Cationic Polyaspartamide Research Articles

Hydrophobicity Tuning of Cationic Polyaspartamide Derivatives for Enhanced Antisense Oligonucleotide Delivery.

Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.

The interaction between mucin and poly(amino acid)s with controlled cationic group content in bulk phase and in thin layers

The type and concentration of charged groups in polymers have a key role in mucoadhesive interactions. A series of cationic poly(amino acid)s with different charge densities was designed to unravel the correlation between chemical structure and mucin-polymer interactions. Colloidal interactions between the mucin protein and synthetic polyaspartamides were tested by dynamic light scattering, zeta potential measurements and turbidimetric titration as a function of polymer-to-mucin mass ratio. The mucoadhesive interactions displayed a strongly non-linear change with polymer composition. The attractive interactions between mucin and the polyaspartamides with at least 50 % cationic groups caused increased light scattering of dispersions due to the aggregation of mucin particles upon their charge reversal. Interactions were further analysed in a thin mucin layer to model life-like situations using a quartz crystal microbalance (QCM) in flow mode. Results pointed out that the fully cationic polyaspartamide is not necessarily superior to derivatives with lower cationic group content. The maximum of adsorbed mass of polymers on mucin was experienced at medium cationic group contents. This emphasizes the relevance of cationic polyaspartamides as mucoadhesive excipients due to their multiple functionalities and the possibility of fine-tuning their interactions with mucin via straightforward chemical steps.

Nucleic Acid Delivery across Biological Barriers

One of the current critical issues in nucleic acid delivery is the efficient mRNA delivery into target cells, directed toward clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genome editing. To this end, we have developed a variety of cationic polyaspartamide derivatives with varying side chain structures because they can form nanocomplexes, termed polyplexes, with mRNA through electrostatic interactions. Interestingly, the delivery functions were highly affected by the chemical structures of the polyaspartamide side chains. Therefore, we review our previous research and provide a rationale for designing polypeptides for mRNA delivery.

Cell-Modulating Effect of Poly(Aspartic Acid) and Its Complex with Cationic Polyaspartamide

In this communication, poly(aspartic acid) (pAsp), its fluorescently labeled conjugate with lucifer yellow ethylenediamine (pAsp–LY), and 2,2-dimethyl-1,3-propanediamine-derived cationic polyaspartamide (pDmpa) were synthesized by liquid-phase method. Octamer of L-aspartic acid (L-Asp8) was also obtained by solid-phase synthesis. pDmpa interacted with both pAsp and model plasmid DNA to form compact nanosized interpolymer complexes, which showed different colloidal properties. Effective cellular uptake of pAsp–LY by NIH 3T3 fibroblasts was detected by confocal microscopy. Pre-complexation of pAsp–LY with pDmpa did not noticeably increase intracellular accumulation of the conjugate. Both pAsp and L-Asp8 were found to increase viability of murine 3T3 cells and human skin fibroblasts at μg/mL concentrations according to the MTT assay (24 h). This effect was observed along with moderate prooxidant activity of the peptides in the cells according to the DCFDA fluorescence assay. The results suggest that aspartic acid-based peptides per se are capable of penetrating mammalian cells and affecting their metabolic activity. The peptides can be complexed with their cationic derivative, i.e., pDmpa polyaspartamide, to develop nanosized formulations of pAsp and its conjugates.

Biodegradable Supramolecular Materials Based on Cationic Polyaspartamides and Pillar[5]arene for Targeting Gram‐Positive Bacteria and Mitigating Antimicrobial Resistance

AbstractThe increasing number of infections caused by pathogenic bacteria has severely affected human society, for instance, numerous deaths are from Gram‐positive methicillin‐resistant Staphylococcus aureus (MRSA) each year. In this work, four biodegradable antibacterial polymer materials based on cationic polyaspartamide derivatives with different lengths of side chains are synthesized through the ring‐opening polymerization of β‐benzyl‐l‐aspartate N‐carboxy anhydride, followed by an aminolysis reaction and subsequent methylation reaction. The cationic quaternary ammonium groups contribute to the insertion of the catiomers into the negatively charged bacterial membranes, which leads to membranolysis, the leakage of bacterial content, and the death of pathogens. Except for wiping out MRSA readily, the biodegradable polymers possessing alterable antibacterial potency can minimize the possibility of microbial resistance and mitigate drug accumulation by virtue of their cleavable backbone. To manipulate the poor biocompatibility of these polycations, carboxylatopillar[5]arene (CP[5]A) is introduced to the polymeric antibacterial catiomers through the supramolecular host–guest approach to obtain novel antibacterial materials with pH‐sensitive characteristics (with CP[5]A departure from cationic quaternary ammonium compounds under acid conditions) and selective targeting of Gram‐positive bacteria. Finally, the facile and robust antibacterial system is successfully applied to in vivo MRSA‐infected wound healing, providing a significant reference for the construction of advanced antibacterial biomaterials.

Tunable nonenzymatic degradability of N-substituted polyaspartamide main chain by amine protonation and alkyl spacer length in side chains for enhanced messenger RNA transfection efficiency

ABSTRACT Degradability of polycations under physiological conditions is an attractive feature for their use in biomedical applications, such as the delivery of nucleic acids. This study aims to design polycations with tunable nonenzymatic degradability. A series of cationic N-substituted polyaspartamides were prepared to possess primary amine via various lengths of alkyl spacers in side chains. The degradation rate of each polyaspartamide derivative was determined by size exclusion chromatography under different pH conditions. The N-substituted polyaspartamide containing a 2-aminoethyl moiety in the side chain (PAsp(AE)) showed considerable degradability under physiological conditions (pH 7.4, 37 °C). In contrast, the N-substituted polyaspartamides bearing a longer alkyl spacer in the side chain, i.e. the 3-aminopropyl (PAsp(AP)) and 4-aminobutyl moieties (PAsp(AB)), more strongly suppressed degradation. Further, a positive correlation was observed between the degradation rate of N-substituted polyaspartamides and a deprotonation degree of primary amines in their side chains. Therefore, we conclude that the deprotonated primary amine in the side chain of N-substituted polyaspartamides can induce the degradation of the main chain through the activation of amide nitrogen in the side chain. When N-substituted polyaspartamides were utilized as a messenger RNA (mRNA) delivery vehicle via formation of polyion complexes (PICs), degradable PAsp(AE) elicited significantly higher mRNA expression efficiency in cultured cells compared to PAsp(AP) and PAsp(AB). The higher efficiency of PAsp(AE) might be due to the facilitated destabilization of PICs within the cells, directed toward mRNA release. Additionally, degradation of PAsp(AE) considerably reduced its cytotoxicity. Thus, our study highlights a useful design of well-defined cationic poly(amino acid)s with tunable nonenzymatic degradability.

Self-Assembly of siRNA/PEG- b-Catiomer at Integer Molar Ratio into 100 nm-Sized Vesicular Polyion Complexes (siRNAsomes) for RNAi and Codelivery of Cargo Macromolecules.

Vesicular polyion complexes (PICs) were fabricated through self-assembly of rigid cylindrical molecules, small interfering RNAs (siRNAs), with flexible block catiomers of poly(ethylene glycol) (2 kDa) and cationic polyaspartamide derivative (70 units) bearing a 5-aminopentyl side chain. 100 nm-sized siRNA-assembled vesicular PICs, termed siRNAsomes, were fabricated in specific mixing ranges between siRNA and block catiomer. The siRNAsome membrane was revealed to consist of PIC units fulfilling a simple molar ratio (1:2 or 2:3) of block catiomer and siRNA. These ratios correspond to the minimal integer molar ratio to maximally compensate the charge imbalance of PIC, because the numbers of charges per block catiomer and siRNA are +70 and -40, respectively. Accordingly, the ζ-potentials of siRNAsomes prepared at 1:2 and 2:3 were negative and positive, respectively. Cross-section transmission electron microscopic observation clarified that the membrane thicknesses of 1:2 and 2:3 siRNAsomes were 11.0 and 17.2 nm, respectively. Considering that a calculated long-axial length of siRNA is 5.9 nm, these thickness values correspond to the membrane models of two (11.8 nm) and three (17.7 nm) tandemly aligned siRNAs associating with one and two block catiomers, respectively. For biological application, siRNAsomes were stabilized through membrane-cross-linking with glutaraldehyde. The positively charged and cross-linked siRNAsome facilitated siRNA internalization into cultured cancer cells, eliciting significant gene silencing with negligible cytotoxicity. The siRNAsome stably encapsulated dextran as a model cargo macromolecule in the cavity by simple vortex mixing. Confocal laser scanning microscopic observation displayed that both of the payloads were internalized together into cultured cells. These results demonstrate the potential of siRNAsomes as a versatile platform for codelivery of siRNA with other cargo macromolecules.

Effect of side groups on the properties of cationic polyaspartamides

Polyaspartamides with dialkylaminoalkyl and short chain alkyl side groups were synthesized and characterized in order to prepare polymer films. Their structure was confirmed by nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FTIR) spectroscopy and their thermal decomposition temperature (Td) was determined by thermogravimetric analysis. The composition of the polymers was adjusted to obtain polyaspartamides with glass transition temperatures (Tg) at around room temperature and the relationship between the structure and the properties was examined. The dissolution profile of polymer films made of polyaspartamides was measured with the help of fluorescent marking to show that dissolution rate of the films depends on the pH and can be controlled by the composition of the polymers.

Structure–biocompatibility and transfection activity relationships of cationic polyaspartamides with (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups

A series of 14 cationic derivatives of poly(aspartic acid) i.e. cationic polyaspartamides with different (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups was synthesized by nucleophilic addition on polysuccinimide. The resulting polyaspartamides have moderate amphiphilic properties. Relationships between the structure and ratio of side groups and in vitro properties of polyaspartamides, including their cytotoxic and membrane-damaging activity towards human cell lines, primary skin fibroblasts and erythrocytes, were established and discussed. Cationic polyaspartamides vary in their DNA-binding, condensing and nuclease-protecting characteristics depending on the concentration ratio of (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups. Effective cell transfection was achieved upon polyaspartamide-mediated plasmid DNA delivery in serum-free medium in the presence of chloroquine. Effect of serum proteins adsorption onto polyaspartamide based polyplexes, and the role of concentration of polyplexes in culture medium in their colloidal stability and transfection process were demonstrated. Synthesized polyaspartamides are biocompatible and long-acting gene carriers, which are applied to cells after dilution and without washing, thus providing transfection level comparable to that of commercial transfection reagent.

Fine‐Tuning of Charge‐Conversion Polymer Structure for Efficient Endosomal Escape of siRNA‐Loaded Calcium Phosphate Hybrid Micelles

For efficient delivery of siRNA into the cytoplasm, a smart block copolymer of poly(ethylene glycol) and charge-conversion polymer (PEG-CCP) is developed by introducing 2-propionic-3-methylmaleic (PMM) amide as an anionic protective group into side chains of an endosome-disrupting cationic polyaspartamide derivative. The PMM amide moiety is highly susceptible to acid hydrolysis, generating the parent cationic polyaspartamide derivative at endosomal acidic pH 5.5 more rapidly than a previously synthesized cis-aconitic (ACO) amide control. The PMM-based polymer is successfully integrated into a calcium phosphate (CaP) nanoparticle with siRNA, constructing PEGylated hybrid micelles (PMM micelles) having a sub-100 nm size at extracellular neutral pH 7.4. Ultimately, PMM micelles achieve the significantly higher gene silencing efficiency in cultured cancer cells, compared to ACO control micelles, probably due to the efficient endosomal escape of the PMM micelles. Thus, it is demonstrated that fine-tuning of acid-labile structures in CCP improves the delivery performance of siRNA-loaded nanocarriers.

N-acetylglucosamine-conjugated block copolymer consisting of poly(ethylene oxide) and cationic polyaspartamide as a gene carrier for targeting vimentin-expressing cells

Gene therapy is not successful due to lack of safe gene delivery vector, low transfection efficiency and inability to target the particular cells. Here, we synthesized a biocompatible block copolymer (abbreviated as PASPG) which consists of cationic poly[(aspartamide)(spermine)] for complexation with DNA and enhancing transfection efficiency due to buffering ability of spermine, poly(ethylene oxide)(PEO) for stability after systemic administration of the gene and N-acetylglucosamine (GlcNAc) as a specific ligand to target vimentin-expressing cells. Primarily, PASPG showed efficient complexation with DNA. Cell viability assay demonstrated that PASPG had low toxicity compared to polyethylenimine 25K. Furthermore, PASPG showed higher transfection efficiency in vimentin-expressing cells than vimentin-deficient ones due to the recognition of GlcNAc in the polymeric gene carrier by vimentin in the cells for the receptor-mediated endocytosis of PASPG. Favorably, the serum had no effect on transfection efficiency of PASPG due to the presence of hydrophilic PEO in the block copolymer. This study reveals that GlcNAc-coupled biocompatible block copolymer can specifically deliver gene to vimentin-expressing cells.

Enhanced stability and gene silencing ability of siRNA-loaded polyion complexes formulated from polyaspartamide derivatives with a repetitive array of amino groups in the side chain

The delivery of siRNA therapeutics owes its success to the development of carrier systems with high efficacy and minimum toxicity. Here, cationic polyaspartamide derivatives with a regulated number and spacing of positively charged amino groups in the side chain were prepared from a single platform polymer of poly(β-benzyl l-aspartate) to assess their availability as siRNA carriers through polyion complex (PIC) formation. These polymers have 1,2-diaminoethane, 1,3-diaminopropane, and N,N’-bis(2-aminoethyl)-1,2-diaminoethane moieties in the side chain, and are termed as PAsp(DET), PAsp(DPT), and PAsp(TEP), respectively. siRNA-loaded PICs stable in serum-containing media were formed from PAsp(TEP) and PAsp(DPT) with two positive charges in the side chain at pH 7.4, whereas no such stable PIC was obtained from PAsp(DET) with only a single charge in the side chain, suggesting facilitated multivalent interactions with siRNA molecules to increase the PIC stability. The PAsp(DPT) and PAsp(TEP) PICs stable in the serum-containing media underwent an appreciably enhanced uptake into cultured cells through endocytosis, and subsequently exerted effective endosomal escape for the significant silencing of target gene expression. Notably, PAsp(TEP) PIC displayed negligible cytotoxicity in sharp contrast to the highly toxic feature of PAsp(DPT) PIC. This cytotoxicity is apparently correlated with the minimal damage to the cytoplasmic membrane of cells exposed to PAsp(TEP) at pH 7.4 evidenced from the fluorescent dye (YO-PRO-1) permeation assay. There was, in turn, a significant increase in YO-PRO-1 permeability at endosomal pH of 5.5 for PAsp(TEP)-exposed cells, indicating that PAsp(TEP) exerts membrane damage in a pH-selective manner, and eventually facilitates the translocation of siRNA-loaded PIC from the acidic endosomal compartment into the cytoplasm for effective gene silencing without any severe toxicity at physiological conditions. This acidic pH modulated enhancement in membrane damage of PAsp(TEP) may be explained by an increased protonation of the arrayed amino groups in the side chain that strongly perturb the endosomal membrane integrity. Eventually, PAsp(TEP) with a side chain array of pH-sensitive amino groups was demonstrated to be a promising component for constructing siRNA carriers exerting effective gene silencing in a less toxic context.

PEG-detachable cationic polyaspartamide derivatives bearing stearoyl moieties for systemic siRNA delivery toward subcutaneous BxPC3 pancreatic tumor

For systemic siRNA delivery into tumor tissues, a safe and efficient vehicle is strongly required. Therefore, we designed a block copolymer of detachable poly(ethylene glycol) (PEG) polycation bearing low pKa amines and hydrophobic moieties in the side chain to develop a smart siRNA complex possessing biocompatibility, high complex stability, and endosomal escaping functionality. A disulfide linkage (-SS-) was inserted as a linker between PEG and a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), with a flanking stearoyl moiety, where PAsp(DET) segment provides the excellent ability of endosome destabilization by direct interaction with the membrane. The resulting polymer, stearoyl PEG-SS-PAsp(DET), was confirmed to form the siRNA complex with an environment-responsive PEG palisade, which was detached from the complex surface under reductive conditions mimicking tumor tissues and cytoplasm because of the disulfide cleavage. The smart siRNA complex allowed significant gene silencing against cultured pancreatic cancer cells without considerable cytotoxicity and erythrocyte disruption, whereas such significant gene silencing was not observed in a control siRNA complex without the disulfide linkage. This enhanced gene silencing activity might be because of the enhanced cellular uptake and subsequent translocation of siRNA into cytoplasm facilitated by PEG detachment around and/or in the cancer cells. Further, intravital real-time confocal laser scanning microscopic observation revealed the effect of hydrophobic stearoyl modification on the stabilization of the siRNA complex for longevity in the blood. Significant in vivo gene silencing of the smart siRNA complex was achieved by systemic administration of vascular endothelial growth factor siRNA in a mouse model bearing a subcutaneous pancreatic tumor, leading to 40% regression in tumor growth. These results demonstrate the strong potential of stearoyl PEG-SS-PAsp(DET) as a vehicle for systemic delivery of siRNA in cancer therapy.

Introduction of stearoyl moieties into a biocompatible cationic polyaspartamide derivative, PAsp(DET), with endosomal escaping function for enhanced siRNA-mediated gene knockdown

Applications of siRNA for cancer therapy have been spotlighted in recent years, but the rational design of efficient siRNA delivery carriers is still controversial, especially because of possible toxicity of the carrier components. Previously, a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), was reported to exert high transfection efficacy for plasmid DNA with negligible cytotoxicity. However, its direct application for siRNA delivery was fairly limited due to the unstable polymer/siRNA complex formation. In this study, to overcome such instability, stearic acid as a hydrophobic moiety was conjugated to the side chain of PAsp(DET) with various substitution degrees. The stearoyl introduction contributed not only to siRNA complex formation with higher association numbers but also to complex stabilization. The obtained stearoyl PAsp(DET)/siRNA complex significantly accomplished more efficient endogenous gene (BCL-2 and VEGF) knockdown in vitro against the human pancreatic adenocarcinoma (Panc-1) cells than did the unmodified PAsp(DET) complex and commercially available reagents, probably due to the facilitated cellular internalization. This finding suggests that the hydrophobic PAsp(DET)-mediated siRNA delivery is a promising platform for in vivo siRNA delivery.

Synthesis and in vitro Property Study of Polyaspartamides

AbstractCationic polyaspartamides including poly‐α,β‐[N′‐(2‐aminoethy1)‐L‐aspartamide] (PAEA), poly‐α,β‐[N′‐(4‐aminobutyl)‐L‐aspartamide] (PABA), poly‐α,β‐[N′‐(6‐aminohexyl)‐L‐aspartamide] (PAHA), poly‐α,β‐[N′‐(5‐amino‐3‐azapentyl)‐L‐aspartamide] (PAAPA) and poly‐α,β‐[N′‐(8‐amino‐3,6‐diazaoctyl)‐L‐aspartamide] (PADAOA) were synthesized from polysuccinimide. Their properties were evaluated by 1H NMR, IR, GPC, fluorescence measurement and in vitro cytotoxicity assays. The molecular weights per primary amine charge group of PAEA(1) (Mn=2229), PAAPA and PADAOA are 212, 279, and 226. Polyaspartamides including PAEA(1), PAAPA, PADAOA and low molecular weight PAHA are markedly less toxic than poly(ethyleneimine) and poly(L‐lysine), however, PABA and higher molecular weight PAHA are slightly less toxic than poly(L‐lysine). Cell cytotoxicity of PAHA was seen to decrease with increasing molecular weight of PAHA, due to water solubility reduction. The negatively charged plasmid DNA has been found to be completely neutralized and complexed by the cationic polyaspartamides at an N/P ratio of 5:1 to 10:1, forming self‐assembled polyplexes via ionic interactions. These polyaspartamide/DNA complexes possess stable zeta potentials and mean particle diameters of about 180 nm for PAEA (1)/DNA and PAAPA/DNA complexes and 280 nm for PADAOA/DNA complexes.

Study of the quantitative aminolysis reaction of poly(β-benzyl l-aspartate) (PBLA) as a platform polymer for functionality materials

A facile and quantitative aminolysis of poly(β-benzyl l-aspartate) (PBLA) as well as the solution properties of the prepared cationic polyaspartamide were investigated in this study. The reaction was found to proceed in good yield without undesired side reactions via the formation of a succinimide intermediate in the polymer backbone, which was efficiently converted to polyaspartamide accompanying the α,β isomerization of the main chain. The polarity of solvents and the secondary structure of the polymer strand were closely related to each other in terms of reactivity and stereoselectivity. The aminolysis of PBLA treated with one equivalent amine against benzyl ester groups resulted in the complete conversion at 35 °C in random-coil solvents within 1 h. The racemization that accompanied this reaction was observed in random-coil solvents, but was efficiently suppressed in helicogenic solvents, with 95% of the optical purity maintained in CH 2Cl 2. In addition, the quantitative introduction of N, N-diisopropylethylenediamine (DIP) led to the formation of cationic polyaspartamide, poly[ N-( N′, N′-diisopropylaminoethyl)aspartamide] (PAsp(DIP)), which showed pH and thermo-sensitivities in aqueous media. This systematic investigation of the aminolysis of PBLA with DIP demonstrates the feasibility of a PBLA-aminolysis system for designing functionalized polyaspartamides which can be useful as biomaterials.

Preparation and Properties of Novel Biodegradable Hydrogel based on Cationic Polyaspartamide Derivative

Novel copolymers consisting of poly(2-hydroxyethyl aspartamide-co-N,N'-dimethyl-1,3-propane aspartamide) (PHEA-DPA) were prepared from polysuccinimide (PSI), which is the thermal polycondensation product of aspartic acid, via a ring-opening reaction with N,N'-dimethyl-1,3-propane diamine (DPA) and ethanolamine. The prepared water-soluble copolymer was then crosslinked by reacting it with hexamethylene diisocyanate to provide the corresponding gel. The swelling behavior and morphology of the crosslinked hydrogels were investigated. The degree of swelling decreased with increasing crosslinking reagent due to the higher crosslinking density. It was also confirmed that the swelling property is affected by pH. At low pH (< pH 4), swelling is increased due to the ionization of DPA with a tertiary amine moiety. In addition, a reversible swelling and de-swelling behavior was demonstrated by adjusting the pH of the solution. The prepared hydrogels showed a well-interconnected microporous structure with regular 5-20 <TEX>$\mu$</TEX>m sized pores.

Novel cationic polyaspartamide with covalently linked carboxypropyl-trimethyl ammonium chloride as a candidate vector for gene delivery

The non-viral gene vector properties of a protein-like polymer, the α,β-poly( N-2-hydroxyethyl)- d, l-aspartamide (PHEA) were investigated after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing cationic groups, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with hydrazide pendant groups by reaction with hydrazine monohydrate (HYD), obtaining the polyhydrazide α,β-poly( N-2-hydroxyethyl/carbazate)- d, l-aspartamide (PHEA–HYD). In this paper we reported that polymer functionalization degree can be easily modulated by varying reaction conditions, so allowing us to produce two PHEA derivatives at different molar percentage of hydrazide groups. Subsequently, condensation reaction of PHEA–HYD copolymers with CPTA yielded α,β-poly( N-2-hydroxyethyl)- N-carbazate[ N′-(3-trimethylammonium chloride)propylhydrazide]- d, l-aspartamide (PHEA–HYD–CPTA) polycation derivatives. In vitro studies were carried out to evaluate polycations ability to complex DNA and to protect it from nuclease degradation. Obtained results demonstrated the good efficiency of our new PHEA-polycations derivatives, PHEA–HYD–CPTA, to complex and condense genomic material even at very low polycation/DNA weight ratio.