Polymeric microspheres as protein transduction reagents

Abstract

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.

Scheme 1.

Derivatization of core-shell microspheres 1 with a cell-cleavable linker, subsequent internal labeling, and protein attachment. Reaction scheme showing the derivatization of core-shell microspheres 1. Reaction conditions: (i) TBTU, N,N-diisopropylethylamine, DMF; (ii) 3-mercaptopropionic acid, DMF; (iii) DY-590-maleimide, DMF; (iv) GFP, EDAC, MES, pH 6.0, NaOH.

Fig. 1.

Indirect immunofluorescence analysis of beadfected, fixed HeLa cells. HeLa cells were beadfected with DY-630/GFP derivatized core-shell microspheres. Samples of the cells were either fixed or fixed and permeabilized. They were then examined via indirect immunofluorescence using mouse anti-GFP antibody and an anti-mouse/phycoerythrin secondary antibody conjugate. Confocal images of GFP and/or phycoreythrin fluorescence illustrate (a) non-permeabilized cells, (b) permeabilized cells, (i) GFP fluorescence, (ii) phycoerythrin fluorescence, (iii) co-localization of GFP/phycoerythrin fluorescence, and (iv) combined fluorescent/bright field image to show cellular location. The white arrow indicates non-internalized microspheres. (Note: microspheres shown in this figure were internally labeled with DY-630, which was selected solely to imbue the microspheres with chemical characteristics similar to those used in subsequent fluorescence microscopy studies. It was first confirmed that DY-630 was undetectable by the filter set/wavelengths employed within these experiments.)

Fig. 2.

Epifluorescent microscopy of beadfected HeLa cells conjugated to GFP via cell-cleavable and non-cleavable linkers. Samples of HeLa cells were beadfected with DY-590-labeled microspheres conjugated to GFP via a cell-cleavable linker (microspheres 6) or via a non-cleavable linker (1). All images represent a z-slice taken at a focal plane approximating the center of the cells. A, combined fluorescent/bright field image. B, DY-590 fluorescence (internal bead label) and GFP fluorescence. C, GFP fluorescence only. D, DY-590 fluorescence only.

Fig. 3.

CellTiter-Blue® analysis of cell viability after beadfection. HeLa cells from five different passage numbers were beadfected in quadruplet with microspheres 5 (derivatized with the cell-cleavable linker only) and 6 (loaded with GFP, linked via the cell-cleavable linker), cultured for 50 h, and compared with non-beadfected controls by means of CellTiter-Blue® analysis, as described under “Experimental Procedures.” Data represent means of resazurin reduction, expressed as a percentage of nontreated controls ± S.D.

Fig. 4.

Epifluorescent microscopy to examine the extracellular stability of the cell-cleavable linker. GFP-loaded microspheres 6 (cell-cleavable linker) were examined via epifluorescent microscopy after incubation at 37 °C for 96 h in used DMEM medium. (i) GFP fluorescence only. (ii) DY-590 fluorescence (internal bead label) only. (iii) Combined GFP and DY-590 fluorescence. (iv) Combined fluorescent/bright field image.