RESUMEN
Pulmonary delivery and formulation of biologics are among the more complex and growing scientific topics in drug delivery. We herein developed a dry powder formulation using disordered mesoporous silica particles (MSP) as the sole excipient and lysozyme, the most abundant antimicrobial proteins in the airways, as model protein. The MSP had the optimal size for lung deposition (2.43 ± 0.13 µm). A maximum lysozyme loading capacity (0.35 mg/mg) was achieved in 150 mM PBS, which was seven times greater than that in water. After washing and freeze-drying, we obtained a dry powder consisting of spherical, non-aggregated particles, free from residual buffer, or unabsorbed lysozyme. The presence of lysozyme was confirmed by TGA and FT-IR, while N2 adsorption/desorption and SAXS analysis indicate that the protein is confined within the internal mesoporous structure. The dry powder exhibited excellent aerodynamic performance (fine particle fraction <5 µm of 70.32%). Lysozyme was released in simulated lung fluid in a sustained kinetics and maintaining high enzymatic activity (71-91%), whereas LYS-MSP were shown to degrade into aggregated nanoparticulate microstructures, reaching almost complete dissolution (93%) within 24 h. MSPs were nontoxic to in vitro lung epithelium. The study demonstrates disordered MSP as viable carriers to successfully deliver protein to the lungs, with high deposition and retained activity.
Asunto(s)
Pulmón , Muramidasa , Tamaño de la Partícula , Polvos , Dióxido de Silicio , Dióxido de Silicio/química , Muramidasa/administración & dosificación , Muramidasa/química , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Porosidad , Polvos/química , Portadores de Fármacos/química , Administración por Inhalación , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Humanos , Excipientes/química , Animales , Química Farmacéutica/métodos , Espectroscopía Infrarroja por Transformada de Fourier , LiofilizaciónRESUMEN
The present study was conducted to investigate the effects of dietary recombinant human lysozyme (RHL) on the growth, immune response, anti-oxidative activity, intestinal morphology, intestinal microflora and disease resistance of shrimp Litopenaeus vannamei. Shrimps with an initial body weight of 2.36 ± 0.02 g were fed diets supplemented with 0 (control group, R0), 0.0025% (R1), 0.005% (R2), 0.01% (R3), 0.02% (R4) and 0.04% (R5) of RHL, respectively. After a 10-week feeding trial, the final body weight, survival rate, weight gain ratio and protein efficiency rate of the shrimps in dietary RHL supplemented groups were significantly higher than that in the control group, while feed conversion ratio was significantly lower (P < 0.05). The total haemocyte count, total anti-oxidative capacity, respiratory burst, activities of phagocytosis, nitric oxide synthase, phenol oxidase and lysozyme in serum were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Meanwhile, the intestinal pile height and wall thickness were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Dietary RHL significantly improved the expressions of immune-related genes in gill, such as lipopolysaccharide-ß-glucan binding protein, Toll, immune deficiency, heat shock protein 70 and Crustin (P < 0.05). The abundance of proteobacteria and bacteroidetes in intestine was higher, while the abundance of firmicutes and cyanobacteria was lower than those in the control group at the phylum level. In addition, dietary RHL supplementation significantly improved the protective ability of shrimp against V. parahaemolyticus infection (P < 0.05). Based on the broken-line model analysis for weight gain ratio after the feeding trial, the optimal level of dietary RHL supplementation for shrimp was estimated to be 0.006375%.
Asunto(s)
Dieta , Resistencia a la Enfermedad , Inmunidad Innata , Muramidasa/administración & dosificación , Penaeidae , Animales , Peso Corporal , Dieta/veterinaria , Suplementos Dietéticos , Humanos , Intestinos/crecimiento & desarrollo , Intestinos/microbiología , Penaeidae/crecimiento & desarrollo , Penaeidae/inmunología , Aumento de PesoRESUMEN
Colitis is characterized by colonic inflammation and impaired gut health. Both features aggravate obesity and insulin resistance. Host defense peptides (HDPs) are key regulators of gut homeostasis and generally malfunctioning in above-mentioned conditions. We aimed here to improve bowel function in diet-induced obesity and chemically induced colitis through daily oral administration of lysozyme, a well-characterized HDP, derived from Acremonium alcalophilum.C57BL6/J mice were fed either low-fat reference diet or HFD ± daily gavage of lysozyme for 12 weeks, followed by metabolic assessment and evaluation of colonic microbiota encroachment. To further evaluate the efficacy of intestinal inflammation, we next supplemented chow-fed BALB/c mice with lysozyme during Dextran Sulfate Sodium (DSS)-induced colitis in either conventional or microbiota-depleted mice. We assessed longitudinal microbiome alterations by 16S amplicon sequencing in both models.Lysozyme dose-dependently alleviated intestinal inflammation in DSS-challenged mice and further protected against HFD-induced microbiota encroachment and fasting hyperinsulinemia. Observed improvements of intestinal health relied on a complex gut flora, with the observation that microbiota depletion abrogated lysozyme's capacity to mitigate DSS-induced colitis.Akkermansia muciniphila associated with impaired gut health in both models, a trajectory that was mitigated by lysozyme administration. In agreement with this notion, PICRUSt2 analysis revealed specific pathways consistently affected by lysozyme administration, independent of vivarium, disease model and mouse strain.Taking together, lysozyme leveraged the gut microbiota to curb DSS-induced inflammation, alleviated HFD-induced gastrointestinal disturbances and lowered fasting insulin levels in obese mice. Collectively, these data present A. alcalophilum-derived lysozyme as a promising candidate to enhance gut health.
Asunto(s)
Acremonium/enzimología , Colitis/tratamiento farmacológico , Microbioma Gastrointestinal/efectos de los fármacos , Muramidasa/administración & dosificación , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Colitis/inducido químicamente , Colitis/microbiología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muramidasa/metabolismoRESUMEN
In this study, cellulose nanocrystals (CNC) as functional cross-linker and Pickering emulsifier was used to stabilize Lysozyme (Lys) encapsulated in quaternary ammonium chitosan nanoparticles (QC NPs) via ionic gelation method. Physicochemical, structural, and antibacterial properties of the CNC stabilized Lys loaded QC NPs were also evaluated. Particle size, particle size distribution, Zeta potential (ZP), and spectroscopic analyses showed the successful encapsulation of Lys. Antibacterial activity of NPs against Staphylococcus aureus and Vibrio parahaemolyticus was investigated on the basis of inhibition zone (IZ), minimum inhibitory concentration (MIC), and minimum bacterial concentration (MBC). MIC and MBC of CNC stabilized Lys loaded HQC NPs against S. aureus were 0.094 and 0.188 while corresponding values for CNC stabilized Lys loaded LQC NPs V. parahaemolyticus were 0.156 and 0.312 mg/mL, respectively. Therefore, CNC stabilized Lys loaded QC NPs have potential implications in the food industry for food preservation and packaging.
Asunto(s)
Antibacterianos/administración & dosificación , Celulosa/análogos & derivados , Muramidasa/administración & dosificación , Nanopartículas/química , Compuestos de Amonio Cuaternario/química , Antibacterianos/farmacología , Muramidasa/farmacología , Staphylococcus aureus/efectos de los fármacos , Vibrio parahaemolyticus/efectos de los fármacosRESUMEN
Corneal transplantation is an effective treatment for corneal blindness. However, it brings risk factors for the occurrence of bacterial keratitis, which can affect the repair effect and even lead to transplantation failure. The difficulty in re-epithelialization is also a main problem faced by corneal transplantation. Herein, a collagen-GelMA composite membrane containing lysozyme (CGL) was developed as an antibacterial corneal implant to fill stromal defect and support re-epithelialization. Characterizations of physicochemical properties and in vitro biocompatibility revealed that the composite membranes have proper water content, light transmittance and mechanical strength as well as good biocompatibility. Particularly, the cell adhesion force and adhesion-related genes expression were evaluated and exhibited an improvement after the addition of GelMA. Furthermore, the formed CGL membrane could continuously release lysozyme and exhibited a bactericidal rate of 96% and 64% after 2 h and 72 h, respectively. The results demonstrated that this CGL membrane has promising application in corneal repair.
Asunto(s)
Antibacterianos/química , Colágeno/química , Trasplante de Córnea/métodos , Membranas Artificiales , Muramidasa/química , Animales , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Adhesión Celular , Células Cultivadas , Trasplante de Córnea/instrumentación , Reactivos de Enlaces Cruzados/química , Liberación de Fármacos , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/fisiología , Muramidasa/administración & dosificación , Muramidasa/farmacología , Conejos , Staphylococcus aureus/efectos de los fármacosRESUMEN
The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses.
Asunto(s)
Adyuvantes Inmunológicos/química , Antígenos/inmunología , Vacunas de Subunidad/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adsorción , Animales , Antígenos/administración & dosificación , Antígenos/química , Cationes/administración & dosificación , Cationes/química , Femenino , Glucolípidos/administración & dosificación , Glucolípidos/química , Inmunogenicidad Vacunal , Liposomas , Ratones , Modelos Animales , Muramidasa/administración & dosificación , Muramidasa/química , Muramidasa/inmunología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/química , Células TH1 , Células Th17 , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/químicaRESUMEN
BACKGROUND: The ability of a high level of dietary Arthrospira platensis, individually or in combination with two exogenous carbohydrate-degrading enzymes (lysozyme and Rovabio®), to improve systemic antioxidant potential and hepatic lipid metabolism was tested in piglets. Forty male post-weaned piglets, sons of Large White × Landrace sows crossed with Pietrain boars, were allocated into 4 groups (n = 10) and fed during 28 days one of the following diets: 1) a control basal diet (cereal and soybean meal); 2) a basal diet with 10% of A. platensis (AP); 3) the AP diet supplemented with 0.005% of Rovabio® (AP + R); 4) the AP diet supplemented with 0.01% of lysozyme (AP + L). RESULTS: Arthrospira platensis decreased BW gain of piglets, regardless the addition of feed enzymes. The majority of plasma metabolites were affected by diets. A. platensis increased total lipids, total cholesterol and LDL-cholesterol, without changing hepatic fatty acid content or modulating, in an expressive manner, the transcriptional profile of lipid sensitive mediators. The antioxidant potential in general, and total carotenoids in particular, were improved by the microalga, regardless lysozyme or Rovabio®. CONCLUSIONS: Summing up, A. platensis, individually and combined with feed enzymes, impacts negatively on piglets' growth but improves the systemic antioxidant potential and changes plasma lipids with a minor modulation on related hepatic metabolic pathways.
Asunto(s)
Alimentación Animal/análisis , Dieta/veterinaria , Spirulina , Sus scrofa/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Antioxidantes/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Complejos Multienzimáticos/administración & dosificación , Muramidasa/administración & dosificación , Sus scrofa/crecimiento & desarrolloRESUMEN
The low efficacy of current conventional treatments for bacterial infections increases mortality rates worldwide. To alleviate this global health problem, we propose drug-free enzyme-based nanomotors for the treatment of bacterial urinary-tract infections. We develop nanomotors consisting of mesoporous silica nanoparticles (MSNPs) that were functionalized with either urease (U-MSNPs), lysozyme (L-MSNPs), or urease and lysozyme (M-MSNPs), and use them against nonpathogenic planktonic Escherichia coli. U-MSNPs exhibited the highest bactericidal activity due to biocatalysis of urea into NaHCO3 and NH3, which also propels U-MSNPs. In addition, U-MSNPs in concentrations above 200 µg/mL were capable of successfully reducing 60% of the biofilm biomass of a uropathogenic E. coli strain. This study thus provides a proof-of-concept, demonstrating that enzyme-based nanomotors are capable of fighting infectious diseases. This approach could potentially be extended to other kinds of diseases by selecting appropriate biomolecules.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Muramidasa/farmacología , Nanopartículas/química , Dióxido de Silicio/química , Ureasa/farmacología , Antibacterianos/administración & dosificación , Biocatálisis , Biopelículas/efectos de los fármacos , Canavalia/enzimología , Portadores de Fármacos/química , Escherichia coli/fisiología , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Muramidasa/administración & dosificación , Ureasa/administración & dosificación , Infecciones Urinarias/tratamiento farmacológicoRESUMEN
Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, administration of the currently available oral formulation results in systemic drug levels that are too low for the inhibition of SARS-CoV-2. We hypothesized that the co-formulation of NIC with an endogenous protein, human lysozyme (hLYS), could enable the direct aerosol delivery of the drug to the respiratory tract as an alternative to oral delivery, thereby effectively treating COVID-19 by targeting the primary site of SARS-CoV-2 acquisition and spread. To test this hypothesis, we engineered and optimized composite particles containing NIC and hLYS suitable for delivery to the upper and lower airways via dry powder inhaler, nebulizer, and nasal spray. The novel formulation demonstrates potent in vitro and in vivo activity against two coronavirus strains, MERS-CoV and SARS-CoV-2, and may offer protection against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to SARS-CoV-2 infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure rapid clinical development and wide-spread utilization.
Asunto(s)
Antivirales/administración & dosificación , Infecciones por Coronavirus/tratamiento farmacológico , Muramidasa/administración & dosificación , Niclosamida/administración & dosificación , Administración por Inhalación , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/instrumentación , Inhaladores de Polvo Seco , Humanos , Ratones Transgénicos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Muramidasa/farmacología , Muramidasa/uso terapéutico , Rociadores Nasales , Niclosamida/farmacología , Niclosamida/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
The objective of this study was to investigate the individual and combined effects of essential oils (EO; comprised of thymol and carvacrol) and lysozyme on experimental NE in broiler chickens. A total of 320 1-day-old chicks were randomly assigned to five treatment groups: no-challenge control (NC), NC + C. perfringens challenge (CC), CC + 120 mg/kg of EO, CC + 100 mg/kg of lysozyme, and CC + 120 mg/kg of EO + 100 mg/kg of lysozyme. The results showed that EO or lysozyme decreased the mortality, alleviated the gut lesions, inhibited the liver Enterobacteriaceae carriage, and increased the villus height of the ileum compared with CC (p < .05), although the proliferation of C. perfringens in the ileum was not inhibited (p > .05). Moreover, EO or lysozyme was found to decrease the ileal concentration of sialic acid and the Mucin2 mRNA expression (p < .05). However, the blend of EO and lysozyme did not display significant effect on the NE-associated mortality or gut damage in contrast with CC (p > .05). In conclusion, these findings suggest the similar protective effects of EO and lysozyme in NE-associated mortality and intestinal impairment, but their blend did not exhibit ameliorative effect.
Asunto(s)
Dieta/veterinaria , Suplementos Dietéticos , Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/veterinaria , Muramidasa/administración & dosificación , Aceites Volátiles/administración & dosificación , Enfermedades de las Aves de Corral/tratamiento farmacológico , Animales , Pollos , Infecciones por Enterobacteriaceae , Enterocolitis Necrotizante/microbiología , Enterocolitis Necrotizante/prevención & control , Masculino , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/prevención & controlRESUMEN
Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina , Duodeno , Inflamación/prevención & control , Muramidasa/administración & dosificación , Peptidoglicano , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Bacterias/metabolismo , Pared Celular/metabolismo , Células Cultivadas , Pollos/metabolismo , Cromatografía Liquida , Duodeno/microbiología , Muramidasa/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Peptidoglicano/metabolismo , Espectrometría de Masas en TándemRESUMEN
Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.
Asunto(s)
Proteínas de Peces/sangre , Proteínas de Peces/inmunología , Oncorhynchus mykiss/sangre , Oncorhynchus mykiss/inmunología , Proteoma/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Proteínas Aviares/administración & dosificación , Proteínas Aviares/inmunología , Proteínas Sanguíneas/clasificación , Proteínas Sanguíneas/inmunología , Proteínas del Huevo/administración & dosificación , Proteínas del Huevo/inmunología , Femenino , Proteínas de Peces/clasificación , Inmunización/veterinaria , Masculino , Muramidasa/administración & dosificación , Muramidasa/inmunología , Filogenia , ProteómicaRESUMEN
Biopharmaceuticals are usually administered intravenously with frequent dosing regimens which may decrease patient compliance. Controlled-release formulations allow to reduce the frequency of injections while providing a constant dosing of the biopharmaceutical over extended periods. These formulations are typically produced by emulsions, requiring high amounts of organic solvents and have limited productivity. Hot-melt extrusion (HME) is an alternative technology to produce controlled drug delivery systems. It is a continuous solvent-free process, leading to a small ecological footprint and higher productivity. However, it may induce thermolabile compounds' degradation. In this work, the impact of the formulation and extrusion temperature on lysozyme's bioactivity and release profile of poly(lactic-co-glycolic acid) (PLGA)-based extended release formulations were evaluated using a design-of-experiments (DoE) approach. The lysozyme-loaded PLGA microparticles were produced by HME followed by milling. It was observed that the in vitro release (IVR) profile was mainly affected by the drug load; higher drug load led to higher burst and total lysozyme release after 14 days. HME temperature seemed to decrease lysozyme's activity although this correlation was not statistically significant (p value = 0.0490). Adding polyethylene glycol 400 (PEG 400) as a plasticizer to the formulation had no significant impact on the lysozyme release profile. The burst release was effectively mitigated with the inclusion of a washing step. Washing the microparticles with water reduced the burst release by 80% whereas washing them with a poly(vinyl alcohol) (PVA) aqueous solution eliminated it. In conclusion, HME is demonstrated to be suitable in producing controlled-release microparticles of small biopharmaceuticals. Graphical abstract.
Asunto(s)
Preparaciones de Acción Retardada , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos , Tecnología de Extrusión de Fusión en Caliente/métodos , Muramidasa/química , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Emulsiones , Muramidasa/administración & dosificación , Solventes/químicaRESUMEN
Plant viruses are biologically safe for mammals and can be successfully used as a carrier/platform to present foreign epitopes in the course of creating novel putative vaccines. However, there is mounting evidence that plant viruses, their virus-like and structurally modified particles may also have an immunopotentiating effect on antigens not bound with their surface covalently. Here, we present data on the adjuvant properties of plant viruses with various shapes (Tobacco mosaic virus, TMV; Potato virus X, PVX; Cauliflower mosaic virus, CaMV; Bean mild mosaic virus, BMMV) and structurally modified TMV spherical particles (SPs). We have analysed the effectiveness of immune response to individual model antigens (ovalbumin, OVA/hen egg lysozyme, HEL) and to OVA/HEL in compositions with plant viruses/SPs, and have shown that CaMV, TMV and SPs can effectively induce total IgG titers to model antigen. Some intriguing data were obtained when analysing the immune response to the plant viruses/SPs themselves. Strong immunity was induced to CaMV, BMMV and PVX, whereas TMV and SPs stimulated considerably lower self-IgG titers. Our results provide new insights into the immunopotentiating properties of plant viruses and can be useful in devising adjuvants based on plant viruses.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Epítopos/inmunología , Inmunización/métodos , Muramidasa/inmunología , Ovalbúmina/inmunología , Virus de Plantas/clasificación , Virus de Plantas/inmunología , Animales , Ratones , Muramidasa/administración & dosificación , Ovalbúmina/administración & dosificaciónRESUMEN
Poor mechanical performances severely limit the application of hydrogels in vivo; for example, it is difficult to perform a very common suturing operation on hydrogels during surgery. There is a growing demand to improve the mechanical properties of hydrogels for broadening their clinical applications. Natural polyphenols can match the potential toughening sites in our previously reported PEG-lysozyme (LZM) hydrogel because polyphenols have unique structural units including a hydroxyl group and an aromatic ring that can interact with PEG via hydrogen bonding and form hydrophobic interactions with LZM. By utilizing polyphenols as noncovalent crosslinkers, the resultant PEG-LZM-polyphenol hydrogel presents super toughness and high elasticity in comparison to pristine PEG-LZM with no obvious changes in the initial shape, and it can even withstand the high pressure from sutures. At the same time, the mechanical properties could be widely adjusted by varying the polyphenol concentration. Interestingly, the PEG-LZM-polyphenol hydrogel has a higher water content than other polyphenol-toughened hydrogels, which may better meet the clinical needs for hydrogel materials. Besides, the introduction of polyphenols endows the hydrogel with improved antibacterial and anti-inflammatory abilities. Finally, the PEG-LZM-polyphenol (tannic acid) hydrogel was demonstrated to successfully patch a rabbit myocardial defect by suturing for 4 weeks and improve the wound healing and heart function recovery compared to autologous muscle patches.
Asunto(s)
Antibacterianos/administración & dosificación , Antiinflamatorios/administración & dosificación , Hidrogeles/administración & dosificación , Muramidasa/administración & dosificación , Polietilenglicoles/administración & dosificación , Polifenoles/administración & dosificación , Taninos/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/química , Antiinflamatorios/química , Línea Celular , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Femenino , Lesiones Cardíacas/tratamiento farmacológico , Hemólisis/efectos de los fármacos , Humanos , Hidrogeles/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Muramidasa/química , Polietilenglicoles/química , Polifenoles/química , Conejos , Ratas Sprague-Dawley , Taninos/químicaRESUMEN
Photoaging, the premature aging of skin induced by ultraviolet rays, is characterized by wrinkling, roughness, laxity, and pigmentary changes. Various natural and synthetic retinoids have been explored for the treatment of aging. Among retinoids, adapalene (Ada, 0.3%) is one of the most potent and widely used drugs to treat photoaging. However, it causes irritant reactions that limit its acceptance by patients. Several studies have shown the applicability of Lysozyme (Lys)-shelled microbubbles (MBs) for drug delivery through sonophoresis, and recently we have shown its efficiency to treat inflammatory skin disease. Here, we report the construction of novel Ada-LysMBs based on opposite electric charges for combined effects to treat photoaging. The Ada-LysMBs were self-assembled and had a mean diameter of 2857 nm. The maximum loading efficiency of Ada onto LysMBs was 13.99 ± 0.59%. An acoustic power density of 3 W/cm2 for 1 min revealing maximum penetration depth of LysMBs was optimized for further in vitro and in vivo studies of Ada-LysMBs. It was observed that in vitro Ada release from Ada-LysMBs at 6 h after ultrasound (US) treatment was more rapid at pH 7.4 (82%) than at pH 5.5 (73%). Franz diffusion experiments on isolated porcine skin indicated that US approximately doubled Ada delivery by Ada-LysMBs and Ada + LysMBs at 12 h and six-fold Lys permeation by LysMBs at 6 h, compared to these treatments alone. A 5-week in vivo study in mice identified significant wrinkle reduction in animals treated with US plus Ada-LysMBs. Our findings indicate that US may be used with Ada-LysMBs in the water phase to treat photoaging by normalizing hyperkeratinization and promoting collagen synthesis.
Asunto(s)
Adapaleno/administración & dosificación , Fármacos Dermatológicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Microburbujas/uso terapéutico , Muramidasa/administración & dosificación , Retinoides/administración & dosificación , Envejecimiento de la Piel/efectos de los fármacos , Ondas Ultrasónicas , Adapaleno/farmacología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Muramidasa/farmacología , Retinoides/farmacología , Porcinos , Rayos Ultravioleta/efectos adversosRESUMEN
This study assessed the effect of Spirulina (Arthrospira platensis), individually and in combination with exogenous enzymes, on growth performance, carcass traits, and meat quality of broiler chickens. One hundred and twenty Ross 308 male chickens were allocated into 40 battery brooders, with 3 birds per cage, and fed ad libitum a corn-based diet during the first 21 D of the trial. The experimental period lasted from day 21 to 35, during which birds were fed 4 different diets: a corn-soybean basal diet, taken as the control group, a basal diet containing 15% Spirulina (MA), a basal diet containing 15% Spirulina plus 0.005% Rovabio Excel AP (MAR), and a basal diet containing 15% Spirulina plus 0.01% lysozyme (MAL). Body weight gain (P < 0.001) and feed conversion rate (P < 0.001) were improved in control chickens, when compared with those fed with Spirulina. In addition, Spirulina increased the length of duodenum plus jejunum in relation to the other treatment (P < 0.01). Chickens on the MAL diet showed a considerable increase in digesta viscosity (P < 0.05) compared with the control group. Breast and thigh meats from chickens fed with Spirulina, with or without the addition of exogenous enzymes, had higher values of yellowness (b*) (P < 0.001), total carotenoids (P < 0.001), and saturated fatty acids (P < 0.001), whereas n-3 polyunsaturated fatty acid (P < 0.01) and α-tocopherol (P < 0.001) decreased, when compared with the control. In conclusion, the incorporation of 15% Spirulina in broiler diets, individually or combined with exogenous enzymes, reduced birds' performance through a higher digesta viscosity, which is likely associated with the gelation of microalga indigestible proteins. In addition, cell wall of Spirulina was successfully broken by the addition of lysozyme, but not by Rovabio Excel AP. Therefore, we anticipate that the combination of lysozyme with an exogenous specific peptidase could improve the digestibility of proteins from this microalga and avoid their detrimental gelation.
Asunto(s)
Alimentación Animal/análisis , Pollos/fisiología , Carne/análisis , Muramidasa/administración & dosificación , Spirulina/química , Animales , Pollos/crecimiento & desarrollo , Dieta/veterinaria , MasculinoRESUMEN
The aim of this work was to develop medicated chewing gums (MCGs) containing 10 mg of lysozyme hydrochloride (LH) and 20 mg of ascorbic acid (AsA) obtained by the compression method with Health in Gum® (HiG®) PWD 01 as a compressible gum base. Because of a low content of active ingredients, it was essential to choose the way of adding them to the tableting mass and evaluate their distribution homogeneity in the dosage units. The blends for compression were prepared by two methods: the first one was simple mixing of all components; the second one included the step of wet granulation of a three-component mixture - LH, sucralose and a taste additive. Flow properties of LH, AsA, HiG®, LH granules and blends for compression were studied. MCGs were evaluated according to Ph.Eur. 9.0 Chapters 2.9.5, 2.9.6 and 2.9.40. AsA and HiG® were characterized as free flowing, while LH had insufficient flow properties. Compared with a simple mixed blend, the granulation step allowed significantly improving flow properties of the final blend for compression. Unlike MCGs compressed from the simple mixed blend, MCGs prepared through the granulation step met Ph.Eur. 9.0 Chapter 2.9.40 requirements. The propriety of MCG preparation method involving the step of wet granulation also has been confirmed by mass and drug content uniformity tests.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Goma de Mascar , Portadores de Fármacos , Muramidasa/administración & dosificaciónRESUMEN
Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of incompletely understood pathophysiology predominantly affecting premature infants. While NEC is associated with microbial invasion of intestinal tissues, and mucus modulates interactions between microbes and underlying tissues, variations in mucus barrier properties with NEC-associated risk factors have not been investigated. This study explored differences in mucus composition (total protein, DNA, mucin content, sialic acid, and immunoregulatory proteins), as well as structural and transport properties, assessed by tracking of particles and bacteria (E. coli and E. cloacae) with developmental age and exposure to NEC stressors in Sprague Dawley rats. Early developmental age (5 day old) was characterized by a more permeable mucus layer relative to 21 day old pups, suggesting immaturity may contribute to exposure of the epithelium to microbes. Exposure to NEC stressors was associated with reduced mucus permeability, which may aid in survival. Feeding with breastmilk as opposed to formula reduces incidence of NEC. Thus, NEC-stressed (N-S) rat pups were orally dosed with breastmilk components lysozyme (N-S-LYS) or docosahexaenoic acid (N-S-DHA). N-S-LYS and N-S-DHA pups had a less permeable mucus barrier relative to N-S pups, which suggests the potential of these factors to strengthen the mucus barrier and thus protect against disease.
Asunto(s)
Envejecimiento/patología , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/uso terapéutico , Enterocolitis Necrotizante/tratamiento farmacológico , Moco/metabolismo , Muramidasa/administración & dosificación , Muramidasa/uso terapéutico , Estrés Fisiológico , Administración Oral , Animales , ADN/metabolismo , Ácidos Docosahexaenoicos/farmacología , Enterobacter cloacae/fisiología , Enterocolitis Necrotizante/microbiología , Escherichia coli/fisiología , Fucosa/metabolismo , Íleon/patología , Íleon/ultraestructura , Inmunoglobulina G/metabolismo , Mucinas/metabolismo , Moco/efectos de los fármacos , Muramidasa/farmacología , Ácido N-Acetilneuramínico/metabolismo , Permeabilidad , Polietilenglicoles/química , Ratas Sprague-Dawley , Estrés Fisiológico/efectos de los fármacosRESUMEN
Inhalation of proteins/peptides has recently received attention as various biopharmaceuticals have emerged on the market. Novel lyophilisates for dry powder inhalation (LDPIs), which are aerosolized by air impact, have been reported and LDPIs are considered an attractive option for the pulmonary administration of biopharmaceuticals. However, desirable disintegration and aerosolization properties have been unavailable in high-dose formulations, which has been a critical issue. This study aimed to investigate high-dose LDPIs and their optimization. In the present study, lysozyme (Lysoz) was used as a stable model protein and formulated with various amino acids. Furthermore, response surface methodology (RSM) and time-of-flight measurement were applied for efficient optimization. Superior disintegration and aerosolization properties were confirmed in the LDPIs with phenylalanine (Phe) and leucine (Leu). RSM results revealed that 0.5 mg/vial of Phe and 1.0 mg/vial of Leu are the optimal quantities for high-dose formulation. Based on these optimum quantities, high-dose LDPI formulations were prepared and the maximum formulable quantity of Lysoz with acceptable inhalation performance was confirmed to be 3.0 mg/vial. The results suggest that LDPI can cover the milligram-order pulmonary administration of proteins/peptides. LDPIs are expected to have biopharmaceutical applications.