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1.
Pharm Res ; 37(3): 33, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31942659

RESUMO

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.


Assuntos
Carboidratos/química , Carboidratos/toxicidade , Polímeros/química , Animais , Apoptose/efeitos dos fármacos , Carboximetilcelulose Sódica/química , Carboximetilcelulose Sódica/toxicidade , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Glucose/química , Glucose/toxicidade , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/toxicidade , Camundongos , Microinjeções , Agulhas , Preparações Farmacêuticas/química , Polissacarídeos/química , Polissacarídeos/toxicidade , Solubilidade , Trealose/química , Trealose/toxicidade
2.
Pharm Res ; 37(1): 11, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873825

RESUMO

PURPOSE: Loss of vaccine potency due to extreme temperature exposure during storage and transport remains a significant obstacle to the success of many vaccines, including the Bacille Calmette-Guérin (BCG) vaccine, the only vaccine available against Mycobacterium tuberculosis. BCG is a live, attenuated vaccine requiring refrigerated storage for viability. In this study, we formulated a temperature-stable BCG dry powder using the spray drying technique. METHODS: We employed a factorial design to optimize our formulation of stabilizing excipients that included L-leucine, bovine serum albumin, polyvinylpyrrolidone, mannitol, and trehalose. Powders were characterized for their particle size, yield, water retention and uptake, glass transition temperature, and aerosol performance. Three optimal powder carrier mixtures were selected from the factorial design for BCG incorporation based on their stability-promoting and powder flow characteristics. Vaccine powders were also assessed for BCG viability and in vivo immunogenicity after long-term storage. RESULTS: Live BCG was successfully spray-dried using the optimized carriers. Dry powder BCG showed no loss in viability (25°C, up to 60% relative humidity; RH) and ~2-log loss in viability (40°C, 75% RH) after one year of storage. The aerodynamic size of the powders was in the respirable range. Further, when healthy mice were immunized intradermally with reconstituted BCG powders (storage for 2 years), the vaccine retained its immunogenicity. CONCLUSION: We developed a spray-dried BCG vaccine that was viable and antigenic after long-term storage. To our knowledge, this is a first study to show room temperature stability of live BCG vaccine without any loss in viability for 12 months.


Assuntos
Vacina BCG/química , Vacina BCG/farmacologia , Composição de Medicamentos/métodos , Excipientes/química , Pós/química , Aerossóis/química , Animais , Linhagem Celular , Sobrevivência Celular , Dessecação/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Feminino , Humanos , Leucina/química , Manitol/química , Camundongos Endogâmicos C57BL , Mycobacterium bovis/citologia , Povidona/química , Soroalbumina Bovina/química , Temperatura Ambiente , Distribuição Tecidual , Trealose/química
3.
Pharm Res ; 37(1): 14, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873808

RESUMO

PURPOSE: The aim of this study is to determine the effects of saccharide-containing excipients on the surface composition of spray-dried protein formulations and their matrix heterogeneity. METHODS: Spray-dried formulations of myoglobin or bovine serum albumin (BSA) were prepared without excipient or with sucrose, trehalose, or dextrans. Samples were characterized by solid-state Fourier-transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while conformational differences were determined by solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). RESULTS: Structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextran-containing formulations. Using ssHDX-MS, the amount of deuterium incorporation and populations present were the largest in the samples processed with dextrans. Linear correlation was found between protein surface coverage and ssHDX-MS peak area (R2 = 0.853) for all formulations with saccharide-containing excipients. CONCLUSIONS: Lower molecular weight species of saccharides tend to enrich the particle surface and reduce protein concentration at the air-liquid interface, resulting in reduced population heterogeneity and improved physical stability, as identified by ssHDX-MS.


Assuntos
Excipientes/química , Mioglobina/química , Soroalbumina Bovina/química , Química Farmacêutica/métodos , Dessecação/métodos , Deutério/química , Dextranos/química , Espectrometria de Massas/métodos , Sacarose/química , Propriedades de Superfície , Trealose/química
4.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 36(5): 803-809, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31631629

RESUMO

Cell freeze-drying can be divided into the freezing and drying processes. Mechanical damage caused by ice crystals and damage from solute during freezing shall not be ignored and lyoprotectants are commonly used to reduce those damages on cells. In order to study the mechanism of lyoprotectants to protect cells and determine an optimal lyoprotectant formula, the thermophysical properties and percentage of unfrozen water of different lyoprotectants in freezing were investigated with differential scanning calorimeter (DSC). The survival rate indicated by trypan blue exclusion test and cell-attachment rate after 24 h using different lyoprotectants to freeze hepatoma Hep-G 2 cells were measured after cell cryopreservation. The results show that 40% (W/V) PVP + 10% (V/V) glycerol + 15% (V/V) fetal bovine serum + 20% (W/V) trehalose formula of lyoprotectant demonstrate the best effect in protecting cells during freezing, for cell-attachment rate after 24 h is 44.56% ± 2.73%. In conclusion, the formula of lyoprotectant mentioned above can effectively protect cells.


Assuntos
Criopreservação , Crioprotetores/química , Congelamento , Trealose/química , Varredura Diferencial de Calorimetria , Liofilização , Células Hep G2 , Humanos
5.
Molecules ; 24(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505895

RESUMO

In animal nutrition, probiotics are considered as desirable alternatives to antibiotic growth promoters. The beneficial effects of probiotics primarily depend on their viability in feed, which demands technical optimization of biomass production, since processing and storage capacities are often strain-specific. In this study, we optimized the production parameters for two broiler-derived probiotic lactobacilli (L. salivarius and L. agilis). Carbohydrate utilization of both strains was determined and preferred substrates that boosted biomass production in lab-scale fermentations were selected. The strains showed good aerobic tolerance, which resulted in easier scale-up production. For the freeze-drying process, the response surface methodology was applied to optimize the composition of cryoprotective media. A quadratic polynomial model was built to study three protective factors (skim milk, sucrose, and trehalose) and to predict the optimal working conditions for maximum viability. The optimal combination of protectants was 0.14g/mL skim milk/ 0.08 g/mL sucrose/ 0.09 g/mL trehalose (L. salivarius) and 0.15g/mL skim milk/ 0.08 g/mL sucrose/ 0.07 g/mL (L. agilis), respectively. Furthermore, the in-feed stabilities of the probiotic strains were evaluated under different conditions. Our results indicate that the chosen protectants exerted an extensive protection on strains during the storage. Although only storage of the strains at 4 °C retained the maximum stability of both Lactobacillus strains, the employed protectant matrix showed promising results at room temperature.


Assuntos
Ração Animal , Lactobacillus/química , Leite/química , Probióticos/química , Fenômenos Fisiológicos da Nutrição Animal , Animais , Galinhas , Crioprotetores , Suplementos Nutricionais , Fermentação , Liofilização , Viabilidade Microbiana , Trealose/química
6.
Eur J Pharm Biopharm ; 144: 139-153, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536784

RESUMO

Trehalose is commonly used as a protein stabilizer in spray dried protein formulations delivered via the pulmonary route. Spray dried trehalose formulations are highly hygroscopic, which makes them prone to deliquescence and recrystallization when exposed to moisture, leading to impairment in aerosolization performance. The main aim of this study was to investigate and compare the effect of hydrophobic amino acids (i.e. L-leucine and L-isoleucine) in enhancing aerosolization performance and in mitigating moisture-induced changes in spray dried trehalose formulations. Trehalose was spray dried with 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine). The spray dried formulations were stored at 25 °C/50% RH for 28 days. Solid state characterization and in vitro aerosolization performance studies were performed on the spray dried formulations before and after storage. The addition of 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine) improved the emitted fractions of spray dried trehalose formulations from a dry powder inhaler. However, ≥ 40% w/w of L-leucine/L-isoleucine was needed to prevent recrystallization of trehalose in the formulations when exposed to 25 °C/50% RH for 28 days. X-ray photoelectron spectroscopy (XPS) demonstrated that samples with 40-60% w/w L-isoleucine had more amino acid on the surfaces of the particles compared to their L-leucine counterparts. This may explain the greater ability of the L-isoleucine (40-60% w/w) samples to cope with elevated humidity compared to L-leucine samples of the same concentrations, as observed in the dynamic vapour sorption (DVS) studies. In conclusion, this study demonstrated that both L-leucine and L-isoleucine were effective in enhancing aerosolization performance and mitigating moisture-induced reduction in aerosolization performance in spray dried trehalose formulations. L-isoleucine proved to be superior to L-leucine in terms of its moisture protectant effect when incorporated at the same concentration in the formulations.


Assuntos
Aminoácidos/química , Trealose/química , Administração por Inalação , Aerossóis/química , Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Inaladores de Pó Seco/métodos , Umidade , Interações Hidrofóbicas e Hidrofílicas , Leucina/química , Pós/química , Molhabilidade/efeitos dos fármacos
7.
Int J Pharm ; 569: 118601, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31394183

RESUMO

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p < 0.05) titer reduction than spray drying using a vibrating mesh nebulizer. The use of centrifugation and dilution of filtered bacteriophage lysate in the formulation step resulted in a significantly greater (p < 0.05) proportion of bacteriophages remaining active relative to use of no centrifugation and dilution. The spray-dried bacteriophage powder generated using leucine and trehalose as excipients was flowable, non-cohesive, and exhibited a high manufacturing yield. The powder retained its titer with no significant differences (p > 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 km, including with a 38 °C temperature excursion. The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure.


Assuntos
Bacteriófagos , Campylobacter/virologia , Química Farmacêutica , Dessecação , Excipientes/química , Leucina/química , Pós , Trealose/química
8.
Int J Pharm ; 569: 118608, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31415881

RESUMO

With respect to all biopharmaceuticals marketed to date, monoclonal antibodies represent the largest fraction with more than 48% market share (2012). However, the development of biopharmaceutical formulations is a challenging task, and time-consuming and cost-intensive high-throughput screenings are still state-of-the-art in formulation design. These screening techniques are almost exclusively based on heuristic decisions thus the benefit in terms of mechanistic understanding is often unclear. It requires novel, physical-sound methods to enhance/optimize future formulation development, ideally by understanding molecular interactions in these complex solutions. A suitable and evaluated measure-of-choice to characterize protein-protein interactions in aqueous protein solutions is the second osmotic virial coefficient B22 which can be measured using static light scattering techniques. Furthermore B22 can be modeled/predicted via the extended mxDLVO model for protein-protein interactions in the presence of single excipients and excipient-mixtures. Building up on this approach, giving an additional insight into water-water and water-excipient interactions, the thermodynamic equation-of-state ePC-SAFT is used to calculate water activity coefficients in the presence of excipient-mixtures. Immunoglobulin G (IgG) was chosen as a model protein to predict B22-values for IgG in the presence of model excipient-mixtures (trehalose-NaCl, l-histidine-trehalose, l-histidine-NaCl). The combination of water activity coefficients and B22 allows to quickly identify a first guess on suitable formulation conditions that then can be further evaluated with existing methods/knowledge.


Assuntos
Excipientes/química , Imunoglobulina G/química , Água/química , Composição de Medicamentos , Histidina/química , Cloreto de Sódio/química , Trealose/química
9.
Eur J Pharm Biopharm ; 143: 61-69, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31445157

RESUMO

Localized aerosol delivery of gene therapies is a promising treatment of severe pulmonary diseases including lung cancer, cystic fibrosis, COPD and asthma. The administration of drugs by inhalation features multiple benefits including an enhanced patient acceptability and compliance. The application of a spray dried powder formulation has advantages over solutions due to their increased stability and shelf life. Furthermore, optimal sizes of the powder can be obtained by spray drying to allow a deep lung deposition. The present study optimized the parameters involved with spray drying polyplexes formed by polyethylenimine (PEI) and nucleic acids in inert excipients to generate a nano-embedded microparticle (NEM) powder with appropriate aerodynamic diameter. Furthermore, the effects of the excipient matrix used to generate the NEM powder on the biological activity of the nucleic acid and the ability to recover the embedded nanoparticles was investigated. The study showed that bioactivity and nucleic acid integrity was preserved after spray drying, and that polyplexes could be reconstituted from the dry powders made with trehalose but not mannitol as a stabilizer. Scanning electron microscopy (SEM) showed trehalose formulations that formed fused, lightly corrugated spherical particles in the range between 1 and 5 µm, while mannitol formulations had smooth surfaces and consisted of more defined particles. After redispersion of the microparticles in water, polyplex dispersions are obtained that are comparable to the initial formulations before spray drying. Cellular uptake and transfection studies conducted in lung adenocarcinoma cells show that redispersed trehalose particles performed similar to or better than polyplexes that were not spray dried. A method for quantifying polymer and nucleic acid loss following spray drying was developed in order to ensure that equal nucleic acid amounts were used in all in vitro experiments. The results confirm that spray dried NEM formulations containing nucleic acids can be prepared with characteristics known to be optimal for inhalation therapy.


Assuntos
Nanopartículas/química , Ácidos Nucleicos/química , Polietilenoimina/química , Pós/química , Células A549 , Administração por Inalação , Aerossóis/química , Varredura Diferencial de Calorimetria/métodos , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Dessecação/métodos , Excipientes/química , Humanos , Manitol/química , Microscopia Eletrônica de Varredura/métodos , Tamanho da Partícula , Trealose/química
10.
Carbohydr Polym ; 223: 115065, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31426953

RESUMO

Probiotics are believed to provide benefits to human health; however, a good storage stability is the prerequisite for the probiotic products making function. Herein, we reported a pathway to fabricate the Ca-alginate/cryoprotectants/cellulose composite (ACFP) capsules to protect L. plantarum cells, which showed the minimal loss of viability during the vacuum freeze-drying process. The trehalose and whey protein isolate (WPI) ingredients in the cryoprotectants prolonged the dissolution time of Ca-alginate shell, which contributed to controlling the release of cells in the desired region. The dry ACFP capsules exhibited gradual release of L. plantarum cells in simulated intestinal fluid (SIF), 2.6 × 106 cfu/mL at 210 min. In addition, 0.1 g of the dry ACFP capsules showed the viable release amount of 3.3 × 106 cfu/mL after the storage of 160 days at 4 °C. The promising results provided a strategy of encapsulating probiotic cells to achieve long-term storage stability and enhanced controlled release behavior in simulated intestinal fluid in the meantime.


Assuntos
Alginatos/química , Celulose/química , Lactobacillus plantarum , Probióticos , Cápsulas/química , Crioprotetores/química , Preparações de Ação Retardada/química , Composição de Medicamentos/métodos , Glicerol/química , Viabilidade Microbiana/efeitos dos fármacos , Trealose/química , Proteínas do Soro do Leite/química
11.
Biochimie ; 165: 196-205, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31408673

RESUMO

Chemical chaperones are a class of small molecules which enhance folding and prevent aggregation of proteins. Investigation of their effects on the processes of protein aggregation is of importance for further understanding of implication of protein aggregation in neurodegenerative diseases, as well as for solving biotechnological tasks. The effects of chemical chaperones trehalose and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) at 37 °C have been studied. The process of thermal aggregation of UV-Phb includes a slow stage of structural reorganization of the UV-Phb molecule, nucleation stage and fast attachment of structurally reorganized UV-Phb molecules to nuclei formed during the nucleation stage. It was shown that both trehalose and HP-ß-CD increased the duration of the nucleation phase and slowed down the rate of structural reorganization of the UV-Phb molecule. This conclusion has been confirmed by the circular dichroism data. In the absence of chaperones, 82% UV-Phb aggregates, whereas in the presence of HP-ß-CD or trehalose the portion of aggregated protein decreases to 70 and 66%, respectively. The data on analytical ultracentrifugation demonstrated that in the presence of these additives the size of protein aggregates decreased. Analysis of the combined effect of trehalose and HP-ß-CD on UV-Phb aggregation showed that protein aggregation was independently affected by trehalose and HP-ß-CD.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Glicogênio Fosforilase Muscular/química , Agregados Proteicos , Trealose/química , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia
12.
Chem Commun (Camb) ; 55(57): 8227-8230, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268107

RESUMO

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.


Assuntos
Nanoestruturas/química , Plasmídeos/química , Tensoativos/química , Transfecção/métodos , Trealose/química , Animais , Linhagem Celular , Humanos , Camundongos , Plasmídeos/metabolismo
13.
Carbohydr Res ; 482: 107739, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31288124

RESUMO

Solvent-free synthesis encourages the design of processes and products that reduce the use and generation of hazardous chemicals. Given the importance of developing greener methodologies, we sought to determine the factors influencing the reaction temperature required for solvent-free, enzymatic synthesis of sugar esters such as trehalose (TRE) esters, using Novozyme 435 as the enzyme catalyst. The use of lauric acid (La) and ethyl laurate (LaEt) as acyl donors did not affect the activation temperature for the generation of trehalose diesters (TDEs), despite the differences in corresponding by-products (water and ethanol). However, when glucose (GLU) and La were employed as reaction substrates as a comparison, glucose monoester (GME) generation readily occurred at much lower temperatures than with the TRE esters, even without a water collection device. Moreover, when the glass transition temperature (Tg) of the sugar substrates increased, a higher reaction temperature was required. These results suggest that while the activation temperature of the reaction did not correlate with the boiling point of the by-product, it did correlate with the glass transition temperature (Tg) of the trehalose substrates. Thus, our work demonstrates the importance of the physical state of amorphous matrices in determining the optimal reaction temperature of a solvent-free sugar synthesis.


Assuntos
Ésteres/química , Temperatura Ambiente , Trealose/química , Trealose/síntese química , Técnicas de Química Sintética , Glucose/química , Concentração de Íons de Hidrogênio , Lauratos/química
14.
J Chem Phys ; 151(1): 015101, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31272172

RESUMO

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.


Assuntos
Luz , Muramidase/química , Espalhamento de Radiação , Análise Espectral/métodos , Trealose/química , Água/química
15.
Cryo Letters ; 40(4): 200-208, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31278400

RESUMO

BACKGROUND: Glycerol, sucrose and trehalose are used as protectants for membrane, protein, cell and tissue preservation. The undercooled state (glassy or rubbery) of their solutions may also offer protection for protein, cells and tissues against radiation damage upon sterilization. OBJECTIVE: The study aimed to examine the protective effects of glycerol, sucrose and trehalose on cryopreserved acellular human dermis against gamma irradiation damage. MATERIALS AND METHODS: Acellular human dermis was cryopreserved at -80°C in glycerol, sucrose and trehalose solutions or their combinations with a base citrate-phosphate buffer (pH 6.0). Cryopreserved acellular dermis was then subjected to 13 kGy gamma irradiation at -78.5°C, and radiation damage was assessed by histological evaluation. RESULTS: Freeze and thaw alone do not alter the structure of acellular dermis, but gamma irradiation at -78.5°C results in significant structural changes in acellular dermis, including the formation of large holes, the damage of collagen fibers and the loss of overall dermis tissue histology. The incorporation of glycerol, sucrose and trehalose into cryopreservation solutions reduces gamma irradiation-induced tissue structural damage considerably. When used alone, trehalose (0.5 M) provided better protection against gamma irradiation damage than did sucrose (0.5 M) and glycerol (1.0 M). When used in combination, the glycerol and trehalose combination provides the best tissue protection. Significant donor-to-donor variation exists in tissue damage after gamma irradiation. For donor dermis that is less sensitive to gamma irradiation damage, glycerol, sucrose or trehalose alone is able to provide good protection. However, for more sensitive donor dermis, only the glycerol and trehalose combination is able to provide sufficient tissue protection. CONCLUSION: Glycerol, sucrose and trehalose protects cryopreserved acellular human dermis against gamma irradiation damage. Cryopreservation solutions can be optimized to permit tissues for gamma sterilization to increase the safety human tissue implants.


Assuntos
Derme Acelular/efeitos dos fármacos , Crioprotetores/química , Raios gama/efeitos adversos , Glicerol/química , Sacarose/química , Trealose/química , Derme Acelular/efeitos da radiação , Criopreservação , Humanos
16.
Cryo Letters ; 40(4): 247-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31278406

RESUMO

BACKGROUND: Data are scarce on thermophysical properties of the glassy trehalose solution at low temperatures. OBJECTIVE: Water vapor pressure above the glassy trehalose solution and the relaxation behavior were studied at temperatures from -57°C to -40°C and at concentrations from 71% to 78% (w/w). MATERIALS AND METHODS: Glassy trehalose solutions were prepared by quenching in liquid nitrogen. Vapour pressure was measured using the static method. RESULTS: Vapour pressure above the glassy trehalose solution was slightly lower than above the glassy sucrose solution. The relaxation of the glassy state can be described by the stretched exponential Kohlrausch-Williams-Watts (KWW) function. The characteristic times of water relaxation (Τp) were compared with those of enthalpy relaxation (Τe). When the difference (∆T) between the glass transition temperature (Tg) and ageing temperature was relatively small, i.e. ∆T ≤ 10 K, Τp is close to Τe. If ∆T ≥ 15 K, Τe will be much greater than Τp. CONCLUSION: The difference of water vapor pressure above the trehalose glassy solution and sucrose glassy solution could lead to significant distinction between their drying kinetics.


Assuntos
Crioprotetores/química , Trealose/química , Pressão de Vapor , Criopreservação , Sacarose , Temperatura Ambiente , Termodinâmica
17.
Int J Pharm ; 569: 118568, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31352055

RESUMO

In freeze-dried protein formulations, the composition governs the physical forms of the excipients and hence their functionality. It is also necessary to understand the effect of composition on the molecular relaxation behavior, a key factor influencing protein stability. Mannitol (bulking agent) - trehalose (lyoprotectant) - bovine serum albumin (BSA) lyophiles with varying trehalose to BSA mass ratios were investigated. The crystalline phases were characterized by X-ray diffractometry. The secondary structure of albumin in lyophiles and reconstituted solutions was evaluated by IR spectroscopy and circular dichroism, respectively. Dielectric spectroscopy was used to obtain the relaxation time of freeze-dried samples. When trehalose to BSA ratio was 0.2, while mannitol crystallized predominantly as the δ-anhydrous polymorph, trehalose remained amorphous. At lower concentrations of BSA, mannitol crystallized in both hemihydrate and anhydrous forms, and trehalose as dihydrate. The extent of dehydration during subsequent drying was dictated by the trehalose to BSA ratio in the formulation. A gradual increase in the Johari-Goldstein relaxation time was observed as the concentration of trehalose increased in the formulation. BSA was more susceptible to stresses from thawing than drying.


Assuntos
Excipientes/química , Manitol/química , Soroalbumina Bovina/química , Trealose/química , Cristalização , Estabilidade de Medicamentos , Liofilização
18.
Chem Biodivers ; 16(9): e1900353, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31329336

RESUMO

Five known secondary metabolites, chrysophanol (1), 7,7'-biphyscion (2), secalonic acid D (3), mannitol (4) and trehalose (5) were isolated for the first time from the extracts of the fungus Phialomyces macrosporus. Their structures were elucidated by NMR methods (1D and 2D NMR analysis), optical activity and ESI-MS. Complete 1 H and 13 C assignments were performed for compound 2. The antimicrobial activity was evaluated by serial microdilution assay for compounds 2 and 3 and results showed that compound 3 exhibited a significant growth inhibition at concentrations of 15.6 mg/ml (S. aureus and S. choleraesius) and 0.97 mg/mL (B. subtilis), comparable to the positive control.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Salmonella/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antraquinonas/química , Antraquinonas/isolamento & purificação , Antraquinonas/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacillus subtilis/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância Magnética , Manitol/química , Manitol/isolamento & purificação , Manitol/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Salmonella/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Trealose/química , Trealose/isolamento & purificação , Trealose/farmacologia , Xantonas/química , Xantonas/isolamento & purificação , Xantonas/farmacologia
19.
Eur J Pharm Biopharm ; 142: 216-221, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31233863

RESUMO

The specific surface area (SSA) of freeze-dried biologics (FD) is usually measured via a Brunauer-Emmett-Teller (BET) analysis of volumetric nitrogen adsorption isotherms. However, this technique has accuracy limitations for materials <0.5 m2/g, requires dry samples, must be measured at 77 K and has slow sample preparation times (drying/degassing). Inverse gas chromatography (IGC) is chromatographic characterization technique which can be used to analyse the SSA (down to ≈0.1 m2/g) of various solid-state materials including powders using organic molecules such as octane at ambient temperatures/pressure for a range of relative humidities. This study presents a comprehensive comparison between the N2 BET adsorption versus octane BET adsorption using IGC methods for determining the SSA's for a range of freeze dried biological materials. These include IgG 5% w/w, an influenza antigen standard, sucrose 5% w/w and trehalose 5% w/w. IGC provided comparable SSA values to the N2 BET method with better reproducibility (lower RSDs %). Large variations in average SSA within manufactured FD batches were observed for both IGC and volumetric determinations. IGC was also used to measure the change in SSA with increasing humidity, with FD cakes shrinking and losing their structural integrity with increasing moisture content. Such information highlights the importance of moisture content in determining the physical stability of FD cakes as exemplified by their SSA. In conclusion, IGC is a suitable alternative method for determining the SSA of freeze-dried biological materials which are generally strongly dependent on their moisture content.


Assuntos
Produtos Biológicos/química , Adsorção , Química Farmacêutica/métodos , Cromatografia Gasosa/métodos , Dessecação/métodos , Excipientes/química , Liofilização/métodos , Umidade , Pós/química , Reprodutibilidade dos Testes , Sacarose/química , Temperatura Ambiente , Trealose/química
20.
Prep Biochem Biotechnol ; 49(9): 846-857, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31244369

RESUMO

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22 ± 1.35% with the viable cell number of 9.5 ± 0.07 × 109 CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6 × 109 CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k-18 = 8.04 × 10-6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k4 = 4.48 × 10-4 and k25 = 9.72 × 10-3 under 4 and 25 °C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.


Assuntos
Liofilização/métodos , Saccharomyces boulardii/citologia , Crioprotetores/química , Ácido Glutâmico/química , Lactose/química , Viabilidade Microbiana , Probióticos/análise , Trealose/química
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