Cationic Polymers Enable Internalization of Negatively Charged Chemical Probes into Bacteria.

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate (D) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis. We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

Blended Block Polycation Micelles Enhance Antisense Oligonucleotide Delivery.

Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an n-butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.

Enhanced ASO-Mediated Gene Silencing with Lipophilic pH-Responsive Micelles.

Herein, we examine the ASO-mediated gene silencing efficiency of pH-responsive micelles, by incorporating 2-(diisopropylamino)ethyl methacrylate (DIP) into the micelle core and comparing physical and biological properties with non-pH-responsive micelles. Additionally, the lipophilic effect of the micelle cores was examined in both types of micelles. Varying lipophilicity was achieved by varying alkyl monomer chain lengths─butyl (4), lauryl (12), and stearyl (18) methacrylate. Each of the micelles formed within our family offered the added benefit of well-defined and uniform templates for loading antisense oligonucleotide (ASO) payloads. Overall, the micelles followed previously established trends of outperforming their linear polymer (nonmicelle) analogs and ASO only control. More specifically, the highest performing micelles were the pH-responsive micelles with longer alkyl chains or higher lipophilicity─D-DIP+LMA and D-DIP+SMA (∼90% silencing). These two micelles demonstrated silencing efficiencies similar to Jet-PEI and Lipofectamine 2000 and caused lower toxicity than Lipofectamine 2000. The shortest alkyl chain pH-responsive micelle, D-DIP+BMA (64%), displayed strong gene silencing similar to that about that of its non-pH-responsive micelle, D-BMA (68%), and the pH-responsive micelle without an alkyl chain incorporated, D-DIP (59%). This work illuminates a minimum alkyl chain length dependence to allow gene silencing within our micelle family. However, including only longer alkyl chains into the micelle core without the pH-responsive unit DIP had a hindering effect, thus demonstrating the requirement of the DIP unit when including longer alkyl chain lengths. This work demonstrates the exemplary gene silencing efficiencies of polymeric micelles and uncovers the relationship between pH responsiveness and performance with lipophilic polymer micelles for enhancing ASO-mediated gene silencing.

Cationic Micelles Outperform Linear Polymers for Delivery of Antisense Oligonucleotides in Serum: An Exploration of Polymer Architecture, Cationic Moieties, and Cell Addition Order.

Antisense oligonucleotides (ASOs) are an important emerging therapeutic; however, they struggle to enter cells without a delivery vehicle, such as a cationic polymer. To understand the role of polymer architecture for ASO delivery, five linear polymers and five diblock polymers (capable of self-assembly into micelles) were synthesized with varying cationic groups. After complexation of each polymer/micelle with ASO, it was found that less bulky cationic moieties transfected the ASO more effectively. Interestingly, however the ASO internalization trend was the opposite of the transfection trend for cationic moiety, indicating internalization is not the major factor in determining transfection efficiency for this series. Micelleplexes (micelle-ASO complexes) generally enable higher transfection efficacy as compared to polyplexes (linear polymer-ASO complexes). Additionally, the order of addition of cells and complexes was explored. Linear polyplexes showed better transfection efficiency in adhered cells, whereas micelleplexes delivered the ASO more efficiently when the cells and micelleplexes were added simultaneously. This phenomenon may be due to increased cell-complex interactions as micelleplexes have increased colloidal stability compared to polyplexes. These findings emphasize the importance of polymer composition and architecture in governing the cellular interactions necessary for transfection, thus allowing advancement in the design principles for nonviral nucleic acid delivery formulations.

Polycation Architecture Affects Complexation and Delivery of Short Antisense Oligonucleotides: Micelleplexes Outperform Polyplexes.

Herein, we examine the complexation and biological delivery of a short single-stranded antisense oligonucleotide (ASO) payload with four polymer derivatives that form two architectural variants (polyplexes and micelleplexes): a homopolymer poly(2-dimethylaminoethyl methacrylate) (D), a diblock polymer poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate) (ObD), and two micelle-forming variants, poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)methylether methacrylate-block-poly(2-dimethylaminoethyl methacrylate)-block-poly(n-butyl methacrylate) (ObDB). Both polyplexes and micelleplexes complexed ASOs, and the incorporation of an Ob brush enhances colloidal stability. Micellplexes are templated by the size and shape of the unloaded micelle and that micelle-ASO complexation is not sensitive to formulation/mixing order, allowing ease, versatility, and reproducibility in packaging short oligonucleotides. The DB micelleplexes promoted the largest gene silencing, internalization, and tolerable toxicity while the ObDB micelleplexes displayed enhanced colloidal stability and highly efficient payload trafficking despite having lower cellular uptake. Overall, this work demonstrates that cationic micelles are superior delivery vehicles for ASOs denoting the importance of vehicle architecture in biological performance.

Polymeric Delivery of Therapeutic Nucleic Acids.

The advent of genome editing has transformed the therapeutic landscape for several debilitating diseases, and the clinical outlook for gene therapeutics has never been more promising. The therapeutic potential of nucleic acids has been limited by a reliance on engineered viral vectors for delivery. Chemically defined polymers can remediate technological, regulatory, and clinical challenges associated with viral modes of gene delivery. Because of their scalability, versatility, and exquisite tunability, polymers are ideal biomaterial platforms for delivering nucleic acid payloads efficiently while minimizing immune response and cellular toxicity. While polymeric gene delivery has progressed significantly in the past four decades, clinical translation of polymeric vehicles faces several formidable challenges. The aim of our Account is to illustrate diverse concepts in designing polymeric vectors towards meeting therapeutic goals of in vivo and ex vivo gene therapy. Here, we highlight several classes of polymers employed in gene delivery and summarize the recent work on understanding the contributions of chemical and architectural design parameters. We touch upon characterization methods used to visualize and understand events transpiring at the interfaces between polymer, nucleic acids, and the physiological environment. We conclude that interdisciplinary approaches and methodologies motivated by fundamental questions are key to designing high-performing polymeric vehicles for gene therapy.