An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles

Abstract

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Fig. 1

Sequential delivery is effective across all CPP types. Polymer dose-dependent uptake of the MK2i peptide (co-delivery of pre-complexed polymer/peptide) fused to a three separate cationic, non-amphipathic CPPs and b two different amphipathic CPPs. Sequential polymer then peptide delivery polymer dose-dependent uptake of the MK2i peptide fused to c three separate cationic, non-amphipathic CPPs and d two different amphipathic CPPs. e Polymer dose-dependent uptake of the YARA CPP fused to two separate therapeutic peptide sequences (MK2i and VASP) when co-delivered. f Polymer dose-dependent uptake of the VASP peptide without a CPP. The mass ratios used for all data shown are 3:1, 1:1, 1:3, 1:5, 1:10, and 1:20 peptide:polymer. Error bars represent SEM. Non-normalized uptake for all data presented in this figure is shown in Supplementary Fig. 4

Fig. 2

PPAA outperforms commercially available peptide delivery reagents. Comparison of peptide delivery reagent-mediated a uptake (numbers above the bars denote the fold increase in peptide uptake compared with the peptide alone) and b retention of the YARA-MK2i peptide over time following treatment removal. c Representative colocalization images and d quantification of peptide colocalization with the endosomal dye LysoTracker Red demonstrating PPAA-mediated endosomal escape and intracellular YARA-MK2i peptide delivery; *p < 0.05 vs. all other treatment groups. e Evaluation of delivery reagent-mediated cytotoxicity compared with delivery of the YARA-MK2i peptide alone (10 µM peptide); *p < 0.05 vs. treatment with the peptide alone; statistical analyses—one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM graphically

Fig. 3

Mechanism of action for PPAA-mediated CPP uptake. a Inhibition of rhodamine labeled PPAA polymer uptake in HCAVSMCs. b The effect of adding inhibitors during PPAA pretreatment on subsequent uptake of Alexa-488 labeled YARA-MK2i peptide in HCAVSMCs (inhibitors applied during PPAA treatment only). c Representative Z-stack images (images have x and y projections of 3-dimensional z-stack images below and to the right of each image, respectively) of the dose-dependent effects of dynasore during PPAA pretreatment on fluorescently labeled polymer and peptide uptake. d The effects of adding inhibitors throughout the entire treatment for both co-delivery and sequential delivery of PPAA and Alexa-488 labeled YARA-MK2i peptide in HCAVSMCs. For a, b, d, *p < 0.05, **p < 0.01, ***p < 0.001 vs. no inhibitor. e Zeta potential measurement of PPAA and YARA-MK2i peptide mediated changes in extracellular surface charge of HCAVSMCs, *p < 0.05. f Schematic representation of the proposed mechanism of action for PPAA-mediated CPP-peptide uptake and intracellular delivery. Poly(propylacrylic acid) (PPAA) interacts with the cell membrane via hydrophobic interactions with the propyl moiety of PPAA. This interaction increases the net negative charge of the cell membrane due to the carboxylate anion present on the polymer. This increase in net negative charge enhances cell surface electrostatic interactions with cationic CPPs, leading to enhanced peptide uptake through both macropinocytic and endocytic pathways. Following uptake, the decrease in endosomal pH results in protonation of the PPAA polymer. This pH change triggers the polymer to dissociate from the CPP-peptide cargo and interact with and destabilize the endosomal membrane, resulting in cytosolic peptide delivery. Statistical analyses were performed with a one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM graphically

Fig. 4

PPAA facilitates endosomal escape and is subsequently trafficked to autophagosomes. a Galectin-8 recruitment measured as the average Galectin-8 intensity per cell 2 h post-treatment with 0 or 10 µM PPAA in the presence or absence of the endosomal acidification inhibitor bafilomycin A and its control nocodazole. Data are presented as means ± 95% CI; two-way ANOVA followed by Tukey’s post hoc test. b Representative microscopy images of the treatment groups presented in A. Images are 712.8 × 712.8 µm. c Tracking in real-time indicates that PPAA causes significant (*p < 0.05) endosomal disruption compared with no treatment after 1 h which increases over the timeframe measured. Data are presented as means ± standard deviation. For a, c, *p < 0.05, ****p < 0.0001 vs. no inhibitor, ^####p < 0.0001. d Comparison of delivery reagent-mediated intracellular peptide bioavailability through a quantitative split-GFP fluorescence transduction assay (numbers above bars denote the fold increase in GFP fluorescence compared with delivery of the peptide corresponding to the eleventh β-strand of the GFP protein alone); *p < 0.05; one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. e PPAA dose-dependent intracellular delivery of TAT-CRE to Ai9 fibroblasts expressing a loxP flanked stop cassette upstream of a tdTomato transgene. Data are expressed as the percentage of treated cells positive for tdTomato expression as a measure of the intracellular bioavailability of the TAT-CRE cargo (12.5 ng/µL = 0.56 µM, 200 ng/µL = 9.1 µM PPAA). Two-way ANOVA testing of the data revealed a significant effect of PPAA dose on TAT-CRE mediated gene recombination (p < 0.0001). Tukey’s post hoc multiple comparisons testing for simple effects within each TAT-CRE dose revealed significant differences in gene recombination with increasing doses of PPAA; *p < 0.05. Data are presented as means ± standard deviation. Representative images of Ai9 fibroblasts pretreated with f 0 and g 200 ng/mL PPAA prior to treatment with 40 units/mL TAT-CRE. Images are 712.8 × 712.8 µm. h Representative still frame (from Supplementary Movie 4) demonstrating that LC3B (green) co-localizes (white arrows) with PPAA (purple) following cellular uptake

Fig. 5

PPAA broadly enhances delivery of cationic biomacromolecules. a Structure of a vivo-morpholino constituted by phosphorodiamidite morpholino oligomer conjugated to a cationc octa-guanidine dendrimer. b Quantification and c representative confocal microscopy images of polymer dose-dependent CD47 vivo-morpholino uptake in human microvascular endothelial cells. d Quantification and e representative confocal microscopy images of vivo-morpholino (green) colocalization with the endolysosomal dye lysotracker (red). *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM

Fig. 6

PPAA pretreatment enhances cationic nanoparticle uptake and bioactivity. a Schematic representation of cationic, amine-surface modified 200 nm polystyrene nanoparticles, synthesis of Cy5-labeled DNA-loaded D-DPB polymeric micellar nanoparticles, and sequential delivery of these nanoparticle formulations following PPAA pretreatment. The effects of dose-dependent PPAA pretreatment on the uptake of b cationic polysterene nanoparticles and c Cy5-labeled DNA-loaded D-DPB nanoparticles in A7r5 cells; *p < 0.05, vs. treatment with nanoparticles alone (i.e., 0 nM PPAA). d The effects of dose-dependent PPAA pretreatment on luciferase gene silencing in luciferase expressing A7r5s; *p < 0.05, **p < 0.01, vs. D-DPB nanoparticles loaded with scrambled siRNA (scramble). For b–d, statistical analyses were performed with a one-way ANOVA followed by a Tukey’s post hoc test. Data are presented as means ± SEM. e PPAA dose-dependent effects on uptake vs. bioactivity

Fig. 7

PPAA enhances endosomal escape and gene editing efficiency. Comparison of a Galectin-8 recruitment and b gene editing efficiency with various delivery systems with and without PPAA pretreatment (50 ng/mL); *p < 0.05. **p < 0.01; one-way ANOVA followed by a Tukey’s post hoc test. Data are presented as means ± SEM. Successful CRISPR/Cas9-mediated gene editing leads to removal of a termination cassette upstream of a tdTomato transgene in Ai9 fibroblasts, leading to activation of tdTomato expression. c Representative microscopy images of galectin-8-YFP recruitment to disrupted endosomes and tdTomato expression following CRISPR/Cas9-mediated gene editing in engineered Ai9 fibroblasts. All Gal8 micrographs shown are 458.6 × 458.6 µm; all Ai9 micrographs shown are 1404 × 1404 µm. 50 ng/mL = 2.27 nM PPAA