Cationic Poly Amino Acid Research Articles

Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers

The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80–90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for >4h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptides.

Protein-Poly(amino acid) Nanocore-Shell Mediated Synthesis of Branched Gold Nanostructures for Computed Tomographic Imaging and Photothermal Therapy of Cancer.

Anisotropic noble metal nanoparticles especially branched gold nanoparticles with a large absorption cross-section and high molar extinction coefficient have promising applications in biomedical field. However, sophisticated and cumbersome methodologies of synthesis along with toxic precursors pose serious concern for its use. Herein, we report the synthesis of branched gold nanostructures from protein (albumin) nanoparticles by a simple reduction method. Albumin nanoparticles were synthesized by a modified desolvation technique with poly-l-arginine (cationic poly amino acid) substituting the conventional toxic cross-linker, glutaraldehyde. In silico molecular docking was carried out to study the interaction of poly-l-arginine with albumin which revealed its binding to Pocket 1B of the A-chain of albumin. The poly-l-arginine-albumin core-shell nanoparticles of ∼100 nm in size served as a base for attachment of gold ions and its reduction to form 140 nm sized branched gold nanostructures conjugated with glutathione. These gold nanostructures exhibited near-infrared absorption λmax at 800 nm with extreme compatibility toward non cancerous (NIH 3T3), oral epithelial carcinoma (KB) cell lines, and human blood (red blood cells, platelets, and coagulation mechanisms) even up to a high concentration of 250 μg/mL. These structures demonstrated superior computed tomographic (CT) contrast ability and marked photothermal cytotoxicity on KB cells. This study reports for the first time a method to develop blood and cell compatible branched gold nanostructures from protein nanoparticles as a dual CT diagnostic and photothermal therapeutic agent.

Development of a Multi-functional Nano-device for Safe and Effective Gene Delivery to Target Organs.

Nucleic acids are expected as novel effective medicines, although they require a drug delivery system (DDS). Complexes of nucleic acids with cationic liposomes and cationic polymers have been mainly used as DDS for clinical use. However, most cationic complexes have disadvantages such as strong cytotoxicity and low biocompatibility. We previously found that a plasmid DNA (pDNA) complex coated with biodegradable γ-polyglutamic acid (γ-PGA) provided adequate gene expression without cytotoxicity. Based on these results, we developed a new DDS (multi-functional Nano-device) of pDNA using biodegradable polyamino acids. A typical cationic polyamino acid, poly-L-lysine (PLL), was complexed with pDNA. The binary complexes, however, showed low gene expression and high cytotoxicity. Gene expression was enhanced by addition of poly-L-histidine (PLH) to the binary complexes. PLH can increase endosome escape of the complexes by inducing pH-buffering effects. The quaternary complexes (pDNA-PLL-PLH-γ-PGA complexes) exhibited high gene expression and low cytotoxicity. Furthermore, we used dendrigraft poly-L-lysine (DGL) instead of PLL and PLH to enhance gene expression. DGL had sterically congested cations and was biodegradable. The ternary complexes (pDNA-DGL-γ-PGA complexes) exhibited markedly high gene expression and low cytotoxicity. The pDNA-DGL-γ-PGA complexes also had high gene expression in the marginal zone (rich dendritic cells) of the spleen after intravenous injection into mice. These results indicate that pDNA-DGL-γ-PGA complexes may be useful as vaccine vectors. Therefore we prepared a novel malaria DNA vaccine using Plasmodium yoelii GPI8p-transamidase-related protein pDNA (PyTAM). The PyTAM-DGL-γ-PGA complexes markedly improved survival time of model mice infected with malaria.

Complexation of DNA with Cationic Polyamino Acid for Biomaterial Purposes

Complexation of DNA with Cationic Polyamino Acid for Biomaterial Purposes

Application of fluorescence polarization to enzyme assays and single nucleotide polymorphism genotyping: some recent developments.

This article describes some recent developments in the field of fluorescence polarization (FP) as applied to enzyme assays and single nucleotide polymorphism (SNP) genotyping. First, we present our recent progress on the application of fluorescence polarization to high throughput screening (HTS). We show how the use of a 2-thiopyridinone-based, mixed disulfide biotinylation reagent can shorten the assay time of our recently reported kinase assay method based on thiophosphorylation and biotinylation from several hours to a few minutes. We also summarize our recent findings on a new approach for HTS of kinases, proteases and phosphatases based on the use of a cationic poly-amino acid such as polyarginine. We show how the careful selection of the polyarginine concentration and the ionic strength of the solution during the FP measurement allow one to expand the range of substrates that can be assayed. Both of these methods are valuable additions to the existing techniques for HTS. Most importantly, both of these methods can be applied to the assay of kinases without the need for any antibodies. In the area of genomics, we present some results from our studies on a new single nucleotide polymorphism typing approach based on the polymerase catalyzed extension of 3' fluorescein labeled primers. Contrary to our initial expectations, we observed that the enzymatic extension of these primers results in a significant decrease of the fluorescence polarization value. Possible explanations of this phenomenon are discussed.

Polylysine and Polyornithine Gene Transfer Complexes: A Study of Complex Stability and Cellular Uptake as a Basis for their Differential in-vitro Transfection Efficiency

Gene transfer vectors formed between the cationic polyamino acid, poly- (l) -ornithine (PLO) and plasmid DNA (pDNA) have demonstrated superior transfection efficiency (up to × 10-fold) compared to equivalent polylysine-based systems in-vitro. The mechanism(s) underlying this observation remains to be elucidated. We previously reported no significant difference in colloidal particle size or zeta potential of polycation/pDNA complexes formed with poly- (l) -lysine (PLL), poly- (d) -lysine (PDL) or PLO. Here we report spectrofluorometric analysis indicating that PLO condenses pDNA at lower charge (+/ −) ratios than PLL or PDL (cf. 0.8:1, 1.2:1 and 1.5:1). Moreover, PLO/pDNA complexes proved more stable to disruption by the polyanions, poly- (l) -aspartic acid (PAA) and heparin. There were no qualitative differences in the ability of the polycations to protect complexed pDNA from enzymatic degradation both in the presence and in the absence of polyanions. The superior transfection efficiency of PLO/pDNA complexes did not appear to be mediated by an increased cellular delivery of pDNA. The data suggests a greater affinity of PLO for pDNA as an important parameter for the observed superior in-vitro transfection efficiency.

Detection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysine

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9–13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.

Evaluation of cationic polymer-coated nanocapsules as ocular drug carriers

New colloidal systems for ocular application were developed and their capacity for increasing the corneal penetration of drugs investigated. Chitosan (CS)-coated and poly- l-lysine (PLL)-coated poly- ϵ-caprolactone (PECL) nanocapsules, were designed based on a strategy that combines the features of PECL nanocapsules as ocular carriers with the advantages of a cationic mucoadhesive coating. Using this approach, an improved interaction of the carrier with the negatively charged corneal epithelium was attempted. The cationic polyaminoacid PLL was directly adsorbed onto preformed PECL nanocapsules whereas the cationic polysaccharide CS was included in the nanocapsules formation medium. The CS and PLL coatings conferred to nanocapsules a high positive surface charge, nevertheless, they did not modify the release profile of the model drug indomethacin from the colloidal system. In vivo studies showed that the systems investigated (uncoated, PLL-coated and CS-coated nanocapsules) increased significantly the concentration of indomethacin in the cornea and aqueous humor with respect of a commercial eye drops. Nevertheless, the ability of PLL-coated and CS-coated nanocapsules of enhancing the ocular penetration of indomethacin was substantially different: the CS coating increased twice, whereas the PLL coating failed to increase the ocular bioavailability of indomethacin when compared to the uncoated particles. Therefore, it is not the positive surface charge but the specific nature of CS that is responsible for the particularly enhanced uptake of the CS-coated nanocapsules. In addition, the PLL-coated and CS-coated nanocapsules displayed a good ocular tolerance. To summarize, the CS-coated nanocapsules represent a useful approach for increasing the ocular bioavailability of drugs.