamic mechanism of action.The main research contents are as follows:(1).Three common peptides(HAIYPRH(T7),YHWYGYTPQNVI(GE11),and RGD targeting the corresponding receptors(Transferrin receptor(TfR),epidermal growth factor receptor(EGFR),and?_??₃ integrin receptor)are selected as example to study the targeting action at the single-molecule level by SMFS based on AFM to study targeting interactions on the surface of living cells at the single-molecule level,and to evaluate T7-TfR,GE11-EGFR,and RGD-?_??₃ integrins receptor in the absence of external force dissociation activation energy(?G_?,0)and other parameters.The results showed that:in A549 cells,the targeting effect of T7 and TfR was the best.The dissociation kinetic rate constant at zero force(K_off,6.80×10^-4 S^-1)is smaller,the binding kinetic rate constant(K_on,0.79 M^-1S^-1)is larger,and the interaction time(?,0.22 s)is shorter.Furthermore,after A549 cells were treated with the drug(Doxorubicin),T7 could still effectively target TfR on the cell membrane.As the same time,the fluorescence experiments also further confirmed the results of SMFS.(2).AFM based force tracing technique was used to track the ultrafast dynamic process of T7-modified gold nanoparticles(Au NP-T7)entering different cell lines at the single-molecule level in real time and compare the dynamic parameters.The results showed that A549 cells with high TfR overexpressed,the average uptake force of Au NP-T7 was 66.70±22.86 pN,the corresponding uptake time was 85.00±30.13 ms,and the average uptake speed was 0.396?m/s;For HeLa cells with lower degree of TfR overexpression than A549,the uptake force was almost unchanged with the average value of 62.58±22.41 pN,however,the uptake time was significantly increased to 100.93±45.55ms with the average speed of 0.332?m/s;However,in normal cells without TfR overexpression(Vero),both average uptake force and time increased to 74.69±34.10 pN,112.06±62.30 ms,and the average speed of 0.302?m/s.The experimental results demonstrated that Au NP-T7 was more readily uptake in cells with a high degree of TfR overexpression.At the same time,the results of fluorescence experiment further confirmed the conclusion of force tracing.(3).The T7 peptide target the TfR and then enter cells easily with the help of transferrin(Tf),it has been widely used as a ligand for the construction of tumor-targeted nanodrug delivery systems.However,the dynamic mechanism by which Tf promotes the entry of T7-conjugated nanocarriers into cells remains unclear.Therefore,the force tracing technology based on AFM was used to reveal the dynamic mechanism by which Tf promotes the entry of Au NP-T7 entering cells at the single-molecule level.The results showed that due to the promoting effect of Tf,the average force required to uptake Au NP-T7 by A549 cells was 53.49±16.83 pN,the corresponding time was 67.24±27.87 ms,and the average uptake speed was 0.496?m/s;For HeLa cells with a lower degree of TfR overexpression than A549,the average uptake force was 59.00±36.81 pN,the corresponding uptake time was 81.53±39.80 ms,and the average speed was 0.410?m/s;In Vero cells without TfR overexpression,the average uptake force and time were69.17±25.46 pN and 148.59±11.96 ms,respectively,and the average speed was 0.227?m/s.The experimental results demonstrate that the promoting effect of Tf helps to decreased the endocytic force and increase the endocytic speed of Au NP-T7 in A549 cells.However,the promoting effect of Tf only increased the endocytic speed of Au NP-T7 uptake by HeLa cells.In contrast,in Vero cells,the addition of Tf decreased the endocytosis speed of Au NP-T7.(4).Novel coronavirus pneumonia(COVID-19)is a worldwide pandemic disease caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)infection,which seriously threatens human health.Understanding the endocytosis process of SARS-Co V-2 entry into cells is crucial,which helps to deeply understand the kinetic mechanism of virus entry into cells and provides a theoretical basis for the treatment of SARS-Co V-2 virus infections.Here,the dynamic process of SARS-Co V-2 virus-like particle entry into cells was measured at the single-particle level using AFM-based force tracing technology.The experimental results showed that the average force required for A549 cells to endocytose a single SARS-Co V-2 virus-like particle was 57±20 pN,the corresponding time was 186±80 ms,and the average speed was 0.18?m/s.The results of the simulation calculation also confirmed the correctness of the experimental results of the force tracing.At the same time,the transmembrane transport mechanism of the SARS-Co V-2 virus-like particle entering A549 cells was studied using different endocytic pathway blocking reagents.The ACE2-derived peptide P4 could significantly reduce the efficiency of virus infection of A549 cells,while the peptide 2019-n Co V-HR2P targeting the HR1(S2 subunit)domain of the virus S protein cannot prevent the virus from entering A549 cells,the results confirmed that SARS-Co V-2 virus-like particle entry into A549cells is dependent on clathrin-mediated endocytosis rather than membrane fusion.In addition,the way the SARS-Co V-2 virus-like particle enters cells varies among cell lines.