Single-cell membrane drug delivery using porous pen nanodeposition

Abstract

Delivering molecules onto the plasma membrane of single cells is still a challenging task in profiling cell signaling pathways with single cell resolution. We demonstrated that a large quantity of molecules could be targeted and released onto the membrane of individual cells to trigger signaling responses. This is achieved by a porous pen nanodeposition (PPN) method, in which a multilayer porous structure, serving as a reservoir for a large amount of molecules, is formed on an atomic force microscope (AFM) tip using layer-by-layer assembly and post processing. To demonstrate its capability for single cell membrane drug delivery, PPN was employed to induce a calcium flux triggered by the binding of released antibodies to membrane antigens in an autoimmune skin disease model. This calcium signal propagates from the target cell to its neighbors in a matter of seconds, proving the theory of intercellular communication through cell-cell junctions. Collectively, these results demonstrated the effectiveness of PPN in membrane drug delivery for single cells; to the best of our knowledge, this is the first technique that can perform the targeted transport and delivery in single cell resolution, paving the way for probing complex signaling interactions in multicellular settings.

Figure 1.. Fabrication and characterization of porous pens.

a) The fabrication process of Layer-by-Layer (LbL) assembly-formed porous structures on the surface of an AFM cantilever and tip. b) SEM observation of porous pens on the entire cantilever. c) SEM observation of tips of porous pens. d) Zoom-in SEM observation of the surface of AFM tips. e) Zoom-in SEM observation of the surface of cantilevers. f-g) Frequency analysis of pores sizes on the surface of porous pens fabricated with different deposition times: for pore sizes smaller than 110 nm (f) and larger than 110 nm (g). Scale bars in (b) 50 μm, (c) 2 μm, (d-e) 1 μm.

Figure 2.. Characterization of protein loading on porous pens and APTES functionalized AFM cantilevers.

a) Amount of protein loading on an APTES functionalized AFM probe and porous-structured AFM probes fabricated with three deposition times: 10 seconds, 1 minute, and 5 minutes. b) The intensity level of the four experimental conditions. c) The amount of protein loading at the substrate of probes, the cantilever and the tip of porous pens. Scale bar in (a): 30 μm.

Figure 3.. Diffusion of loaded protein molecules from porous pens fabrication with different deposition times.

a-c) Representative fluorescence images of porous pen samples during protein diffusion of 114 hours. Porous pens are fabricated with 5-minute (a), 1-minute (b), and 10-second (c) deposition times, respectively. d) The fluorescence intensity of the three experimental conditions over 114 hours. e) A diagram illustrates the two types of protein interactions with the porous structure. Scale bars in (a), (b), and (c): 100 μm.

Figure 4.

Porous pen nanodeposition (PPN) of protein molecules onto a substrate. (a-c) Representative results for PPN experiments after porous pens are incubated (a) or rinsed (b) in PBS, or directly after protein loading (c). d) The fluorescence intensity of deposited protein dots in the three experimental conditions. Scale bars in (a), (b), and (c): 5 μm.

Figure 5.. PPN enabled spatial profiling of antibody-induced cell signaling in keratinocyte monolayer.

a) A diagram of targeting and releasing of loaded antibody molecules onto the membrane of a single cell in a cell monolayer. b) The gradient of Calcium signal within the monolayer around the targeted cell. c) Time-lapse fluorescence images of cells stimulated by an AK23 loaded porous pen. d) Time-lapse fluorescence images of cells stimulated by negative control of a porous pen. Scale bars in (c)(d): 100 μm.

Figure 6.. Quantitative analysis of the spatial profile of AK23-induced Calcium signals within a keratinocyte monolayer.

a) The fluorescence intensity of experimental and control groups 32 seconds after stimulations. b) The induction time of Calcium signal of neighboring cells is linearly related with the distance between this cell to the target cell. c-d) The Calcium wave signals of three cells with different distances from the stimulated cell in experimental (c) and negative control (d) groups.