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.

Hydrophilic Surface Modification of Cationic Unimolecular Bottlebrush Vectors Moderate pDNA and RNP Bottleplex Stability and Delivery Efficacy.

A cationic unimolecular bottlebrush polymer with chemically modified end-groups was synthesized to understand the impact of hydrophilicity on colloidal stability, nucleic acid delivery performance, and toxicity. The bottlebrush polymer template was synthesized using grafting-through techniques and was therefore composed of a polynorbornene backbone with poly(2-(dimethylamino)ethyl methacrylate) side chains with dodecyl trithiocarbonate end-groups. Postpolymerization modification was performed to fully remove the end-groups or install hydroxy and methoxy poly(ethylene glycol) functional groups on the bottlebrush exterior. The bottlebrush family was preformulated with biological payloads of pDNA and CRISPR-Cas9 RNP in both water and PBS to understand binding, aggregation kinetics, cytotoxicity, and delivery efficacy. Increasing end-group hydrophilicity and preformulation of bottleplexes in PBS increased colloidal stability and cellular viability; however, this did not always result in increased transfection efficiency. The bottlebrush family exemplifies how formulation conditions, polymer loading, and end-group functionality of bottlebrushes can be tuned to balance expression with cytotoxicity ratios and result in enhanced overall performance.

Design and synthesis of a shikimoyl-functionalized cationic di-block copolypeptide for cancer cell specific gene transfection.

Targeted and efficient gene delivery systems hold tremendous potential for the improvement of cancer therapy by enabling appropriate modification of biological processes. Herein, we report the design and synthesis of a novel cationic di-block copolypeptide, incorporating homoarginine (HAG) and shikimoyl (LSA) functionalities (HDA-b-PHAGm-b-PLSAn), tailored for enhanced gene transfection specifically in cancer cells. The di-block copolypeptide was synthesized via sequential N-carboxyanhydride (NCA) ring-opening polymerization (ROP) techniques and its physicochemical properties were characterized, including molecular weight, dispersity, secondary conformation, size, morphology, and surface charge. In contrast to the cationic poly-L-homoarginine, we observed a significantly reduced cytotoxic effect of this di-block copolypeptide due to the inclusion of the shikimoyl glyco-polypeptide block, which also added selectivity in internalizing particular cells. This di-block copolypeptide was internalized via mannose-receptor-mediated endocytosis, which was investigated by competitive receptor blocking with mannan. We evaluated the transfection efficiency of the copolypeptide in HEK 293T (noncancerous cells), MDA-MB-231 (breast cancer cells), and RAW 264.7 (dendritic cells) and compared it with commonly employed transfection agents (Lipofectamine). Our findings demonstrate that the homoarginine and shikimoyl-functionalized cationic di-block copolypeptide exhibits potent gene transfection capabilities with minimal cytotoxic effects, particularly in cancer cells, while it is ineffective for normal cells, indicative of its potential as a promising platform for cancer cell-specific gene delivery systems. To evaluate this, we delivered an artificially designed miRNA-plasmid against Hsp90 (amiR-Hsp90) which upon successful transfection depleted the Hsp90 (a chaperone protein responsible for tumour growth) level specifically in cancerous cells and enforced apoptosis. This innovative approach offers a new avenue for the development of targeted therapeutics with an improved efficacy and safety profile in cancer treatment.

Synthesis of Phospho-Polypeptides via Phosphate-Containing N-Carboxyanhydride: Application in Enzyme-Induced Self-Assembly, and Calcium Carbonate Mineralization.

An easy synthetic strategy was developed to synthesize the phosphate-functionalized amino acid N-carboxyanhydride (NCA), using simple primary amine initiators to obtain homo and block phospho-polypeptides with controlled molecular weight and molecular weight distribution. The methodology was extended to the synthesis of the end-functionalized homo polypeptides (15 to 50 repeat unit) and block co-polypeptides with PEG (0.7 K, 2 K, and 5 K) and glycopolypeptide (15-unit mannose glycopolypeptide) as one of the blocks. The deprotected fully water-soluble anionic phosphate-based polypeptides showed pH-dependent helical conformation with a helical content of 20 %, which further changed to ß-sheets upon addition of the enzyme alkaline phosphatase (ALP) due to dephosphorylation. The block co-polypeptide containing PEG as one of the blocks led to its self-assembly into colloidal structures, such as vesicles with a hydrodynamic diameter of ∼250 nm, due to the formation of amphiphilic block co-polymer upon dephosphorylation. The nature of the colloidal structures formed can be temporally controlled by the extent of dephosphorylation. Finally, the phospho-polypeptides serve as a template for the mineralization of calcium carbonate with varying polymorphs and morphologies.

Amphiphilic mannose-6-phosphate glycopolypeptide-based bioactive and responsive self-assembled nanostructures for controlled and targeted lysosomal cargo delivery.

Receptors of carbohydrate mannose-6-phosphate (M6P) are overexpressed in specific cancer cells (such as breast cancer) and are also involved in the trafficking of mannose-6-phosphate labeled proteins exclusively onto lysosomes via cell surface M6P receptor (CI-MPR) mediated endocytosis. Herein, for the first time, mannose-6-phosphate glycopolypeptide (M6PGP)-based bioactive and stimuli-responsive nanocarriers are reported. They are selectively taken up via receptor-mediated endocytosis, and trafficked to lysosomes where they are subsequently degraded by pH or enzymes, leading to the release of the cargo inside the lysosomes. Two different amphiphilic M6P block copolymers M6PGP15-APPO44 and M6PGP15-(PCL25)2 were synthesized by click reaction of the alkyne end-functionalized M6PGP15 with pH-responsive biocompatible azide end-functionalized acetal PPO and azide end-functionalized branched PCL, respectively. In water, the amphiphilic M6P-glycopolypeptide block copolymers self-assembled into micellar nanostructures, as was evidenced by DLS, TEM, AFM, and fluorescence spectroscopy techniques. These micellar systems were competent to encapsulate the hydrophobic dye rhodamine-B-octadecyl ester, which was used as the model drug. They were stable at physiological pH but were found to disassemble at acidic pH (for M6PGP15-APPO44) or in the presence of esterase (for M6PGP15-(PCL25)2). These M6PGP based micellar nanoparticles can selectively target lysosomes in cancerous cells such as MCF-7 and MDA-MB-231. Finally, we demonstrate the clathrin-mediated endocytic pathway of the native FL-M6PGP polymer and RBOE loaded M6PGP micellar-nanocarriers, and selective trafficking of MCF-7 and MDA-MB-231 breast cancer cell lysosomes, demonstrating their potential applicability toward receptor-mediated lysosomal cargo delivery.