Selectively Fluorinated PAMAM-Arginine Conjugates as Gene Delivery Vectors

: Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging

Synthesis of Amphiphilic Hydantoin-based Universal Peptidomimetics as Antibiotic Agents

Three model hydantoin-based universal peptidomimetics are designed and synthetized. Their preferred amphiphilic -turn conformation was assessed by molecular modeling and NMR experiments, and their antibacterial acivity tasted against Gram positive and Gram negative bacteria strains, demostrating that these compounds could be a captivating class of antibiotic to fight emergent drug resistanc

Synthesis and Conformational Analysis of Hydantoin-Based Universal Peptidomimetics

The synthesis of a collection of enantiomerically pure, systematically substituted hydantoins as structural privileged universal mimetic scaffolds is presented. It relies on a chemoselective condensation/cyclization domino process between isocyanates of quaternary or unsubstituted α-amino esters and N-alkyl aspartic acid diesters followed by standard hydrolysis/coupling reactions with amines, using liquid-liquid acid/base extraction protocols for the purification of the intermediates. Besides the nature of the α carbon on the isocyanate moiety, either a quaternary carbon or a more flexible methylene group, conformational studies in silico (molecular modeling), in solution (NMR, circular dichroism (CD), Fourier transform infrared (FTIR)), and in solid state (X-ray) showed that the presented hydantoin-based peptidomimetics are able to project their substituents in positions superimposable to the side chains of common protein secondary structures such as α-helix and β-turn, being the open α-helix conformation slightly favorable according to molecular modeling, while the closed β-turn conformation preferred in solution and in solid state

The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency

Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity

Synthesis ofN-Glycosyl Conjugates through a Multicomponent Domino Process

The development of efficient methods for the combinatorial synthesis of N-glycosyl conjugates is vital for many fields of modern synthetic organic chemistry. Herein, we report a multicomponent domino process for the regioselective synthesis of a large array N-glycosyl-Asp-urea conjugates, which could be further functionalized in a chemoselective way, starting from easily accessible reactants such as N-glycosylamines, fumaric acid monoesters, azides, and isocyanates. The process occurs under very mild conditions, does not require the use of strong bases/acids or high temperature, and is highly versatile, working efficiently with a range of protecting groups and substituents

Aminoglycosides: From Antibiotics to Building Blocks for the Synthesis and Development of Gene Delivery Vehicles

Aminoglycosides are a class of naturally occurring and semi synthetic antibiotics that have been used for a long time in fighting bacterial infections. Due to acquired antibiotic resistance and inherent toxicity, aminoglycosides have experienced a decrease in interest over time. However, in the last decade, we are seeing a renaissance of aminoglycosides thanks to a better understanding of their chemistry and mode of action, which had led to new trends of application. The purpose of this comprehensive review is to highlight one of these new fields of application: the use of aminoglycosides as building blocks for the development of liposomal and polymeric vectors for gene delivery. The design, synthetic strategies, ability to condensate the genetic material, the efficiency in transfection, and cytotoxicity as well as when available, the antibacterial activity of aminoglycoside-based cationic lipids and polymers are covered and critically analyzed

Solid-Phase Synthesis of Gly-Ψ[CH(CF3)NH]-Peptides

The solid-phase synthesis of Gly-Ψ[CH(CF3)NH]-peptides is presented. In order to achieve this goal, the synthesis of Gly-Ψ[CH(CF3)NH]-dipeptides having the C-terminus unprotected, the N-terminus protected as Fmoc- or Teoc-, and possibly side chain functionalities protected with acid-labile protecting groups has been developed. A selected small library of six peptidomimetics, encompassing analogues of biological relevant peptides, have been obtained in high purity

Enantioseparation of chiral (benzylsulfinyl)benzamide sulfoxides by capillary electrophoresis using cyclodextrins as chiral selectors

Sulfur as a stereogenic center can be found in synthetic compounds and natural products. The current study evaluated the enantioseparation of 16 chiral (benzylsulfinyl)benzamide compounds by capillary electrophoresis using charged cyclodextrins (CDs) as chiral selectors in 50 mM sodium acetate buffer, pH 5.5. The sulfoxides varied in the type and position of the substituent of the benzyl moiety as well as the position and methylation of the amide group. Typically, randomly substituted CDs separated the majority of the model analytes in contrast to single isomer CDs. In case of random substitution, γ-CD derivatives displayed higher resolution ability toward the set of model compounds followed by β-CD and α-CD derivatives. Except for a few examples, the (+)-enantiomer of the analytes migrated before the (-)-isomer irrespective of the type of the CD so that the chiral recognition appeared to be also mostly independent on the structure of the sulfoxides. Evaluation of complexation constants and complex mobilities of selected CD-analyte pairs revealed that the separations were based on the stereoselective complexation by the CD expressed as complexation constants but examples for complex mobilities as the determining factor for the enantiomer migration order were also found. In case of 2-(4-bromobenzylsulfinyl)-N-methyl benzamide in the presence of heptakis(2,3-di-O-methyl-6-O-sulfo)-α-CD reversal of the enantiomer migration order as a function of the CD concentration was observed. Using neutral CD derivatives in the presence of sodium dodecyl sulfate-based micelles at pH 9.0 only few sulfoxides could be enantioseparated