Structures and Protonation States of Hydrophilic–Cationic Diblock Copolymers and Their Binding with Plasmid DNA

Abstract

Complexation between plasmid DNA (pDNA) and a set of diblock copolymers, each with one cationic block and one hydrophilic, charge-neutral block, is examined. A range of hydrophilic block structures are explored, whereas the cationic block is fixed as poly( N-(2-aminoethyl) methacrylamide) (PAEMA) with a degree of polymerization of 60 ± 3. The hydrophilic blocks include poly(ethylene glycol) (PEG45), poly(oligo(ethylene glycol) methyl ether methacrylate) (P(OEGMA)51), and poly(2-deoxy-2-methacrylamido glucopyranose) (PMAG52). The numbers represent the degrees of polymerization and are chosen such that the diblock contour lengths are similar (37 ± 2 nm). The three diblock copolymers and a homopolycation control, PAEMA59, are compared with respect to their state of dissolution in aqueous environments, as well as their complexation with pDNA. The diblock copolymers are found to partially aggregate as pH increases above 6, whereas each separate block generally dissolves well over a wide pH range. The hydrophilic block proves to be a critical parameter in determining the structures of pDNA-diblock complexes. When the molar ratio of polycation amines to pDNA phosphates (i.e., N/P) is less than 1, a bulkier hydrophilic block leads to larger resulting complexes. As more polycations are added to the system (N/P > 1.5), colloidal stability becomes an important factor, making more water-soluble systems stabilize at smaller sizes. Further, the charge density effect on the binding thermodynamics is elucidated via calorimetric measurements. P(OEGMA)51- b-PAEMA60 exhibits a greater amount of endothermic pDNA binding per charged amine at higher pH, implying that lower cationic charge density promotes more phosphate pairing per amine on average. Also, the colloidal stability and the circular dichroism spectral evolution of the pDNA-PAEMA59 complexes are dependent on pH, showing noticeable differences between pH = 6.0 vs 7.4. To summarize, controlling the solution pH may be crucial in pDNA-polycation complexation, as it impacts polycation solubility, binding characteristics, and the final complex properties. The findings reported herein should aid researchers in drawing more rigorous structure-function correlations in the field of polymeric gene delivery.

Nucleic Acids Based Polyelectrolyte Complexes: Their Complexation Mechanism, Morphology, and Stability

“…When nucleic acids are mixed with hydrophilic-cationic block copolymers, PCMs of very different compositions are formed. − Assuming the hydrophilic block catiomers are well solvated in aqueous solutions, the cationic blocks interact with negatively charged nucleic acids to form pseudoneutral micellar core while keeping the neutral-hydrophilic blocks as the corona (Figure b). The presence of the hydrophilic shell inhibits intercomplex interaction, leading to PCMs of improved stability even under charge-neutral conditions. ,,, In one example, Jung et al investigated complexation of plasmid DNA with various hydrophilic block catiomers containing poly­( N -(2-aminoethyl) methacrylamide) (PAEMA) as the cationic block . When prepared near charge neutral conditions of 1 ≤ N/P (amine/phosphate) ≤ 2, the complexes formed using PAEMA homopolymer showed poor colloidal stability, while the complexes formed using the block copolymers all showed improved stability with no immediate aggregate formation.…”

“…9,24,38,40 In one example, Jung et al investigated complexation of plasmid DNA with various hydrophilic block catiomers containing poly(N-(2-aminoethyl) methacrylamide) (PAEMA) as the cationic block. 123 When prepared near charge neutral conditions of 1 ≤ N/P (amine/phosphate) ≤ 2, the complexes formed using PAEMA homopolymer showed poor colloidal stability, while the complexes formed using the block copolymers all showed improved stability with no immediate aggregate formation. A similar observation of improved complex stability was also reported by Haladjova et al 9 In their study, short (∼113 bp) and long (∼2000 bp) dsDNA were complexed with either PVBTMAC homopolymer or PVBTMAC-b-POEGMA block copolymers.…”

Nucleic Acid Delivery from Granular Hydrogels

“…We used the common linear poly(ethylene imine) (PEI) polymer to condense plasmid DNA, as these particles are known to be highly charged, unstable at physiological conditions and at concentrations higher than 0.1 µg µL −1 , yet are highly effective at delivering nucleic acids. [ 37,38 ] For in vivo transfection, doses of DNA are generally in the 0.1–1.0 mg kg −1 for IV administration and 0.5–5.0 mg kg −1 for local delivery, such as intramuscular injection. Thus, concentrations above 0.5 µg µL −1 within hydrogels are desirable, which are difficult to achieve with surface‐loading.…”

Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells

“…Our group has studied the effect of DNA size on complexation in a variety of model systems. 43,44 In this work, our goal was to interrogate the role of plasmid size on transfection in two primary human cell types of high interest to cell therapy applications. To this end, we first modeled the transfection efficiency of this functional plasmid payload by engineering both a smaller 4.7 kbp reporter plasmid containing GFP and an identical promoter−reporter sequence that was expanded to 10 kbp in size.…”

“…In this work, we sought to improve our fundamental understanding of polymer-based nucleic acid delivery as a function of plasmid size and to develop improved strategies for the delivery of functional large plasmids that encode CRISPR-Cas9 with primary human cells used directly in treatment applications. Our group has studied the effect of DNA size on complexation in a variety of model systems. , In this work, our goal was to interrogate the role of plasmid size on transfection in two primary human cell types of high interest to cell therapy applications. To this end, we first modeled the transfection efficiency of this functional plasmid payload by engineering both a smaller 4.7 kbp reporter plasmid containing GFP and an identical promoter–reporter sequence that was expanded to 10 kbp in size.…”