Research On Intracellular Delivery Method Of Exogenous Substances Based On Filter Paper And Semiconductor Refrigeration Device

The intracellular delivery of exogenous substances is an essential technical means in the field of biomedical research,and has been widely used in multiple application scenarios such as biomanufacturing,cell therapy,and disease diagnosis and analysis,for example,delivering CAR gene to T lymphocytes for cancer immunotherapy,and delivering Cas 9 protein and single-stranded guide RNA to cells for gene editing.However,direct delivery of exogenous substances requires crossing the plasma membrane barrier,which protects intracellular components from extracellular influences.Even the cryoprotectant trehalose with a molecular weight of only 342 Da and a size of about 1 nm has no ability to enter cells through the membrane lipid bilayer.Ineffective intracellular delivery and toxicity issues hinder the application of exogenous molecules in cells,especially for the delivery of some important cell types(such as stem cells).Although many delivery technologies and strategies are present,each technique has its own limitations.The delivery cost is usually a major limiting factor for general laboratories.In addition,simplifying the operation process and shortening the delivery time are key challenges.Researchers need a portable,simple and low-cost method or a practical platform that can stably,rapidly,and effectively facilitate the intracellular delivery of various exogenous substances.Therefore,this study developed two cell delivery devices for simple and rapid intracellular delivery of various exogenous substances.The main research results of this paper are as follows:1.This study designed and developed the filter paper-syringe(FPS)delivery platform,a new type of cell delivery device based on filter paper.The cells in a syringe are forced to pass through the filter paper quickly.During this process,external pressure forces the cells to collide and squeeze with the fiber matrix of the filter paper,causing the cells to deform rapidly to enhance the permeability of the cell membrane and realizing the efficient delivery of a variety of exogenous substances(dextran,PI,plasmid DNA,antibodies,and siRNA),including difficult-to-transfect primary and stem cells.Moreover,the large gap between the fiber networks of filter paper can prevent the cells from bearing high pressure,thus maintaining high cell vitality.Results show that slow-speed filter paper used can stably and efficiently deliver small molecules(such as 3-5 k Da dextran)repeatedly into different cells,and this method was also applicable to sensitive primary cells.Meanwhile,we found that the FPS method not only does not require a lengthy operating step compared with the widely used lipofectamine transfection reagent(Lipofectamine 3000)delivery of siRNA,but also the delivery efficiency is similar.In conclusion,the FPS approach is a simple,low-cost,versatile,easy-to-operate(single-step)and fast(about 2 s)delivery method and may be an attractive alternative to the membrane destruction-based transfection.2.A rapid temperature change(RTC)platform based on semiconductor refrigeration chips was designed and constructed.The micro-channel designed can accurately control the temperature of the cold and hot sides,and combined with the flexible design of the cold and heat conduction blocks,we can fully and flexibly utilize the cold and hot sides of the device,so as to realize the rapid change of fluid temperature.The results showed that by manipulating the parameters such as flow rate and voltage of the device,the temperature control of 4 ℃ at the cold side and 42 ℃ at the hot side of the device was realized.Using RTC platform,the transformation of E.coli and the expression of GFP were realized,resulting in higher transformation efficiency and shorter time than traditional methods.In addition,efficient delivery of small molecules(PI and trehalose)was achieved using RTC platform,trehalose delivery using RTC effectively improved the cryopreservation activity of cells,with similar post-cryopreservation compared with cells cryopreserved in organic solvent(DMSO).Compared with DMSO cryopreservation,cells cryopreserved after trehalose delivery did not require tedious washing procedures to remove organic solvents.The device has a simple structure and does not require complex external equipment,and its portability is expected to have significant application potential in cell medicine research and industrial processes.