Uptake and function of membrane-destabilizing cationic nanogels for intracellular drug delivery

Abstract

The design of intracellular drug delivery vehicles demands an in-depth understanding of their internalization and function upon entering the cell to tailor the physicochemical characteristics of these platforms and achieve efficacious treatments. Polymeric cationic systems have been broadly accepted to be membrane disruptive thus being beneficial for drug delivery inside the cell. However, if excessive destabilization takes place, it can lead to adverse effects. One of the strategies used to modulate the cationic charge is the incorporation of hydrophobic moieties, thus increasing the hydrophobic content. We have demonstrated the successful synthesis of nanogels based on diethylaminoethyl methacrylate and poly(ethylene glycol) methyl ether methacrylate. Addition of the hydrophobic monomers tert-butyl methacrylate or 2-(tert-butylamino)ethyl methacrylate shows improved polymer hydrophobicity and modulation of the critical swelling pH. Here, we evaluate the cytocompatibility, uptake, and function of these membrane-destabilizing cationic methacrylated nanogels using in vitro models. The obtained results suggest that the incorporation of hydrophobic monomers decreases the cytotoxicity of the nanogels to epithelial colorectal adenocarcinoma cells. Furthermore, analysis of the internalization pathways of these vehicles using inhibitors and imaging flow cytometry showed a significant decrease in uptake when macropinocytosis/phagocytosis inhibitors were present. The membrane-disruptive abilities of the cationic polymeric nanogels were confirmed using three different models. They demonstrated to cause hemolysis in sheep erythrocytes, lactate dehydrogenase leakage from a model cell line, and disrupt giant unilamellar vesicles. These findings provide new insights of the potential of polymeric nanoformulations for intracellular delivery.

Keywords: cationic; drug delivery; intracellular; nanoparticles; polymer.

Figure 1

Cytocompatibility of polycationic nanogels as a function of polymer concentration. Symbols represent PDET (), PDETB20 (), PDETB30 (), PDETBA20 (), or PDETBA30 (). Proliferation of Caco‐2 cells was determined by MTS assay following 90 min nanogel exposure and is expressed as a fraction of the control (untreated) cells. Data are expressed as mean ± SEM, n = 8. Statistical significance determined via pairwise t‐test between cells exposed to PDETB20 and PDET or PDETB30 and PDET (*p < .005)

Figure 2

Uptake inhibition in Caco‐2 cells. Intracellular PDETB30‐OG488 fluorescence relative to noninhibited control. Caco‐2 cells preincubated with inhibitors for 30 min prior to 60 min exposure to 25 μg ml⁻¹ PDETB30‐OG488. Bars represent the mean of two pooled experiments ± SEM *p < .05, **p < .01. Arrow designates control group

Figure 3

Representative fluorescent micrographs of Caco‐2 cells exposed to endocytosis inhibitors and PDETB30‐OG488. Images sampled from median intensity region of OG488 fluorescent histogram. Scale bar represents 7 μm

Figure 4

Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), filipin III (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml⁻¹ PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells

Figure 5

Hemolysis and pyrene fluorescence as a function of nanogel formulation and solution pH. Panel (a) shows contour plots for PDET, PDETB20, and PDETB30 (top) and PDET, PDETBA20, and PDETBA30 (bottom). Panel (b) shows the concentration‐dependent hemolytic activity of PDET (□), PDETB30 (), and PDETBA30 () in 150 mM phosphate buffer at early endosomal pH (pH 6.0). Erythrocytes exposed to various polymer concentrations for 60 min at 37 °C. Data points represent the mean of triplicate samples ± SD. Panels (c) and (d) show the influence of TBMA incorporation on pyrene excitation (I338/I333 ratio) in P(DEAEMA‐co‐TBMA‐g‐PEGMA) nanogels (panel c), and of the inclusion of TBAEMA in the nanogel formulation (panel d). Nanogels suspended at 0.5 mg ml⁻¹ and pyrene dissolved at 6 × 10–7 M in 100 mM phosphate buffers at designated pH values. Panel (e) shows a summary table of the pH transition (pH_app) of cationic nanogel formulations

Figure 6

Representative time‐dependent LDH leakage from Caco‐2 cells following 60 min (●), 180 min (○), or 360 min () exposure to PDET (a), PDETB30 (b), and PDETBA30 (c). Data points represent the sample mean ± SEM (n = 4). LDH leakage calculated relative to untreated cells and surfactant‐lysed cells

Figure 7

Polymer‐mediated LDH leakage from Caco‐2 cells following exposure to PDET (), PDETB10 (), PDETB20 (), or PDETB30 () for 60 min (a) or PDET (), PDETBA10 (), PDETBA20 (), or PDETBA30 () for 60 min (b)

Figure 8

Destabilization of GUV membranes. Intravesical red fluorescence indicates sucrose‐Texas Red. Green fluorescence indicates membrane lipid DHPE‐Bodipy FL. GUVs were suspended in 100 mM phosphate buffer at pH 6.5. PDET (a) or PDETB30 (b) in isosmotic phosphate buffer was added at achieve a final concentration of 50 μg ml⁻¹. GUVs after 30 s incubation (c and d). Images captured using Zeiss spinning disc confocal microscope at 100×