Fabrication Of Intracellular Delivery Platforms Based On Photothermal Surfaces

Intracellular delivery of exogenous molecules(e.g.nucleic acids,proteins)is of vital importance in various areas,including fundamental biological research,industrial production and clinical diagnosis or diasease treatment.So far,the existed intracellular delivery systems,according to different ways for exogenous molecules to penetrate cell membranes,could be divided into two categories,namely,carrier mediated delivery and membrane disruption based delivery.However,carrier mediated intracellular delivery system suffers from several drawbacks,such as safety concerns,low delivery efficiency and low universality.On the other hand,membrane disruption based intracellular delivery system is more advantageous in the types of cells and cargoes that are amenable.The intracellular delivery system based on the photothermal effect of gold nanoparticle layers(GNPLs)allows the delivery of diverse exogenous molecules with efficiencies over 95%into Hela cells without compromising cell viability.However,the gene transfection efficiency of recalcitrant cells is not so satisfying.Moreover,GNPL,with high surface rougness,provides numerous cell adhesion sites and makes it hard to harvest cells carrying exogenous molecules for continuous applications.The main work of this thesis is to improve the overall performance of the intracellular delivery platforms based on photothermal surfaces,including increasing the delivery efficiency of recalcitrant cells,and on the base of which,facilitating the harvest of cells carrying exogenous molecules on the photothermal substrates.The detailed researches are as follows:(1)The transfection efficiency of recalcitrant cells of GNPL based intracellular delivery platform was greatly improved after being combined with low molecular weight polyethyleneimine(LPEI).In this GNPL/LPEI based intracellular delivery system,LPEI acts as the gene carrier condensing the plasmid DNA(pDNA)in the form of pDNA/LPEI complexes,and GNPL acts as a photoporation agent making the cell membrane more permeable and facilitating the entrance of pDNA/LPEI complexes.Moreover,LPEI protects the pDNA from the nuclease degradation in cells.First,a series of GNPLs with different roughness were fabricated with the in situ chemical reduction method,and their performance in gene transfection was determined;then,the dynamic light scattering assay and agarose gel retarding assay were performed to determine the size and zeta potential of pDNA/LPEI complexes as well as the ability of LPEI to immobilize the pDNA at different N/P ratio;finally,the transfection efficiency of recalcitrant cells with GNPL/LPEI based intracellular delivery system was determined.As the result shown that when combining the GNPL with LPEI,it gave significantly higher transfection efficiencies for recalcitrant cells(88.5%for mEFs and 94.0%for HUVECs)compared to using GNPL or LPEI separately.(2)A universal and nondestructive intracellular delivery platform was developed with polydopamine(PDA)coating as the photothermal substrate and poly(N-isopropylacrylamide)(PNIPAAm)as the temperature responsive cell releasing layer,which showed high efficiency in both cargo delivery and cell harvest.In this PDA/PNIPAAm based intracellular delivery system,PDA acts as the photothermal substrate.Under the irradiation of near infrared light,it converts light into heat and makes cell membrane more permeable and thus facilitating the entrance of exogenous molecules;PNIPAAm acts as the temperature responsive cell releasing layer.With temperature being cooled down to its low critical solution temperature(LCST),the surface will be converted from hydrophobicity to hydrophilicity and cells with exogenous molecules could be harvested high efficiently.First,Au slides coated with PDA and PNIPAAm hybrid film(Au-PDA-PNIP)was fabricated with the self-polymerization of dopamine and the successive free radical polymerization with N-isopropylacrylamide.Then,an ellipsometer,scanning electronic microscopy,atomic force microscopy and X-ray photoelectron spectroscopy,thermal camera and water contact angle instrument were utilized to characterize the thickness,surface morphology,elemental composition,photothermal effect and temperature responsive wettability changes of Au-PDA-PNIP.Next,exogenous molecules with different sizes including dextran,bovine serum albumin(BSA)and pDNA were chosen as model cargos,and Hela cells,mEFs,HUVECs and mouse dendritic cells(mDCs)were chosen as model cells to evaluate the performance of Au-PDA-PNIP in intracellular delivery.Moreover,cells with exogenous molecules were harvested from the surface and re-cultured.Finally,HUVECs were transfected with a functional gene pEGFP-ZNF580,and reverse transcription polymerase chain reaction(RT-PCR)as well as wound healing assay were performed to determine the expression of ZNF580 and its effect in promoting the migration rate of HUVECs.As the result shown that this platform could high-efficiently deliver diverse cargos into various cell lines and harvest cells with exogenous molecules for continuous applications.Moreover,the successful delivery of functional pEGFP-ZNF580 and its effect in promoting the migration rate of HUVECs demonstrates its potential in more practical applications.In summary,the overall performance of intracellular delivery platforms based on photothermal substrates was highly improved by combining it with other functional materials.The transfection efficiency of recalcitrant cells was increased after being combined with LPEI as the gene carrier,and based on which,cells with exogenous molecules were harvested by the temperature responsiveness of PNIPAAm.This upgraded intracellular delivery system could deliver diverse cargos with different sizes into various cell lines.It is reasonable to believe that this intracellular delivery system will show its promise in more practical areas.