This article presents the synthesis, self-assembly, and biological activity as transfection agents for pDNA, siRNA, and mRNA of novel pyridinium pseudogemini surfactants, interfacially engineered from the most efficient gemini surfactants and lipids generated in our amphiphile research program. Formulation of novel amphiphiles in water revealed supramolecular properties very similar to those of gemini surfactants, despite their lipidlike charge/mass ratio. This dual character was found also to enhance endosomal escape and significantly increase the transfection efficiency. We were also successful in identifying the parameters governing the efficient delivery of pDNA, siRNA, and mRNA, drawing valuable structure–activity and structure–property relationships for each nucleic acid type, and establishing DNA/siRNA/mRNA comparisons. Several supramolecular complexes identified in this study proved to be extremely efficient nucleic acid delivery systems, displaying excellent serum stability and tissue penetration in three-dimensional organoids.Keywords: amphiphile; DNA; mRNA; pyridinium; siRNA; transfection;

Mixtures of highly curved pyridinium-decorated Au nanoparticles and standard pyridinium cationic lipids efficiently and synergetically transfected DNA in vitro, while displaying an excellent cytotoxic profile.

A series of aromatic/heterocyclic bis-sulfonamides were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores, coupled with either ethylene glycol oligomeric or polymeric diamines to yield bis-sulfonamides with short or long (polymeric) linkers. Testing of novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity in a detailed structure–activity relationship study. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25) able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment.

Self-assembled synthetic gene delivery systems represent the bottom-up approach to gene delivery and gene silencing, in which scientists are designing novel cationic and procationic amphiphiles that can pack, transport, and deliver nucleic acids to various targets in the body in a controlled manner. These supramolecular assemblies are safer than viruses, but they are lagging behind them in efficiency. We are presenting recent progress that has narrowed this gap through better understanding the delivery barriers and incorporation of this knowledge in the design of novel synthetic amphiphiles, formulations, and revolutionary screening and optimization processes. Structure–properties and structure–activity relationships were drawn within each amphiphile class, presenting the cellular and animal models used to generate them. We are also revealing pertinent in vitro/in vivo correlations that emphasize promising amphiphiles and successful formulation optimization efforts for efficient in vivo nucleic acid delivery, together with main organ targets and diseases treatable with these revolutionary technologies.

A series of ethylene bis-imidazoles was synthesized via a novel microwave-mediated synthesis. Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against CA VA and CA VII, also displaying excellent selectivity against other CA isozymes present in this organ.

The study presents the effects of blending a cationic gemini surfactant into cationic lipid bilayers and its impact on the plasmid DNA compaction and delivery process. Using nanoDSC, dynamic light scattering, zeta potential, and electrophoretic mobility measurements, together with transfection (2D- and 3D-) and viability assays, we identified the main physicochemical parameters of the lipid bilayers, liposomes, and lipoplexes that are affected by the gemini surfactant addition. We also correlated the cationic bilayer composition with the dynamics of the DNA compaction process and with transfection efficiency, cytotoxicity, and the internalization mechanism of the resultant nucleic acid complexes. We found that the blending of gemini surfactant into the cationic bilayers fluidized the supramolecular assemblies, reduced the amount of positive charge required to fully compact the plasmid DNA and, in certain cases, changed the internalization mechanism of the lipoplexes. The transfection efficiency of select ternary lipoplexes derived from cationic gemini surfactants and lipids was several times superior to the transfection efficiency of corresponding binary lipoplexes, also surpassing standard transfection systems. The overall impact of gemini surfactants into the formation and dynamic of cationic bilayers was found to depend heavily on the presence of colipids, their nature, and amount present in lipoplexes. The study confirmed the possibility of combining the specific properties of pyridinium gemini surfactants and cationic lipids synergistically to obtain efficient synthetic transfection systems with negligible cytotoxicity useful for therapeutic gene delivery.Keywords: cationic lipid; gemini surfactant; lipoplex; pyridinium; synergy; transfection;

We are reporting a new set of biocompatible, low-toxicity pyridinium cationic lipids based on a dopamine backbone on which hydrophobic alkyl tails are attached via an ether linkage. Due to their optimized hydrophilic/hydrophobic interface and packing parameter, the new lipids are able to strongly self-assemble either alone or when coformulated with colipids DOPE or cholesterol. The supra-molecular assemblies generated with the novel pyridinium amphiphiles were characterized in bulk and in solution via a combination of techniques including DSC, nanoDSC, SAXS, TOPM, TEM, DLS, zeta potential, and electrophoretic mobility measurements. These cationic bilayers can efficiently condense and deliver DNA to a large variety of cell lines, as proven by our self-assembling/physicochemical/biological correlation study. Using the luciferase reporter gene plasmid, we have also conducted a comprehensive structure–activity relationship study, which identified the best structural parameters and formulations for efficient and nontoxic gene delivery. Several formulations greatly surpassed established transfection systems with proved in vitro and in vivo efficiency, being able to transfect a large variety of malignant cells even in the presence of elevated levels of serum. The most efficient formulation was able to transfect selectively primary rat dopaminergic neurons harvested from nucleus accumbens, and neurons from the frontal cortex, a premise that recommends these synthetic vectors for future in vivo delivery studies for neuronal reprogramming.

A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested—valuable lead compounds for physiology and pathology studies involving these isozymes.

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.

A series of ethylene bis-imidazoles was synthesized via a novel microwave-mediated synthesis. Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against CA VA and CA VII, also displaying excellent selectivity against other CA isozymes present in this organ.

A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested—valuable lead compounds for physiology and pathology studies involving these isozymes.

Mixtures of highly curved pyridinium-decorated Au nanoparticles and standard pyridinium cationic lipids efficiently and synergetically transfected DNA in vitro, while displaying an excellent cytotoxic profile.