Amino Acid–Substituted Dextran‐Based Non‐Viral Vectors for Gene Delivery

Abstract

To form bio‐inspired non‐viral vectors for DNA delivery, the polysaccharide dextran is allowed to react with Boc‐amino protected amino acids glycine, β‐alanine, and L‐lysine activated with 1,1’‐carbonyldiimidazole and subsequent dextran ester deprotection. A library of such dextran esters is made available to investigate the relationship between polymer structure, complex formation, stability, toxicity, and transfection. Only dextran esters of β‐alanine and L‐lysine are able to efficiently interact with DNA as shown by dye exclusion assays, to form nanosized complexes (70–110 nm) with positive zeta potential. With increasing substitution degree and complex charge ratios, the L‐lysine esters accomplish more effective binding and protection of DNA against enzymatic degradation than β‐alanine esters. However, luciferase reporter gene assays reveal higher transfection for β‐alanine than for L‐lysine esters due to a more effective DNA release and better suited buffing area of the amino groups triggering the endosomal release. Conclusively, β‐alanine‐substituted dextran derivatives may serve as promising non‐viral vectors.

The Role of Formamidine Groups in Dextran Based Nonviral Vectors for Gene Delivery on Their Physicochemical and Biological Characteristics

“…In comparable experiments Zink et al. [ 14 ] reported that dextran beta‐alanine esters displayed a protection of pDNA from DNase I only at N/P ratio 40 with a DS ≥ 1.16. The delocalization of the charge density and the bivalent binding behavior of the formamidine substituent seems to support a more efficient pDNA protection.…”

“…DEXfa 0.63 and DEXfa 0.92 complexes demonstrated a notable potential for introducing pDNA into eukaryotic CHO‐K1 cells at N/P ratio 20 and especially at N/P 40 which was comparable to the performance of other cationic dextrans. [ 9,14,33,34 ] In contrast, DEXfa 0.45 was found to be less efficient in transfection even at the highest N/P value of 40. When higher amounts of pDNA (4 vs 8 and 10 µg per well) were subjected to transfection using the best performing DEXfa 0.92 /pDNA complexes with N/P ratio 40, their ability to transfer pDNA could even be increased up to 15‐fold (8 µg pDNA) and 23‐fold (10 µg pDNA) (Figure 4).…”

“…This type of dextran was already successfully used as the basis for gene delivery agents in studies before, especially in combination with different cationic amino acids. [ 14 ] It was also chosen since typical pharmaceutically used dextrans for intravenous applications have molar masses between 40–70 000 g mol −1 with elimination half‐lives between 4–8 h to accomplish adequate retention times. Dextrans of those molar masses can be metabolized by enzymatic hydrolysis in the body and eliminated.…”

“…Additionally, a horizontal gel retardation assay was performed according to Zink et al. [ 14 ] Fifty µL complex sample (1 µg pDNA) were mixed with 5 µL loading buffer (40 × 10 −3 m tris(hydroxymethyl)aminomethane, Tris; 50% glycerol (85%); 1 × 10 −3 m ethylenediaminetetraacetic acid, EDTA; pH 7.4; all Carl Roth GmbH). Subsequently, 10 µL of this dilution were applied on a 1% agarose gel (peqGOLD Universal Agarose, Peqlab Biotechnology GmbH, Radnor, USA) containing ethidium bromide (SERVA Electrophoresis GmbH, Heidelberg, Germany).…”

“…[ 12,13 ] Recently, it was shown that dextran esters substituted with amino acids like beta‐alanine and l ‐lysine accomplished effective binding and protection of DNA against enzymatic degradation. [ 14 ] A luciferase reporter gene assay revealed higher transfection for beta‐alanine than for l ‐lysine esters due to a more effective DNA release and a higher buffering capacity triggering the endosomal release.…”