pH-sensitive packaging of cationic particles by an anionic block copolymer shell

Abstract

Cationic non-viral vectors show great potential to introduce genetic material into cells, due to their ability to transport large amounts of genetic material and their high synthetic versatility. However, designing materials that are effective without showing toxic effects or undergoing non-specific interactions when applied systemically remains a challenge. The introduction of shielding polymers such as polyethylene glycol (PEG) can enhance biocompatibility and circulation time, however, often impairs transfection efficiency. Herein, a multicomponent polymer system is introduced, based on cationic and hydrophobic particles (P(nBMA46-co-MMA47-co-DMAEMA90), (PBMD)) with high delivery performance and a pH-responsive block copolymer (poly((N-acryloylmorpholine)-b-(2-(carboxy)ethyl acrylamide)) (P(NAM72-b-CEAm74), PNC)) as shielding system, with PNAM as alternative to PEG. The pH-sensitive polymer design promotes biocompatibility and excellent stability at extracellular conditions (pH 7.4) and also allows endosomal escape and thus high transfection efficiency under acidic conditions. PNC shielded particles are below 200 nm in diameter and showed stable pDNA complexation. Further, interaction with human erythrocytes at extracellular conditions (pH 7.4) was prevented, while acidic conditions (pH 6) enabled membrane leakage. The particles demonstrate transfection in adherent (HEK293T) as well as difficult-to-transfect suspension cells (K-562), with comparable or superior efficiency compared to commercial linear poly(ethylenimine) (LPEI). Besides, the toxicity of PNC-shielded particles was significantly minimized, in particular in K-562 cells and erythrocytes. In addition, a pilot in vivo experiment on bone marrow blood cells of mice that were injected with PNC-shielded particles, revealed slightly enhanced cell transfection in comparison to naked pDNA. This study demonstrates the applicability of cationic hydrophobic polymers for transfection of adherent and suspension cells in culture as well as in vivo by co-formulation with pH-responsive shielding polymers, without substantially compromising transfection performance. Graphical Abstract

Improving Gene Delivery: Synergy between Alkyl Chain Length and Lipoic Acid for PDMAEMA Hydrophobic Copolymers

“…In our previous work, the synergistic effect of copolymerizing nBMA and MMA with DMAEMA also proved advantageous in enhancing transfection. [22,35] Consequently, copolymers P1-P3 were designed and synthesized, incorporating nBMA, EMA, and MMA, respectively, to utilize their performance benefits, while simultaneously investigating the influence of pendant acyclic alkyl chain length on overall performance. On the other hand, polymers P4-P7 were devised as controls to investigate the specific impacts of LAMA (P4 and P5) and MMA (P6 and P7).…”

Optimization of Mixed Micelles Based on Oppositely Charged Block Copolymers by Machine Learning for Application in Gene Delivery

“…Furthermore, it has to be kept in mind that the guanidinium group of GPAm is fully charged independently of the pH‐value due to its high apparent p K a (>12), [ 49 ] while only 45% of the carboxy group of CEAm is charged at pH = 5 and 92% are charged at pH = 7 (apparent p K a ≈ 5.1). [ 56 ] DLS measurements revealed monomodal size distributions for all initially mixed micelles with an excess of Gua (Mix‐19/16, Mix‐25/19, Mix‐27/17, Mix‐19/11, Mix‐30/10) with Z ‐Average values larger than the purely cationic micelles (32–68 nm) and narrow PDIs < 0.25 (Table S5, Supporting Information). Interestingly, Mix‐19/16 revealed the largest Z ‐Average value (68 nm) with the smallest PDI (0.033) of all assemblies, pointing toward the formation of larger morphologies than micelles.…”

“…An optimal amount of cationic charge is required for cellular uptake and intracellular distribution and, thus, high efficiency. [ 56 ] Therefore, the hit mixed micelles and their micelleplexes were characterized by DLS measurements and compared to the Gua 100 homopolymer. Overall, the hit mixed micelles reveal a hydrodynamic size below 40 nm (31–38 nm) with a PDI ≤ 0.3 ( Figure A).…”

Tailoring Gene Transfer Efficacy through the Arrangement of Cationic and Anionic Blocks in Triblock Copolymer Micelles

“…These “stealth” polymers induce a hydration layer, which reduces interactions with serum proteins and increases circulation time in blood . However, the improved biosafety profile is often accompanied by a reduced efficiency, known as the toxicity-efficiency dilemma. , Recent studies show that this dilemma can be circumvented by inserting negatively charged functionalities into the polymeric carrier instead, which partially compensate the positive charges. ,− Anionic groups can be integrated in the form of a polymer either (i) electrostatically as a coating of a cationic polyplex or (ii) by covalent incorporation into a (block) copolymer structure. − The first approach is more straightforward in terms of the synthesis of the polymers, but requires optimization of the formulation conditions (mixing order, ratio of opposite charges). − By contrast, the challenge of the second approach is the controlled synthesis of (block) copolymers containing positively and negatively charged functionalities. To date, only a few studies exist, where anionic functionalities have been covalently incorporated into polymeric micelles for gene delivery. ,, Thus, the potential of anionic charges in nanocarrier systems has not yet been fully exploited due to limited knowledge of advantageous compositions and monomer sequences.…”

“…Ultrapure water was added slowly, followed by dialysis against 20 mM sodium acetate buffer (pH 5), generating micelles with a hydrophobic P­( n BA) core and a hydrophilic shell. Since the guanidinium group is fully charged independent of the used pH-value (apparent p K a > 12), the pH-responsive element at physiological pH is the anionic block containing CEAm and NAM (apparent p K a (PCEAm) ≈ 5.1) . At a pH of 5, only about half of the carboxy groups are charged, while at pH 7.4 almost all are charged (92%).…”