Nanoparticle mediated laser-induced photoporation is a physical cell membrane disruption approach to directly deliver extrinsic molecules into living cells, which is particularly promising in applications for both adherent and suspension cells. In this work, we explored surface modifications of graphene quantum dots (GQD) and reduced graphene oxide (rGO) with polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance colloidal stability while retaining photoporation functionality. After photoporation with FITC-dextran 10 kDa (FD10), the percentage of positive HeLa cells (81% for GQD-PEG, 74% for rGO-PEG and 90% for rGO-PEI) increased approximately two-fold compared to the bare nanomaterials. While for Jurkat suspension cells, the photoporation efficiency with polymer-modified graphene-based nanomaterial reached as high as 80%. Cell viability was >80% in all these cases. In addition, polymer functionalization proved to be beneficial for the delivery of larger macromolecules (FD70 and FD500) as well. Finally, we show that rGO is suitable for photoporation using a near-infrared laser to reach 80% FD10 positive HeLa cells at 80% cell viability. We conclude that modification of graphene-based nanoparticles with PEG and especially PEI provide better colloidal stability in cell medium, resulting in more uniform transfection and overall increased efficiency.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.