Arginine Ethyl Ester Research Articles

Crystal structure of the Michaelis complex of trypsin with N‐α‐benzoyl‐l‐arginine ethyl ester

AbstractThe crystal structure of Michaelis complex of the bovine trypsin with N‐α‐benzoyl‐l‐arginine ethyl ester (BAEE) was determined at 2.30 Å resolution. The structure of enzyme‐substrate complex was solved in space group H32. The active site of trypsin was found to be occupied with the N‐α‐benzoyl‐l‐arginine ethyl ester. The hydrolyzed product of substrate molecules was also crystallized with trypsin. The substrate was embedded within the S1 binding site of the enzyme, and showed contacts with Gly‐195, Ser‐216, and Ser‐192. Some water molecules were also found within the vicinity of catalytic residues of enzyme. Interestingly, the hydrolyzed product present within the crystal lattice was found to be with inverted configuration. The crystal structure of trypsin in‐complex with N‐α‐benzoyl‐l‐arginine ethyl ester has never been reported previously.

A combined approach of lauroyl arginine ethyl ester hydrochloride and kojic acid in mitigating fresh-cut potato deterioration

The combinational effects of kojic acid and lauroyl arginine ethyl ester hydrochloride (ELAH) on fresh-cut potatoes were investigated. Kojic acid of 0.6% (w/w) effectively inhibited the browning of fresh-cut potatoes and displayed antimicrobial capacity. The color difference value of samples was decreased from 175 to 26 by kojic acid. In contrast, ELAH could not effectively bind with the active sites of tyrosinase and catechol oxidase at molecular level. Although 0.5% (w/w) of ELAH prominently inhibited the microbial growth, it promoted the browning of samples. However, combining kojic acid and ELAH effectively inhibited the browning of samples and microbial growth during the storage and the color difference value of samples was decreased to 52. This amount of kojic acid inhibited enzyme activities toward phenolic compounds. The results indicated that combination of kojic acid and ELAH could provide a potential strategy to extend the shelf life of fresh-cut products.

Development and characterization of starch/PVA antimicrobial active films with controlled release property by utilizing electrostatic interactions between nanocellulose and lauroyl arginate ethyl ester

The two nanocellulose (nanofibrillated cellulose (NFC) and carboxylated nanofibrillated cellulose (C-NFC)) could interact with lauryl arginine ethyl ester hydrochloride (LAE) through electrostatic bonding. The zeta potential (absolute value) of C-NFC (−27.80 mV) was higher than that of NFC (−10.07 mV). The starch/polyvinyl alcohol active films with controlled release property by utilizing electrostatic interactions between nanocellulose and LAE were prepared and their properties were investigated. For incorporation of the NFC or C-NFC, the cross-section of the films became slightly uneven and some fibrils were observed, the films exhibited an increase in strength, while the film water vapor and oxygen barrier properties decreased. The release of LAE from the films to food simulants (10 % ethanol) decelerated with increasing of NFC or C-NFC. These might be mainly attributed to the enhanced electrostatic interaction between NFC or C-NFC and LAE. It demonstrated that nanocellulose with higher negative charges would exhibit stronger electrostatic interaction with LAE, thus slowing the release of LAE. The film with highest C-NFC content exhibited smallest inhibition zone among LAE-containing films, which was related with its slowest release rate of LAE. It showed a great prospect to develop controlled release active packaging films by utilizing electrostatic interactions between substances.

A comprehensive evaluation of arginine and its derivatives as protein formulation stabilizers

Arginine and its derivatives (such as arginine ethyl ester and acetyl arginine) have varying degrees of protein aggregation suppressor effect across different protein solutions. To understand this performance ambiguity, we evaluated the activity of arginine, acetyl arginine, and arginine ethyl ester for aggregation suppressor effect against human intravenous immunoglobulin G (IgG) solution at pH 4.8. Both arginine and its cationic derivative arginine ethyl ester in their hydrochloride salt forms significantly reduced the colloidal and conformational stability (reduced kd and Tm) of IgG. Consequently, the monomer content was decreased with an increase in subvisible particulates after agitation or thermal stress. Furthermore, compared to arginine, arginine ethyl ester with one more cationic charge and hydrochloride salt form readily precipitated IgG at temperatures higher than 25 °C. On the contrary, acetyl arginine, which mostly exists in a neutral state at pH 4.8, efficiently suppressed the formation of subvisible particles retaining a high amount of monomer owing to its higher colloidal and conformational stability. Concisely, the charged state of additives significantly impacts protein stability. This study demonstrated that contrary to popular belief, arginine and its derivatives may either enhance or suppress protein aggregation depending on their net charge and concentration.

The mechanism of thermal aggregation of glutamate dehydrogenase. The effect of chemical chaperones

Chemical chaperones are low-molecular compounds counteracting protein aggregation. Understanding of the mechanism of their effects is key to their potential use in biotechnology. The aggregation of bovine liver glutamate dehydrogenase (GDH) was studied at 40 °C and 50 °C using dynamic light scattering, analytical ultracentrifugation, size-exclusion chromatography and differential scanning calorimetry. At 40 °C the GDH aggregation proceeds through the slow stages of hexamer dissociation and formation of small oligomeric aggregates. At 50 °C these stages are transient. The rate-limiting stage of the overall aggregation process is unfolding of the protein molecule; the order of aggregation with respect to protein, n = 1. The test system based on GDH aggregation at 50 °C was used to quantify the anti-aggregation activity of chemical chaperones by comparing their half-saturation concentrations [L]0.5. Arginine ethyl ester had the highest anti-aggregation activity, with [L]0.5 = 4 ± 1 mM. For other additives, [L]0.5 was 22 ± 1 mM (arginine), 18 ± 1 mM (argininamide) and 95 ± 12 mM (proline). Arginine at concentrations up to 300 mM, argininamide at concentrations higher than 300 mM and arginine ethyl ester at concentrations higher than 500 mM enhance aggregate-aggregate sticking. These results explain the mechanism of heat-induced GDH aggregation and its peculiarities at different temperatures or in the presence of chemical chaperones.

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid–liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

Arginolipid: A membrane-active antifungal agent and its synergistic potential to combat drug resistance in clinical Candida isolates.

Antifungal drug resistance exhibits a major clinical challenge for treating nosocomial fungal infections. To find a possible solution, we synthesized and studied the antifungal activities of three different arginolipids (Nα -acyl-arginine ethyl ester) against clinical drug-resistant isolates of Candida. The most active arginolipid, oleoyl arginine ethyl ester (OAEE) consisting of a long unsaturated hydrophobic chain, was tested for its mode of action, which revealed that it altered ergosterol biosynthesis and compromised the fungal cell membrane. Also, OAEE was found to exhibit synergistic interactions with fluconazole (FLU) or amphotericin B (AmB) against planktonic Candida cells, wherein it reduced the inhibitory concentrations of these drugs to their in vitro susceptible range. Studies conducted against theC. tropicalis biofilm revealed that the OAEE+AmB combination synergistically reduced the metabolic activity and hyphal density in biofilms, whereas OAEE+FLU was found to be additive against most cases. Finally, the evaluated selective toxicity of OAEE toward fungal cells over mammalian cells could establish it as an alternative treatment for combating drug-resistant Candida infections.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.

Rational Design of Cholesterol Derivative for Improved Stability of Paclitaxel Cationic Liposomes.

This work explores synthesis of novel cholesterol derivative for the preparation of cationic liposomes and its interaction with Paclitaxel (PTX) within liposome membrane using molecular dynamic (MD) simulation and in-vitro studies. Cholesteryl Arginine Ethylester (CAE) was synthesized and characterized. Cationic liposomes were prepared using Soy PC (SPC) at a molar ratio of 77.5:15:7.5 of SPC/CAE/PTX. Conventional liposomes were composed of SPC/cholesterol/PTX (92:5:3M ratio). The interaction between paclitaxel, ligand and the membrane was studied using 10ns MD simulation. The interactions were studied using Differential Scanning Calorimetry (DSC) and Small Angle Neutron Scattering analysis. The efficacy of liposomes was evaluated by MTT assay and endothelial cell migration assay on different cell lines. The safety of the ligand was determined using the Comet Assay. The cationic liposomes improved loading efficiency and stability compared to conventional liposomes. The increased PTX loading could be attributed to the hydrogen bond between CAE and PTX and deeper penetration of PTX in the bilayer. The DSC study suggested that inclusion of CAE in the DPPC bilayer eliminates Tg. SANS data showed that CAE has more pronounced membrane thickening effect as compared to cholesterol. The cationic liposomes showed slightly improved cytotoxicity in three different cell lines and improved endothelial cell migration inhibition compared to conventional liposomes. Furthermore, the COMET assay showed that CAE alone does not show any genotoxicity. The novel cationic ligand (CAE) retains paclitaxel within the phospholipid bilayer and helps in improved drug loading and physical stability. Graphical Abstract ᅟ.

Dental plaque regrowth studies to evaluate chewing gum formulations incorporating magnolia bark extract

The plaque inhibiting properties of magnolia bark extract (MBE) were assessed in a volunteer trial following the consumption of various sugar-free chewing gum formulations over a period of 4-days. Paired t-tests demonstrated significant (p<0.15) differences between the placebo and a gum containing MBE (0.4%) plus lauramide arginine ethyl ester (LAE) (0.5%) with respect to% plaque coverage (36.3% vs 34.0%) and area of plaque fluorescence (109.4mm2 vs 75.2mm2). These findings were supported by microbiological counts of total salivary bacteria (7.77log10cfu/ml vs 7.45log10cfu/ml) as well as Streptococcus spp. (6.76log10cfu/ml vs 6.29log10cfu/ml). MBE (0.4%)+LAE (0.5%) delivered by chewing gum had a moderate inhibitory effect on plaque formation and salivary bacteria. Limiting the formation of dental plaque and salivary bacteria, specifically oral streptococci, could contribute towards an improvement in oral health with respect to gum disease and caries.

A change in the aggregation pathway of bovine serum albumin in the presence of arginine and its derivatives

Chemical chaperones including arginine and its derivatives are widely used by biochemists working on the design of agents, which are able to efficiently suppress protein aggregation. To elucidate the mechanisms of anti-aggregation activity of chemical chaperones, methods based on registration of the increment in light scattering intensity must be supplemented with methods for direct detection of the portion of aggregated protein (γagg). For this purpose asymmetric flow field-flow fractionation was used in the present work. It was shown that heat-induced aggregation of bovine serum albumin (BSA) followed the kinetics of the reaction of the second order (0.1 M sodium phosphate buffer, pH 7.0, 70 °C). It was proposed to use Rhvs γagg plots to characterize the aggregation pathway (Rh is the hydrodynamic radius of the protein aggregates, which was calculated from the dynamic light scattering data). The changes in the shape of Rhvs γagg plots in the presence of arginine, arginine amide and arginine ethyl ester are indicative of the changes in the aggregation pathway of BSA aggregation. A conclusion has been made that larger aggregates are formed in the presence of arginine hydrochloride and its derivatives.

L-arginine and arginine ethyl ester enhance proliferation of endothelial cells and preadipocytes - how an arginine ethyl ester-releasing biomaterial could support endothelial cell growth in tissue engineering.

Adipose tissue engineering is a promising solution for the reconstruction of soft tissue defects. An insufficient neovascularisation within the scaffolds that leads to necrosis and tissue loss is still a major shortcoming of current tissue engineering attempts. Biomaterials, which release angiogenic factors such as L-arginine, could overcome this challenge by supporting the neovascularisation of the constructs. L-arginine is insoluble in organic solvents and thus cannot be incorporated into commonly used polymers in contrast to its ethyl ester. Here, we compared the effects of arginine and its ethyl ester on endothelial cells and preadipocytes, and generated an arginine ethyl ester-releasing, angiogenic polymer. We cultivated adipose tissue-derived endothelial cells and preadipocytes in arginine-free medium supplemented with L-arginine or L-arginine ethyl ester and assayed the proliferation rate and the degree of adipogenic differentiation, respectively. Additionally, we prepared arginine ethyl ester-releasing poly(D,L-lactide) foils, and investigated their impact on endothelial cell proliferation. We could demonstrate that arginine ethyl ester like arginine significantly increased the proliferation of endothelial cells and preadipocytes without inhibiting an induced adipogenic conversion of the preadipocytes. Further, we could show that the arginine ethyl ester-releasing polymer significantly increased endothelial cell growth. The present data are helpful guidance for generating angiogenic biomaterials that promote endothelial cell growth, and thereby could support neovascularisation within tissue engineering approaches.

Natural antimicrobials subtilosin and lauramide arginine ethyl ester synergize with conventional antibiotics clindamycin and metronidazole against biofilms of Gardnerella vaginalis but not against biofilms of healthy vaginal lactobacilli.

The purpose of this study was to evaluate the ability of clindamycin and metronidazole to synergize with natural antimicrobials against biofilms of bacterial vaginosis (BV)-associated Gardnerella vaginalis. Minimum bactericidal concentrations for biofilm cells (MBCs-B) were determined for each antimicrobial. The MBCs-B of lauramide arginine ethyl ester (LAE), subtilosin, clindamycin and metronidazole were 50, 69.5, 20 and 500 μg mL(-1), respectively. A checkerboard assay and isobologram were used to analyze the type of interactions between these antimicrobials. The combination of metronidazole with natural antimicrobials did not inhibit planktonic lactobacilli. Clindamycin with either LAE or with subtilosin was inhibitory for planktonic but not for biofilm-associated lactobacilli. All tested antimicrobial combinations were inhibitory for BV-associated Mobiluncus curtisii and Peptostreptococcus anaerobius. LAE and subtilosin synergized with clindamycin and metronidazole against biofilms of G. vaginalis but not biofilm-associated vaginal lactobacilli. The biofilms of BV-associated pathogens can be controlled by synergistically acting combinations of conventional antibiotics and natural antimicrobials which will help better management of current antibiotics, especially considering robust bacterial resistance. Our findings create a foundation for a new strategy in the effective control of vaginal infections.

Evaluation of antimicrobial activities of sequential spray applications of decontamination treatments on chicken carcasses.

The objective of this study was to evaluate the effects of sequential applications of ɛ-polylysine (EPL) or lauramide arginine ethyl ester (LAE) sprays followed by an acidic calcium sulfate (ACS) spray on inoculated chicken carcasses to reduce Salmonella (Salmonella enterica serovars including Salmonella typhimurium and Salmonella enteritidis) contamination during 6 days of storage (4.4°C). Secondly, reductions of the resident microflora were studied on uninoculated chicken carcasses following the sequential application of the treatments, chilling and 10 days of storage at 4.4°C. The treatment of Salmonella inoculated carcasses with 300 mg/L EPL followed by 30% ACS (EPL300-ACS30) sprays reduced Salmonella counts initially by 1.5 log cfu/mL and then by 1.2 log cfu/mL (p<0.05) following 6 days of storage at 4.4°C. Likewise, 200 mg/L LAE followed by 30% ACS (LAE200-ACS30) treatment reduced initial Salmonella counts on poultry carcasses by 1.8, 1.4 and 1.8 log cfu/mL (p<0.05), respectively, after 0, 3, and 6 days storage. Immediately after the treatments, EPL300-ACS30 and LAE200-ACS30 both reduced Escherichia coli counts significantly by 2.6 and 2.9 log cfu/mL, respectively. EPL300-ACS30 and LAE200-ASC30 were effective in lowering psychrotroph counts by 1 log cfu/mL on day 10 when compared to the control and distilled water treatments. This study demonstrated that EPL300-ACS30 and LAE200-ACS30 were effective in reducing Salmonella on inoculated chicken carcasses both after treatment and during the storage at 4.4°C for up to 6 days. In addition, reductions in psychrotroph counts indicated that these treatments might have the potential to increase the shelf-life of poultry carcasses.

The Natural Antimicrobial Subtilosin A Synergizes with Lauramide Arginine Ethyl Ester (LAE), ε-Poly-L-lysine (Polylysine), Clindamycin Phosphate and Metronidazole, Against the Vaginal Pathogen Gardnerella vaginalis.

Bacterial vaginosis (BV) is a common, recurrent vaginal infection linked to increased chances of preterm delivery, incidence of sexually transmitted infections and fertility problems. BV is caused by a shift of the vaginal ecosystem from predominately Lactobacillus to a multispecies Actinomyces biofilm with the most common representatives identified as Gardnerella vaginalis and Prevotella spp. Current treatments have been associated with increased resistance as well as negative effects on healthy microbiota. The objective of this study was to evaluate the synergistic potential of ten two-antimicrobial combinations against G. vaginalis and four representative lactobacilli. The four tested antimicrobials were lauramide arginine ethyl ester, ε-poly-L-lysine, clindamycin phosphate, metronidazole and the bacteriocin subtilosin A. The use of bacteriocins as either synergist or alternative treatment positions bacteriocins as an excellent alternative to current antibiotics. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of each of the antimicrobials individually, and the checkerboard assay was used to evaluate these MICs in combination. Clindamycin and subtilosin (CS), and metronidazole and subtilosin were synergistic against G. vaginalis in terms of fractional inhibitory concentration index (FICI). All tested combinations were found to have Bliss synergy. The combination of clindamycin and polylysine (CP) was identified as antagonistic against L. acidophilus in terms of both FICI and Bliss synergy. The combination of clindamycin and metronidazole (CM) was antagonistic against L. vaginalis for both FICI and Bliss synergy. The combinations of CP, clindamycin and LAE, CS, and LAE and polylysine were identified as Bliss antagonistic against L. vaginalis but did not indicate FICI antagonism.

Tissue Kallikrein Activity, Detected by a Novel Method, May Be a Predictor of Recurrent Stroke: A Case-Control Study.

Aim. Tissue kallikrein (TK) protein content in plasma has been shown to be negatively associated with both incident and recurrent strokes. The aims of this study were to develop a novel method for detecting TK activity and to investigate its association with event-free survival over 5 years in Chinese first-ever stroke patients. Methods. We designed a case-control study with 321 stroke patients (174: ischemic stroke, 147: hemorrhagic stroke) and 323 healthy local controls. TK activity was measured by a novel assay utilizing the immunological characteristics of TK and the catalysis of benzoyl arginine ethyl ester hydrochloride (BAEE). Results. TK protein levels above 0.200 mg/L in plasma were not associated with urinary TK activity or the risk of stroke recurrence. TK activity was significantly lower in stroke patients compared with controls (1.583 ± 0.673 Eu/mL versus 1.934 ± 0.284 Eu/mL, P < 0.001). After adjusting for traditional risk factors, TK activity was negatively associated, in a dose-response manner, with the risk of overall stroke recurrence and positively associated with event-free survival during a 5-year follow-up (relative risk (RR), 0.69; 95% CI, 0.57–0.84; P < 0.001). Conclusions. Our findings suggest that urinary TK activity may be a stronger predictor of stroke recurrence than plasma TK levels.

Montelukast-loaded nanostructured lipid carriers: Part II Pulmonary drug delivery and in vitro–in vivo aerosol performance

The aim of the present study was to establish the potential of montelukast loaded nanostructured lipid carrier (MNLC) for pulmonary application. The formulated nanoparticles were evaluated in vitro for aerodynamic characterization and in vivo for pulmokinetics in Wistar rats. The in vitro cytotoxicity was performed on A549 cell line and compared with montelukast-aqueous solution. MNLC was prepared with montelukast (0.2%), Precirol ATO5 (solid lipid), and Capryol-90 (liquid lipid) in the ratio of 7:3 using melt-emulsification-homogenization method. dl-Pyrrolidonecarboxylic acid salt of l-cocyl arginine ethyl ester (CAE), a biodegradable surfactant in the concentration of 1% was used to stabilize the nanoparticles. The particle size and encapsulation efficiency (EE) were 184.6±2.7nm and >95%, respectively. MNLC-Dry powder for inhalation (DPI) was prepared by lyophilization using 3% mannitol as cryoprotectant and carrier. MNLC-DPI was evaluated for flow, crystallographic and thermal properties. Mass median diameters (MMD) and density for MNLC-DPI were found to be 15.1±1.4μm and 0.051±0.002g/cc, respectively. In vitro aerosol performance study indicated more than 95% of the emitted dose (ED) at both the flow rates studied. Mass median aerodynamic diameters (MMAD) of 3.24±0.67μm with 69.98±1.9% fine particle fraction (FPF) were obtained at 30L/min flow rate, whereas at 60L/min MMAD and FPF were found to be 2.83±0.46μm and 90.22±2.6%, respectively. In vitro cytotoxicity study on A549 cells revealed higher safety of MNLC than pure drug. The pulmonary pharmacokinetic study demonstrated improved bioavailability, longer residence of drug in the lung and targeting factor of 11.76 for MNLC as compared to montelukast-aqueous solution. Thus, the results of the study demonstrated the potential of montelukast lipidic nanoparticulate formulation to improve the efficacy with reduced toxicity leading to better performance of drug as MNLC-DPI for inhalation use.

Montelukast-loaded nanostructured lipid carriers: Part I Oral bioavailability improvement

The purpose of the study was to formulate montelukast-loaded nanostructured lipid carrier (MNLC) to improve its systemic bioavailability, avoid hepatic metabolism and reduce hepatic cellular toxicity due to metabolites. MNLC was prepared using melt-emulsification-homogenization method. Preformulation study was carried out to evaluate drug-excipient compatibility. MNLCs were prepared using spatially different solid and liquid lipid triglycerides. CAE (DL-Pyrrolidonecarboxylic acid salt of L-cocyl arginine ethyl ester), a cationic, biodegradable, biocompatible surfactant was used to stabilize the system. MNLCs were characterized by FTIR, XRPD and DSC to evaluate physicochemical properties. MNLCs having a particle size of 181.4±6.5nm with encapsulation efficiency of 96.13±0.98% were prepared. FTIR findings demonstrated no interaction between the drug and excipients of the formulation which could lead to asymmetric vibrations. DSC and XRPD study confirmed stable amorphous form of the montelukast in lipid matrix. In vitro release study revealed sustained release over a period of 24h. In vivo single dose oral pharmacokinetic study demonstrated 143-fold improvement in bioavailability as compared to montelukast-aqueous solution. Thus, the result of this study implies that developed MNLC formulation be suitable to sustain the drug release with improvement in the bioavailability.

Arginoplexes: an arginine-anchored nanoliposomal carrier for gene delivery

There is a need of an efficient and safe non-viral gene delivery carrier due to promising future of nucleic acid-based therapeutics in the treatment of intractable diseases. Cytotoxicity and cost are the major concerns with current quaternary ammonium-based cationic liposomes. The major aim of current research work was development and in vitro evaluation of arginine-anchored nanoliposomes for gene delivery. l-Arginine–fatty acid conjugate was synthesized and characterized using IR, NMR, and mass spectroscopy. Synthesized conjugate—lauroyl arginine ethyl ester (LAE) was successfully incorporated into liposomes. Effect of nanocarrier composition on DNA binding was evaluated by preparing solid lipid nanoparticle (SLN) and self nanoemulsifying system (SNES) using same LAE concentration. Effect of cationic head on DNA binding was also evaluated. Arginine-anchored nanoliposomes—arginoplexes (APX) showed superior DNA-binding affinity. Surface PEG was expected to cause hindrance in DNA binding in SLNs and SNES. Guanidino group was found to be a better cationic head for DNA binding compared to primary amine or quaternary amine. Gel retardation assay was performed to optimize the ratio of DNA to LAE in nanocarrier. Serum stability, haemolysis, cytotoxicity, and transfection studies were carried out to evaluate APX. Binding of DNA to APX was found to be stable in the presence of serum, and no degradation of DNA was observed. APX containing 2 mg/ml LAE which exhibited particle size of ~72 nm with zeta potential of +57.5 mV, showed lower cytotoxicity and better transfection. APX can be a promising carrier for gene delivery.

Quantification of anti-aggregation activity of chaperones: a test-system based on dithiothreitol-induced aggregation of bovine serum albumin.

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin–target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively).