Studying The Dynamic Mechanism Of Transporting A Single Dendritic Polymer Polyamidoamine Through Cell Membranes By Single-molecule Force Spectroscopy Based On Atomic Force Microscopy

Dendritic macromolecule polymer polyamidoamine(PAMAM)has lots of unique advantages compared with a great many traditional polymeric drug carriers.There are a large number of functional terminal groups on its surface,which could be modified and then connect various drug molecules.Secondly,the spherical distribution of high generation dendritic macromolecules,within the larger cavity,gives the drug molecules more opportunities to embed.Thus a growing numbers of researchers are working on PAMAM applied as drug carrier.However,as the first step in drug carrier into the cell,the process of across the membrane of PAMAM is not very clear because of the research method limited by temporal-spatial resolution.Here,single molecule force tracing technique(can measure the dynamic process on piconewton force and microsecond level)based on atomic force microscope(AFM)was used to study the trans-membrane dynamic process of single G5-PAMAM dendrimers.We recorded the dynamic process of single PAMAM into living cells in real time,and measured the force and duration of the dynamic transporting process,and revealed the endocytosis mechanism.The research provided more theoretical basis of the application of PAMAM as a drug carrier,which has important significance in biomedical field.Research progress is as follows:1.In this paper,this study recorded the dynamic process of a single G5-PAMAM into the living cells,as well as the force and duration needed for trans-membrane.The duration of single G5-PAMAM transporting into a living cell ranged from 2.5 to 65 ms with a mean value of 19 ± 11 ms and the corresponding force distributed in the range of 50-425 pN with a mean value of 172 ± 74 pN.2.In addition,this study revealed the endocytosis mechanism of G5-PAMAM entry into cells.The control and block experiments not only did by force tracing technique in single molecule level,but also by confocal microscopy.The rate of G5-PAMAM entry into Vero cells was quantified as the average fluorescence intensity of cells pretreated by inhibitors relative to that of control cells(without treatment with inhibitors).It is noteworthy that the data gathered from the two techniques showed a similar tendency.Both the single molecule and ensemble results illustrate that the G5-PAMAM transporting through cell membranes relies on the caveolin-mediated/clathrin-mediated endocytosis,and macropinocytosis pathways with the main pathway being clathrin-mediated endocytosis and macropinocytosis.