Validation of the Absorption-Enhancing Ability of Oligoarginines Grafted onto a Backbone of Hyaluronic Acid through Animal Studies from Rodents to Primates.

The purpose of our research is to develop functional additives that enhance mucosal absorption of biologics, such as peptide/protein and antibody drugs, to provide their non-to-poor invasive dosage forms self-managed by patients. Our previous in vivo and in vitro studies demonstrated that the intranasal absorption of biologics in mice was significantly improved when coadministered with oligoarginines anchored chemically to hyaluronic acid via a glycine spacer, presumably through syndecan-4-mediated macropinocytosis under activation by oligoarginines. The present mouse experiments first revealed that diglycine-L-tetraarginine-linked hyaluronic acid significantly enhanced the intranasal absorption of sulpiride, which is a poor-absorptive organic compound with a low molecular weight. However, similar enhancement was not observed for levofloxacin, which has a similarly low molecular weight but is a well-absorptive organic compound, probably because its absorption was mostly dominated by passive diffusion. The subsequent monkey experiments revealed that there was no species difference in the absorption-enhancing ability of diglycine-L-tetraarginine-linked hyaluronic acid for not only organic compounds but also biologics. This was presumably because the expression levels of endocytosis-associated membrane proteins on the nasal mucosa in monkeys were almost equivalent to those in mice, and poorly membrane-permeable/membrane-impermeable drugs were mainly absorbed via syndecan-4-mediated macropinocytosis, regardless of animal species. Drug concentrations in the brain assessed in mice and monkeys and those in the cerebral spinal fluids (CSFs) assessed in monkeys indicated that drugs would be delivered from the systemic circulation to the central nervous system by crossing the blood-brain and the blood-CSF barriers under coadministration with the hyaluronic acid derivative. In line with our original hypothesis, this new set of data supported that our oligoarginine-linked hyaluronic acid would locally perform on the mucosal surface and enhance the membrane permeation of drugs under its colocalization.

Effective mRNA Delivery by Condensation with Cationic Nanogels Incorporated into Liposomes.

The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an ∼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.

L17ER4: A cell-permeable attenuated cationic amphiphilic lytic peptide.

We have developed a series of attenuated cationic amphiphilic lytic (ACAL) peptides that can efficiently bring immunoglobulin G (IgG) and other functional proteins into cells. Delivery is generally achieved through the coadministration of ACAL peptides with cargo proteins. However, conjugation of ACAL peptides with cargos may be a promising approach for in vivo application to link in vivo outcomes of ACAL peptides and cargos. This study describes the creation of a new cell-permeable ACAL peptide, L17ER4. L17E is an optimized prototype of ACAL peptides previously developed in our laboratory for efficient delivery of IgGs into cells. Delivery was improved by functionalizing L17E with a tetra-arginine (R4) tag. Compared to the use of R8, a representative cell-penetrating peptide with high intracellular delivery efficacy, conjugation with L17ER4 afforded approximately four-fold higher cellular uptake of model small-molecule cargos (fluorescein isothiocyanate and HiBiT peptide). L17ER4 was also able to deliver proteins to cells. Fused with L17ER4, Cre recombinase was delivered into cells. Intracerebroventricular injection of Cre-L17ER4 into green red reporter mice, R26GRR, led to significant in vivo gene recombination in ependymal cells, suggesting that L17ER4 may be used as a cell-penetrating peptide for delivering protein therapeutics into cells in vivo.

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

Enhancing the activity of membrane remodeling epsin-peptide by trimerization.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

Biodegradable Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Safe Adjuvant for Mucosal Vaccination.

We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly( N-vinylacetamide- co-acrylic acid) (PNVA- co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA- co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA- co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA- co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery.

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6-14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R6) and nona-arginine (R9)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6-14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.

Elementary processes for the entry of cell-penetrating peptides into lipid bilayer vesicles and bacterial cells.

Cell-penetrating peptides (CPPs) can translocate across the plasma membrane of living eukaryotic cells and enter the cytosol without significantly affecting cell viability. Consequently, CPPs have been used for the intracellular delivery of biological cargo such as proteins and oligonucleotides. However, the mechanisms underlying the translocation of CPPs across the plasma membrane remain unclear. In this mini-review, we summarize the experimental results regarding the entry of CPPs into lipid bilayer vesicles obtained using three methods: the large unilamellar vesicle (LUV) suspension method, the giant unilamellar vesicle (GUV) suspension method, and the single GUV method. The advantages and disadvantages of these methods are also discussed. Experimental results to date clearly indicate that CPPs can translocate across lipid bilayers and enter the vesicle lumen. Three models for the mechanisms and pathways by which CPPs translocate across lipid bilayers are described: (A) through pores induced by CPPs, (B) through transient prepores, and (C) via formation of inverted micelles. Both the pathway of translocation and the efficiency of entry of CPPs depend on the lipid composition of the bilayer and the type of CPP. We also describe the interaction of CPPs with bacterial cells. Some CPPs have strong antimicrobial activities. There are two modes of action of CPPs on bacterial cells: CPPs can induce damage to the plasma membrane and thus increase permeability, or CPPs enter the cytosol of bacterial cells without damaging the plasma membrane. The information currently available on the elementary processes by which CPPs enter lipid bilayer vesicles and bacterial cells is valuable for elucidating the mechanisms of entry of CPPs into the cytosol of various eukaryotic cells.

Investigation of Intracellular Delivery of NuBCP-9 by Conjugation with Oligoarginines Peptides in MDA-MB-231 Cells.

Oligoarginines (Rn) are becoming promising tools for the intracellular delivery of biologically active molecules. NuBCP-9, a peptide that induces apoptosis in B-cell lymphoma 2 (Bcl-2)-expressing cancer cells, has been reported to promote the uptake and non-specific cytotoxicity of R8, also called octaarginine. However, it is unknown whether a similar synergistic effect can be seen with other Rn. In this study, we conjugated NuBCP-9 with various Rn (n=8, 10, 12, 14) to investigate and compare their cellular uptake characteristics. In addition, their non-specific cytotoxicity and apoptosis-inducing abilities were evaluated. We found that NuBCP-9 conjugated with Rn enhanced cellular uptake mainly through clathrin-mediated endocytosis and macropinocytosis, and that the uptake pathways were not different from those used by unconjugated Rn. However, the cytotoxicity study showed that NuBCP-9-R12 and NuBCP-9-R14 conjugates enhanced non-specific cytotoxicity. We found that NuBCP-9-R10 conjugate had the highest uptake efficiency and induced correspondingly high levels of apoptosis, while resulting in a tolerable degree of non-specific toxicity.

Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells.

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature.

Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

Oligoarginine peptide structure and its effect on cell penetration in ocular drug delivery.

Oligoarginine cell-penetrating peptides (CPPs) are short peptides that can enhance drug delivery into cells and are of particular interest in ocular topical formulations for age-related macular degeneration (AMD) treatments. The length and structural characteristics of these peptides are considered crucial for drug delivery. This study investigates how oligoarginine length (Rn) affects their penetration mechanism, drug delivery capabilities, and antimicrobial properties, providing insights into their potential roles in AMD treatment delivery. In this study, oligoarginine peptides showed limited pore-forming abilities in a carboxyfluorescein-containing liposomal model, with R9 being the only oligoarginine length recording a significant pore-formation level. Their antibacterial efficacy depended on both the CPP length and bacterial class, with longer peptides exhibiting stronger antibacterial effects. Importantly, oligoarginine was found nontoxic to relevant mammalian cells for ocular delivery. The membrane translocation abilities of oligoarginine were consistent regardless of cargo presence. Additionally, cargo delivery by oligoarginine across in vitro cellular models for ocular delivery was dependent on peptide length and cell type, with longer chains being more effective at cargo uptake in a corneal epithelium cell line, and with shorter chains proving more effective for cargo delivery in a retinal epithelium cell line. This proposes that the chain length of oligoarginine could be used as a strategic tool in the formulation process to selectively target distinct regions of the eye. Overall, this study expands our understanding of how oligoarginine CPPs can be applied as penetration enhancers to improve the delivery of therapeutics in an ocular topical formulation within the clinical context of AMD.

Inhibition of nicotinic acetylcholine receptors by oligoarginine peptides and polyamine-related compounds.

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed "cyclo"R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C-methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. "Cyclo"R8 showed similar activity to that of R8 against α9α10 nAChR (IC50 ≈ 60 nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica, as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C-methylated derivatives having IC50 of about 30 µM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC50 ≈ 15 µM), but it revealed high potency against rat α9α10 nAChR (IC50 ≈ 200 nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain.

Enhancing the Cellular Delivery of Nanoparticles using Lipo-Oligoarginine Peptides.

Nanoparticles have great potential as nanotherapeutics, delivery vectors, and molecular imaging agents due to their flexible properties. Although intracellular and nuclear delivery of nanoparticles is desirable for therapeutic applications, it remains a challenge. Cell penetrating peptides (CPPs) are a powerful tool for the intracellular delivery of various cargoes. Here we report that functionalization of nanoparticles with a myristoylated oligoarginine CPP promoted cellular uptake without increased toxicity. It was evidenced that the myristoylated CPP is much more effective in transporting nanoparticles than the unmodified CPPs.

Acquisition of absorption-enhancing abilities of cationic oligopeptides with short chain arginine residues through conjugation to hyaluronic acid.

Cell-penetrating peptides such as oligoarginines are one of promising tools that improve mucosal absorption of poorly membrane-permeable biologics. We have already demonstrated that conjugation of L-octaarginine to hyaluronic acid via a tetraglycine spacer resulted in a 3-fold enhancement of nasal absorption of somatropin (Mw: ca. 22.1 kDa) in mice when compared with the unmodified peptide. Here, we evaluated absorption-enhancing abilities and safety profiles of oligopeptides with short chain arginine residues conjugated to hyaluronic acid. Somatropin absorption was hardly ever enhanced by diglycine-L-tetraarginine. The peptide acquired the absorption-enhancing ability through the conjugation; however, it disappeared when arginine residues were halved. In vivo data were consistent to in vitro cellular uptake of somatropin. When somatropin was substituted with exendin-4 (Mw: ca. 4.2 kDa), cellular uptake was significantly enhanced by diglycine-L-diarginine conjugated to hyaluronic acid under comparison with the unmodified peptide. The conjugate also exhibited the enhancement ability in mice, as observed for hyaluronic acid derivatives with four and more arginine residues. Another cell studies revealed that oligoarginine-linked hyaluronic acid tended to be less toxic as arginine residues were reduced. Results indicated that diglycine-L-tetraarginine-linked hyaluronic acid was the most suitable candidate as an absorption enhancer whose Mw-independent enhancement ability and safety were well-balanced.

α-Conotoxin RgIA and oligoarginine R8 in the mice model alleviate long-term oxaliplatin induced neuropathy.

Оligoarginines were recently discovered (Lebedev et al., 2019 Nov) as a novel class of nicotinic acetylcholine receptors (nAChRs) inhibitors, octaoligoarginine R8 showing a relatively high affinity (44 nM) for the α9/α10 nAChR. Since the inhibition of α9/α10 nAChR by α-conotoxin RgIA and its analogs is a possible way to drugs against neuropathic pain, here in a mice model we compared R8 with α-conotoxin RgIA in the effects on the chemotherapy-induced peripheral neuropathy (CIPN), namely on the long-term oxaliplatin induced neuropathy. Tests of cold allodynia, hot plate, Von Frey and grip strength analysis revealed for R8 and α-conotoxin RgIA similar positive effects, expressed most prominently after two weeks of administration. Histological analysis of the dorsal root ganglia sections showed for R8 and RgIA a similar partial correction of changes in the nuclear morphology of neurons. Since α9/α10 nAChR might be not the only drug target for R8, we analyzed the R8 action on rat TRPV1 and TRPA1, well-known nociceptive receptors. Against rTRPV1 at 25 µM there was no inhibition, while for rTRPA1 IC50 was about 20 µM. Thus, involvement of rTRPA1 cannot be excluded, but in view of the R8 much higher affinity for α9/α10 nAChR the latter seems to be the main target and the easily synthesized R8 can be considered as a potential candidate for a drug design.

In Vitro Cellular Uptake and Transfection of Oligoarginine-Conjugated Glycol Chitosan/siRNA Nanoparticles.

Chitosan and its derivatives have been extensively utilized in gene delivery applications because of their low toxicity and positively charged characteristics. However, their low solubility under physiological conditions often limits their application. Glycol chitosan (GC) is a derivative of chitosan that exhibits excellent solubility in physiological buffer solutions. However, it lacks the positive characteristics of a gene carrier. Thus, we hypothesized that the introduction of oligoarginine peptide to GC could improve the formation of complexes with siRNA, resulting in enhanced uptake by cells and increased transfection efficiency in vitro. A peptide with nine arginine residues and 10 glycine units (R9G10) was successfully conjugated to GC, which was confirmed by infrared spectroscopy, 1H NMR spectroscopy, and elemental analysis. The physicochemical characteristics of R9G10-GC/siRNA complexes were also investigated. The size and surface charge of the R9G10-GC/siRNA nanoparticles depended on the amount of R9G10 coupled to the GC. In addition, the R9G10-GC/siRNA nanoparticles showed improved uptake in HeLa cells and enhanced in vitro transfection efficiency while maintaining low cytotoxicity determined by the MTT assay. Oligoarginine-modified glycol chitosan may be useful as a potential gene carrier in many therapeutic applications.

Oligo(para-phenylenes)s-Oligoarginine Conjugates as Effective Antibacterial Agents with High Plasma Stability and Low Hemolysis.

Bacterial infections have become a global threat to human health, and the design of antibacterial agents is always an urgent task for biomedicine. Amphiphilic antibacterial agents with a different mechanism of action from traditional antibiotics have attracted researchers' attention more and more in recent years. In this work, a series of antibacterial conjugates composed of oligo(para-phenylenes)s and oligoarginine were synthesized, and their antibacterial activity was investigated. 2,2'-Biphenyl, 2,2″-terphenyl, and 2,2‴-quaterphenyl were conjugated with one or two triarginines by "click" chemical reactions to form compounds. The conjugates showed antibacterial activity against the typical Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, relatively low cytotoxicity to L929 cell line, and hemolytic activity in a certain range of concentration. Among these conjugates, 2,2‴-quaterphenyl-triarginine conjugate (2,2‴-QP-1) showed the highest antibacterial activity against both E. coli and S. aureus. Besides, it presented better stability in plasma compared with the positive control peixiganan. The antimicrobial mechanism of 2,2‴-QP-1 was also investigated by transmission electron microscopy and confocal laser scanning microscopy, showing that 2,2‴-QP-1 could interact with the bacterial membrane and then disrupt the membrane structure. This work demonstrated a prospective approach for the design of antibacterial agents with highly effective antibacterial activity, high stability in plasma, and low cytotoxicity.