Effects of γ-polyglutamic acid supplementation on alfalfa growth and rhizosphere soil microorganisms in sandy soil.

This study aimed at exploring the effects of γ-polyglutamic acid on the growth of desert alfalfa and the soil microorganisms in the rhizosphere. The study examined the effects of varying concentrations of γ-polyglutamic acid (0%-CK, 2%-G1, 4%-G2, 6%-G3) on sandy soil, the research investigated its impact on the growth characteristics of alfalfa, nutrient content in the rhizosphere soil, and the composition of bacterial communities. The results indicated that there were no significant differences in soil organic matter, total nitrogen, total phosphorus, total potassium, and available phosphorus content among the G1, G2, and G3 treatments. Compared to CK, the soil nutrient content in the G2 treatment increased by 14.81-186.67%, showing the highest enhancement. In terms of alfalfa growth, the G2 treatment demonstrated the best performance, significantly increasing plant height, chlorophyll content, above-ground biomass, and underground biomass by 54.91-154.84%. Compared to the CK treatment, the number of OTUs (operational taxonomic units) in the G1, G2, and G3 treatments increased by 14.54%, 8.27%, and 6.84%, respectively. The application of γ-polyglutamic acid altered the composition and structure of the bacterial community, with Actinobacteriota, Proteobacteria, Chloroflexi, Acidobacteriota, and Gemmatimonadota accounting for 84.14-87.89% of the total bacterial community. The G2 treatment significantly enhanced the diversity and evenness of soil bacteria in the rhizosphere. Redundancy analysis revealed that organic matter, total nitrogen, total potassium, moisture content, and pH were the primary factors influencing the structure of bacterial phyla. At the genus level, moisture content emerged as the most influential factor on the bacterial community. Notably, moisture content exhibited a strong positive correlation with Acidobacteriota, which in turn was positively associated with indicators of alfalfa growth. In summary, the application of γ-polyglutamic acid at a 4% ratio has the potential for improving sandy soil quality, promoting plant growth, and regulating the rhizosphere microbial community.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Poly-γ-glutamic acid nanoparticles as adjuvant and antigen carrier system for cancer vaccination.

Vaccination is an innovative strategy for cancer treatment by leveraging various components of the patients' immunity to boost an anti-tumor immune response. Rationally designed nanoparticles are well suited to maximize cancer vaccination by the inclusion of immune stimulatory adjuvants. Also, nanoparticles might control the pharmacokinetics and destination of the immune potentiating compounds. Poly-γ-glutamic acid (γ-PGA) based nanoparticles (NPs), which have a natural origin, can be easily taken up by dendritic cells (DCs), which leads to the secretion of cytokines which ameliorates the stimulation capacity of T cells. The intrinsic adjuvant properties and antigen carrier properties of γ-PGA NPs have been the focus of recent investigations as they can modulate the tumor microenvironment, can contribute to systemic anti-tumor immunity and subsequently inhibit tumor growth. This review provides a comprehensive overview on the potential of γ-PGA NPs as antigen carriers and/or adjuvants for anti-cancer vaccination.

pH Responsive Poly(Amino Acid) Nanoparticles as Potent Carrier Adjuvants for Enhancing Cellular Immunity.

Adjuvants are widely used in vaccine to improve the protection or treatment efficacy. However, so far they inevitably produce side effects and are hard to induce cellular immunity in practical application. Herein, two kinds of amphiphilic poly(glutamic acid) nanoparticles (α-PGA-F and γ-PGA-F NPs) as nanocarrier adjuvants are fabricated to induce an effective cellular immune response. Amphiphilic PGA are synthesized by grafting phenylalanine ethyl ester to form biodegradable self-assembly nanoadjuvants in a water solution. The model antigen, chicken ovalbumin (OVA), can be loaded into PGA-F NPs (OVA@PGA-F NPs) with the high loading ratio >12%. Moreover, compared with γ-PGA-F NPs, the acidic environment can induce the α-helical secondary structure of α-PGA NPs, promoting membrane fusion and more fast antigen lysosomal escape. Hence, the antigen presenting cells treated with OVA@α-PGA-F NPs show higher secretion of inflammatory cytokines, and higher expression of major biological histocompatibility complex class I and CD80 than those of OVA@γ-PGA-F NPs. Overall, this work indicates that pH responsive α-PGA-F NPs as a carrier adjuvant can effectively improve the ability of cellular immune responses, leading to it being a potent candidate for vaccine applications.

Hydrocarbon Degradation and Microbial Survival Improvement in Response to γ-Polyglutamic Acid Application.

To improve the environmental sustainability of cleanup activities of contaminated sites there is a need to develop technologies that minimize soil and habitat disturbances. Cleanup technologies, such as bioremediation, are based on biological products and processes, and they are important for the future of our planet. We studied the potential of γ-poly glutamic acid (PGA) as a natural component of biofilm produced by Bacillus sp. to be used for the decomposition of petroleum products, such as heavy naphtha (N), lubricating oil (O), and grease (G). The study aimed to assess the impact of the use of different concentrations of PGA on the degradation process of various fractions of petroleum hydrocarbons (PH) and its effect on bacterial population growth in harsh conditions of PH contamination. In laboratory conditions, four treatments of PGA with each of the petroleum products (N, O, and G) were tested: PGA0 (reference), PGA1 (1% PGA), PGA1B (1% PGA with Bacillus licheniformis), and PGA10 (10% PGA). After 7, 28, 56, and 112 days of the experiment, the percentage yield extraction, hydrocarbon mass loss, geochemical ratios, pH, electrical conductivity, and microorganisms survival were determined. We observed an increase in PH removal, reflected as a higher amount of extraction yield (growing with time and reaching about 11% in G) and loss of hydrocarbon mass (about 4% in O and G) in all treatments of the PGA compared to the reference. The positive degradation impact was intensive until around day 60. The PH removal stimulation by PGA was also reflected by changes in the values of geochemical ratios, which indicated that the highest rate of degradation was at the initial stage of the process. In general, for the stimulation of PH removal, using a lower (1%) concentration of PGA resulted in better performance than a higher concentration (10%). The PH removal facilitated by PGA is related to the anionic homopoliamid structure of the molecule and its action as a surfactant, which leads to the formation of micelles and the gradual release of PH absorbed in the zeolite carrier. Moreover, the protective properties of PGA against the extinction of bacteria under high concentrations of PH were identified. Generally, the γ-PGA biopolymer helps to degrade the hydrocarbon pollutants and stabilize the environment suitable for microbial degraders development.

Polyglutamate-based nanoconjugates for image-guided surgery and post-operative melanoma metastases prevention.

Rationale: Cutaneous melanoma is the most aggressive and deadliest of all skin malignancies. Complete primary tumor removal augmented by advanced imaging tools and effective post-operative treatment is critical in the prevention of tumor recurrence and future metastases formation. Methods: To meet this challenge, we designed novel polymeric imaging and therapeutic systems, implemented in a two-step theranostic approach. Both are composed of the biocompatible and biodegradable poly(α,L-glutamic acid) (PGA) nanocarrier that facilitates extravasation-dependent tumor targeting delivery. The first system is a novel, fluorescent, Turn-ON diagnostic probe evaluated for the precise excision of the primary tumor during image-guided surgery (IGS). The fluorescence activation of the probe occurs via PGA degradation by tumor-overexpressed cathepsins that leads to the separation of closely-packed, quenched FRET pair. This results in the emission of a strong fluorescence signal enabling the delineation of the tumor boundaries. Second, therapeutic step is aimed to prevent metastases formation with minimal side effects and maximal efficacy. To that end, a targeted treatment containing a BRAF (Dabrafenib - mDBF)/MEK (Selumetinib - SLM) inhibitors combined on one polymeric platform (PGA-SLM-mDBF) was evaluated for its anti-metastatic, preventive activity in combination with immune checkpoint inhibitors (ICPi) αPD1 and αCTLA4. Results: IGS in melanoma-bearing mice led to a high tumor-to-background ratio and reduced tumor recurrence in comparison with mice that underwent surgery under white light (23% versus 33%, respectively). Adjuvant therapy with PGA-SLM-mDBF combined with ICPi, was well-tolerated and resulted in prolonged survival and prevention of peritoneal and brain metastases formation in BRAF-mutated melanoma-bearing mice. Conclusions: The results reveal the great clinical potential of our PGA-based nanosystems as a tool for holistic melanoma treatment management.

Quantification of the actual composition of polymeric nanocapsules: a quality control analysis.

Nanocapsules (NCs) are drug delivery nanosystems that contain an oily core, stabilized by a surfactant, and surrounded by a polymeric shell. The assembling of the components is based on physical and physicochemical forces, and, hence, usually, only a fraction of each component is finally part of the NCs' structure, while the remaining amount might be solubilized or forming micelles in the NCs' suspending medium. Usually, reports on the characterization of nanostructures simply indicate the association efficiency of the loaded drugs instead of their complete final composition. In this work, we have developed a liquid chromatography (LC) mass spectrometry (MS) methodology that allows the quantification of all the components of a series of NCs prepared by different techniques, namely DL-α-tocopherol; D-α-tocopherol polyethylene glycol 1000 succinate; benzethonium; lecithin; hexadecyltrimethylammonium; 1,2-dioleoyl-3-trimethylammoniumpropane; caprylic/capric triglycerides; macrogol 15-hydroxystearate; polysorbate 80; polysialic acid; hyaluronic acid; and polyethylene glycol polyglutamic acid. The LC-MS method was validated in terms of linearity (0.9383 < r2 < 0.9997), quantification limits, and recoveries of the isolated NCs' and waste fractions. The final composition of the isolated NCs was found to strongly depend on their composition and preparation technique. In our view, the rigorous quantification of the exact composition of nanosystems is essential for the progress of nanotechnology. This quantitative analysis will allow researchers to draw more accurate conclusions about the influence of the nanosystems' composition on their biological performance.

Effect of total glucosides of paeony and Tripterygium wilfordii polyglycosides on erythrocyte methotrexate polyglutamates in rats, analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The aim of the study was to explore the effect of total glucosides of paeony (TGP) and Tripterygium wilfordii polyglycosides (TWP) on erythrocyte methotrexate polyglutamates (MTXPGs), the metabolites of methotrexate (MTX). METHODS: An ultra-high-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method was developed to determine MTXPGs. The effects of MTXPGs were analysed using 24 male Sprague-Dawley rats that were randomly divided into the MTX alone, MTX-TGP combined, and MTX-TWP combined groups. Rats were administered MTX at a dose of 0.9 mg/kg once a week, TGP at 0.054 g/kg and TWP at 1.8 mg/kg three times a day. Venous blood (1.0 ml) was collected at weeks 2, 4, 6, 9, 12 and 15 and then analysed using the developed UPLC-MS/MS method. KEY FINDINGS: Specificity, linear range, inter-and intra-day precision, recovery, matrix effect and stability of MTXPGs met the standard regulations. This method was successfully used for the detection of MTXPGs. After administration of MTX alone, erythrocyte MTXPGs increased and accumulated in a time- and dose-dependent manner. Compared to MTX alone, the combination with TGP significantly decreased the content of total MTXPGs and short-chain MTXPGs (Methotrexate [MTX/MTXPG1] and 4-amino-10-methylpteroyldiglutamic acid [MTXPG2], P < 0.05), but had no significant effect on long-chain MTXPGs (4-amino-10-methylpteroyltriglutamic acid [MTXPG3], P > 0.05) and very long-chain MTXPGs (4-amino-10-methylpteroyltetraglutamic acid [MTXPG4] and 4-amino-10-methylpteroylpentaglutamic acid [MTXPG5], P > 0.05) at week 15. The combination of MTX with TWP had no significant effect on the content of total MTXPGs, short-chain MTXPGs and long-chain MTXPGs (P > 0.05), but it significantly decreased the content of very long-chain MTXPGs (P < 0.05) at week 15. CONCLUSIONS: The UPLC-MS/MS method was successfully used to determine MTXPGs in rat erythrocytes. TGP and TWP in combination with MTX affected the production of MTXPGs of different chain lengths in erythrocytes.

Mass Spectrometry Imaging of Bio-oligomer Polydispersity in Plant Tissues by Laser Desorption Ionization from Silicon Nanopost Arrays.

Mass spectrometry imaging (MSI) enables simultaneous spatial mapping for diverse molecules in biological tissues. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has been a mainstream MSI method for a wide range of biomolecules. However, MALDI-MSI of biological homopolymers used for energy storage and molecular feedstock is limited by, e.g., preferential ionization for certain molecular classes. Matrix-free nanophotonic ionization from silicon nanopost arrays (NAPAs) is an emerging laser desorption ionization (LDI) platform with ultra-trace sensitivity and molecular imaging capabilities. Here, we show complementary analysis and MSI of polyhydroxybutyric acid (PHB), polyglutamic acid (PGA), and polysaccharide oligomers in soybean root nodule sections by NAPA-LDI and MALDI. For PHB, number and weight average molar mass, polydispersity, and oligomer size distributions across the tissue section and in regions of interest were characterized by NAPA-LDI-MSI.

Studies on the intracellular accumulation process of methotrexate and its correlation with the key protein using an LC-MS/MS method: a novel way to realize prospective individualized medication.

High-dose methotrexate (HDMTX) combined with leucovorin (LV) is the first-line drug therapy for many kinds of malignant tumors. However, the specific treatment plans, such as dosage and duration of administration, are usually formulated according to the clinician's experience and therapeutic drug monitoring (TDM) of methotrexate in patients' plasma, which are responsible for strong individual differences of drug usage. A large number of studies have shown that methotrexate targets the inside of the cell. The key cytotoxic component is the methotrexate polyglutamates (MTXPGs) in the cell. The concentration of methotrexate in plasma does not reflect the efficacy and side effects well. Based on mass spectrometry technology, we developed and validated an accurate, sensitive, and stable method to quantify the intracellular MTX (MTXPG1) and its metabolites MTXPG2-7 simultaneously. The lower limit of quantification was 0.100 ng/ml, and the run time was only 3 min. Moreover, our team has already developed two LC-MS/MS-based methods to respectively quantify methotrexate in plasma samples and two key proteins (γ-glutamyl hydrolase [GGH] and folylpolyglutamate synthetase [FPGS]) in peripheral blood mononuclear cells (PBMC). Through these highly sensitive and accurate approaches, we have gained a deep understanding of the whole pharmacokinetic process of MTX and explored the key factors affecting the accumulation process of intracellular active components (MTXPGs). Based on this research, it is possible to find a more effective way to provide an accurate reference for clinical drug use than traditional therapeutic drug monitoring (TDM).

Gelatin-Poly (γ-Glutamic Acid) Hydrogel as a Potential Adhesive for Repair of Intervertebral Disc Annulus Fibrosus: Evaluation of Cytocompatibility and Degradability.

STUDY DESIGN: An in vitro experimental study testing a Gelatin-poly (γ-glutamic acid) hydrogel for disc repair. OBJECTIVE: To evaluate the cytocompatibility and degradability of the above mentioned hydrogel for intervertebral disc annular fibrosis (AF) repair. SUMMARY OF BACKGROUND DATA: No repair strategies for correcting annular defects in lumbar discectomy have been clinically well recognized. Exogenous supplementation of regenerative materials to fill defects is a minimally invasive way to restore compromised mechanical properties. The injected materials, most commonly gelatin-based materials with cross-linking agents, serve as sealants and as a scaffold for incorporating biomaterials for augmentation. However, cytotoxicity of hydrogel crosslinking agents is of concern in developing viable materials. METHODS: This in vitro experimental study evaluated a newly developed gelatin-based hydrogel for intervertebral disc AF repair. Mechanical strength was augmented by γ-PGA, and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) was used for material crosslinking. Isolated bovine tail intervertebral discs (IVDs) were used to test the hydrogel, and hydrogel surface monolayer AF cell culture was used to investigate efficacy in hydrogel constructs of different EDC concentrations. Cell metabolic activity was evaluated with Alamar blue assay, cell viability assay with live/dead stain, and sulfated glycosaminoglycan (GAG) and double strain DNA were quantified to evaluate proliferation of implanted cells and synthesis of extracellular matrix (ECM) proteins. RESULTS: EDC concentrations from 10 to 40 mM resulted in significant decreases in AF cell proliferation without obvious influence on cell viability. Higher EDC concentrations resulted in decreased percentage of Alamar blue reduction and GAG and DNA concentration, but did not affect GAG/DNA and live-dead ratios. Degradation tests revealed that higher EDC concentrations decreased the hydrogel degradation rate. CONCLUSION: The developed gelatin-poly (γ-PGA) hydrogel with 20 mM EDC concentration provides an effective gap-filling biomaterial with good cytocompatibility, suggesting substantial promise for use as a sealant for small AF defects.Level of Evidence: N/A.

Applications and Functions of γ-Poly-Glutamic Acid and its Derivatives in Medicine.

BACKGROUND: γ-Poly-Glutamic Acid (γ-PGA) is a naturally occurring homo-polyamide produced by various strains of Bacillus. It is made from repeating units of L-glutamic acid, D-glutamic acid, or both connected through amide linkages between α-amino and γ-carboxylic acid groups. As a biopolymer substance, the attractive properties of γ-PGA are that it is water-soluble, biodegradable, biocompatible, non-toxic, non-immunogenic, and edible. Therefore, it can be used as a green and environmentally friendly biological material. METHODS: The review concentrates on the reports revealing the functions and potential use of γ-PGA and its derivatives in medicine. RESULTS & DISCUSSION: γ-PGA is described to possess several properties that may be exploited in medicine. The biopolymer reportedly has been successfully applied not only as a metal chelator, drug carrier/ deliverer, and gene vector, but also used safely as a vaccine adjuvant, tissue engineering material, and contrast agent. CONCLUSION: γ-PGA could be potentially considered as a potential biomedical material in the field of medicine.

Stabilization of soybean oil-in-water emulsions using polypeptide multilayers: Cationic polylysine and anionic polyglutamic acid.

Oil-in-water emulsions are used as delivery systems for non-polar functional ingredients in various industries, including foods, cosmetics, personal care products, agrochemicals, and pharmaceuticals. Emulsions, however, tend to breakdown under the conditions found in many commercial products. In this study, the functional performance of the lipid droplets in emulsions was tailored by sequential layer-by-layer electrostatic deposition of oppositely charged polypeptides onto their surfaces. Cationic poly-L-lysine (PLL) and anionic poly-glutamic acid (PGA) were used as a pair of oppositely charged polypeptides (pH 4.0). First, a primary emulsion (10% w/w soybean oil-in-water emulsion) was formed consisting of small lipid droplets (d32 = 500 µm) coated by a natural surfactant (0.05% w/w quillaja saponin). Second, cationic PLL was deposited onto the surfaces of the anionic saponin-coated droplets. Third, anionic PGA was deposited onto the surfaces of the cationic PLL-saponin-coated droplets. We then assessed the ability of the coatings to protect the lipid droplets from aggregation when the pH (2.0-9.0), ionic strength (0-350 mM), or temperature (30-90 °C) were altered. The properties of the primary, secondary, and tertiary emulsions were monitored by measuring the mean particle diameter (d32), electrical characteristics (ζ-potential), and microstructure of the lipid droplets. The electrical characteristics of the droplets could be modulated by controlling the number and type of layers used. The primary emulsion had the best resistance to varying environmental conditions, while the secondary emulsion had the worst, suggesting electrostatic deposition should only be used to obtain specific functionalities. Interestingly, PLL detached from the surfaces of the secondary emulsions at high salt concentrations due to electrostatic screening, which improved their salt stability. This phenomenon may be useful for some food applications, e.g., having cationic droplets during food storage, but anionic ones inside the human body.

The Application and Functional Progress of γ-Poly-Glutamic Acid in Food: A Mini-Review.

γ-Poly-glutamic acid (γ-PGA) is a naturally occurring homo-polyamide produced by various strains of Bacillus. As a biopolymer substance, γ-PGA possesses a few predominant features containing good water solubility, biocompatibility, degradability and non-toxicity. Based on this, γ-PGA can be used in pharmaceutical, such as drug carrier/deliverer, vaccine adjuvant, and coating material for microencapsulation, etc. Moreover, it has also been applied in a broad range of industrial fields including food, medicine, bioremediation, cosmetics, and agriculture. Especially, γ-PGA is an extremely promising food ingredient. In this mini-review, our aim is to review the function and application progress of γ-PGA in the food industry: e.g., improving taste and flavor, enhancing physical property, and promoting health.

Effective Nephroprotection Against Acute Kidney Injury with a Star-Shaped Polyglutamate-Curcuminoid Conjugate.

The lack of effective pharmacological treatments for acute kidney injury (AKI) remains a significant public health problem. Given the involvement of apoptosis and regulated necrosis in the initiation and progression of AKI, the inhibition of cell death may contribute to AKI prevention/recovery. Curcuminoids are a family of plant polyphenols that exhibit attractive biological properties that make them potentially suitable for AKI treatment. Now, in cultured tubular cells, we demonstrated that a crosslinked self-assembled star-shaped polyglutamate (PGA) conjugate of bisdemethoxycurcumin (St-PGA-CL-BDMC) inhibits apoptosis and necroptosis induced by Tweak/TNFα/IFNγ alone or concomitant to caspase inhibition. St-PGA-CL-BDMC also reduced NF-κB activation and subsequent gene transcription. In vivo, St-PGA-CL-BDMC prevented renal cell loss and preserved renal function in mice with folic acid-induced AKI. Mechanistically, St-PGA-CL-BDMC inhibited AKI-induced apoptosis and expression of ferroptosis markers and also decreased the kidney expression of genes involved in tubular damage and inflammation, while preserving the kidney expression of the protective factor, Klotho. Thus, due to renal accumulation and attractive pharmacological properties, the application of PGA-based therapeutics may improve nephroprotective properties of current AKI treatments.

A dual-functional implant with an enzyme-responsive effect for bacterial infection therapy and tissue regeneration.

Biomaterial-associated bacterial infection is one of the major causes of implant failure. The treatment of such an implant infection typically requires the elimination of bacteria and acceleration of tissue regeneration around implants simultaneously. To address this issue, an ideal implanted material should have the dual functions of bacterial infection therapy and tissue regeneration at the same time. Herein, an enzyme-responsive nanoplatform was fabricated in order to treat implant-associated bacterial infection and accelerate tissue regeneration in vivo. Firstly, Ag nanoparticles were pre-encapsulated in mesoporous silica nanoparticles (MSNs) by a one-pot method. Then, poly-l-glutamic acid (PG) and polyallylamine hydrochloride (PAH) were assembled by the layer-by-layer (LBL) assembly technique on MSN-Ag to form LBL@MSN-Ag nanoparticles. Furthermore, the LBL@MSN-Ag nanoparticles were deposited on the surface of polydopamine-modified Ti substrates. PG is a homogeneous polyamide composed of an amide linkage, which can be degraded by glutamyl endonuclease secreted by Staphylococcus aureus. Inductively coupled plasma spectroscopy (ICP) results proved that the LBL@MSN-Ag particles show a significant enzyme responsive release of Ag ions. Furthermore, results of antibacterial experiments in vitro showed that the Ti substrates modified with an LBL@MSN-Ag nanocoating presented an excellent antibacterial effect. As for an animal experiment in vivo, in a bacterium infected femur-defect rat model, the modified Ti implants effectively treated bacterial infection. More importantly, the results of micro-CT, haematoxylin-eosin staining and Masson's trichrome staining demonstrated that the modified Ti implants significantly promoted the formation of new bone tissue after implantation for 4 weeks. The present system paves the way for developing the next generation of implants with the functions of treating bacterial infection and promoting tissue regeneration.

Preparation of water-soluble chitosan/poly-gama-glutamic acid-tanshinone IIA encapsulation composite and its in vitro/in vivo drug release properties.

Some diseases could be treated by Tanshinone IIA (TA), which is an isolated component from the Chinese medicinal herb Tanshen (Salvia miltiorrhiza). However, the poor water solubility and low oral bioavailability of TA limited its clinical application. In this paper, TA was encapsulated by water - soluble chitosan/poly - γ - glutamic acid (WCS-γ-PGA) to improve its dissolution and oral bioavailability. The in vitro dissolution and in vivo metabolism of the encapsulated composite in rats were employed to evaluate the efficiency of the improvement. FTIR spectroscopy was applied to confirm the validity of encapsulation for TA by WCS-γ-PGA. The study's results showed that the optimal ratio of TA to drug carrier (WCS + Î³-PGA) was 1:5.5 in weight with a reaction time of 1 h at room temperature for the encapsulation. The proper concentrations for WCS and TA in preparing the encapsulated composite using γ-PGA 0.125 mg ml-1 were 6 mg ml-1 and 1 mg ml-1, respectively; The encapsulation efficiency and drug loading efficiency of WCS-γ-PGA-TA composite were (93.99 ± 2.20)% and (10.73 ± 0.75)%, respectively. The cumulative release of TA from the WCS-γ-PGA-TA encapsulated composite reached to 81% within 60 min, which was 5.56 times of that of the original TA in vitro dissolution. The peak concentration Cmax of TA from the encapsulated composite in rat blood as measured by an ultracentrifugation test of an intra - gastric administration was 4.43 times that of the original TA concentration, and the area under the drug-time curve AUC (0-t) and AUC (0-∞) (p<0.01) of the WCS-γ-PGA-TA encapsulated composite were 4.56 and 4.20 times that of the original TA, respectively. It indicated that the encapsulation of TA with WCS-γ-PGA improved its solubility and bioavailability significantly.

Poly(γ-Glutamic Acid)/Chitosan Hydrogel Nanoparticles For Effective Preservation And Delivery Of Fermented Herbal Extract For Enlarging Hair Bulb And Enhancing Hair Growth.

INTRODUCTION: Hair growth-promoting herbal extract mixtures (4HGF) exhibits significant anti-inflammatory activities relevant to promoting hair growth; however, its efficacy in patients with hair loss has been limited majorly due to its low penetration ability into hair follicles. Herein, we prepared hydrogels via dropwise addition of poly(γ-glutamic acid) (PGA) solution containing 4HGF into chitosan (CS) solution, resulting in quick formation of ~400 nm-sized hydrogel particles through electrostatic interaction-derived ionic gelation with over 50% encapsulation efficiency of 4HGF (PGA-4HGF). METHODS: The size and morphology of PGA-4HGF were characterized by TEM, SEM, and dynamic light scattering analyses. Encapsulation efficiency and loading capacity of 4HGF within PGA-4HGF, as well as in vitro release profiles were determined by simply measuring the characteristic absorbance of 4HGF. Penetrating efficiency of PGA-4HGF was evaluated by tracking the respective fluorescence through model porcine skin with confocal laser microscope system. By treating PGA-4HGF on telogenic mice and dermal papilla cells (DPCs), we evaluated the size of hair bulbs in mice, as well as morphological changes in DPCs. RESULTS: Negligible and sustained release of entrapped 4HGF from the hydrogel nanoparticles were observed under acidic and physiological pH conditions, respectively, which is quite advantageous to control their release and prolong their hair growth-promoting effect. The hydrogel nanoparticles were penetrable through the porcine skin after incubation with or without shaking. After treating telogenic mice and DPCs with PGA-4HGF, we detected enlargement of hair bulbs and remarkable shape changes, respectively, thereby showing its potential in induction of hair growth. CONCLUSION: These results suggest that the hydrogel nanoparticle formulation developed in this study can be employed as a potential approach for the preservation of hair growth-promoting compounds, their delivery of into hair follicles, and enhancing hair growth.

Hair Growth Promoting Effect of 4HGF Encapsulated with PGA Nanoparticles (PGA-4HGF) by ß-Catenin Activation and Its Related Cell Cycle Molecules.

Poly-γ-glutamic acid (γ-PGA)-based nanoparticles draw remarkable attention as drug delivery agents due to their controlled release characteristics, low toxicity, and biocompatibility. 4HGF is an herbal mixture of Phellinus linteus grown on germinated brown rice, Cordyceps militaris grown on germinated soybeans, Polygonum multiflorum, Ficus carica, and Cocos nucifera oil. Here, we encapsulated 4HGF within PGA-based hydrogel nanoparticles, prepared by simple ionic gelation with chitosan, to facilitate its penetration into hair follicles (HFs). In this study, we report the hair promoting activity of 4HGF encapsulated with PGA nanoparticles (PGA-4HGF) and their mechanism, compared to 4HGF alone. The average size of spherical nanoparticles was ~400 nm in diameter. Continuous release of PGA-4HGF was observed in a simulated physiological condition. As expected, PGA-4HGF treatment increased hair length, induced earlier anagen initiation, and elongated the duration of the anagen phase in C57BL/6N mice, compared with free 4HGF treatment. PGA-4HGF significantly increased dermal papilla cell proliferation and induced cell cycle progression. PGA-4HGF also significantly increased the total amount of ß-catenin protein expression, a stimulator of the anagen phase, through induction of cyclinD1 and CDK4 protein levels, compared to free 4HGF treatment. Our findings underscore the potential of PGA nanocapsules to efficiently deliver 4HGF into HFs, hence promoting hair-growth. Therefore, PGA-4HGF nanoparticles may be promising therapeutic agents for hair growth disorders.

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model.

Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the collagen-induced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of 40 days. At the completion of the study tissue samples were analyzed for MTX and folate content and assessed for their relationship with MTX efficacy. MTX treatment resulted in a reduction in disease activity that was variable and dose-dependent. Erythrocyte accumulation of MTX and its polyglutamate metabolites were dose proportionate, however, polyglutamate metabolites represented a mean ±â€¯S.E.M. of 8.9 ±â€¯0.4% of total erythrocyte MTX, which is markedly lower than previously observed in humans and failed to display any significant association with MTX efficacy. MTX treatment resulted in reductions in erythrocyte 5-methyl-tetrahydrofolate (5mTHF) levels that were similar to those previously observed in human studies. Disease induction was associated with a decrease in liver 5mTHF and increased formyl-tetrahydrofolate (fTHF) that was normalized in MTX treated mice. MTX efficacy was associated with reductions in erythrocyte 5mTHF (P = 0.04) and increases in liver 5mTHF (P = 0.0001). Together, these findings demonstrate a relationship between alterations in tissue folate levels and MTX efficacy, and supports erythrocyte levels of 5mTHF as a marker of MTX efficacy in autoimmune arthritis.