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1.
Nat Chem Biol ; 20(8): 1053-1065, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38424171

RESUMO

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.


Assuntos
Escherichia coli , Estabilidade Proteica , Proteoma , Proteoma/metabolismo , Proteoma/química , Humanos , Escherichia coli/metabolismo , Temperatura , Betaína/química , Betaína/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Trealose/química , Trealose/metabolismo , Proteômica/métodos , Prolina/química , Prolina/metabolismo , Glucose/química , Glucose/metabolismo , Glicerol/química , Glicerol/metabolismo , Metilaminas
2.
Biochem Biophys Res Commun ; 727: 150323, 2024 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-38945065

RESUMO

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.


Assuntos
Saponinas , Trealose , Saponinas/química , Trealose/química , Imunoprecipitação/métodos , Animais , Detergentes/química , Ligação Proteica , Humanos , Octoxinol/química
3.
Mol Pharm ; 21(7): 3634-3642, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38805365

RESUMO

Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine Escherichia coli adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.


Assuntos
Adenilato Quinase , Escherichia coli , Liofilização , Trealose , Liofilização/métodos , Adenilato Quinase/metabolismo , Trealose/química , Soroalbumina Bovina/química , Excipientes/química , Varredura Diferencial de Calorimetria , Maltose/química , Histidina/química , Arginina/química , Espectroscopia de Ressonância Magnética
4.
Mol Pharm ; 21(7): 3163-3172, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38781678

RESUMO

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the ß-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.


Assuntos
Liofilização , Espectroscopia de Ressonância Magnética , Sacarose , Trealose , Trealose/química , Sacarose/química , Liofilização/métodos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Albumina Sérica Humana/química , Albumina Sérica/química , Estabilidade de Medicamentos , Química Farmacêutica/métodos , Excipientes/química , Dissacarídeos/química
5.
Mol Pharm ; 21(5): 2555-2564, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38551918

RESUMO

Poloxamer 188 (P188) was hypothesized to be a dual functional excipient, (i) a stabilizer in frozen solution to prevent ice-surface-induced protein destabilization and (ii) a bulking agent to provide elegant lyophiles. Based on X-ray diffractometry and differential scanning calorimetry, sucrose, in a concentration-dependent manner, inhibited P188 crystallization during freeze-drying, while trehalose had no such effect. The recovery of lactate dehydrogenase (LDH), the model protein, was evaluated after reconstitution. While low LDH recovery (∼60%) was observed in the lyophiles prepared with P188, the addition of sugar improved the activity recovery to >85%. The secondary structure of LDH in the freeze-dried samples was assessed using infrared spectroscopy, and only moderate structural changes were observed in the lyophiles formulated with P188 and sugar. Thus, P188 can be a promising dual functional excipient in freeze-dried protein formulations. However, P188 alone does not function as a lyoprotectant and needs to be used in combination with a sugar.


Assuntos
Varredura Diferencial de Calorimetria , Excipientes , Liofilização , Poloxâmero , Trealose , Liofilização/métodos , Poloxâmero/química , Excipientes/química , Trealose/química , Varredura Diferencial de Calorimetria/métodos , Sacarose/química , Difração de Raios X , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/química , Cristalização/métodos , Química Farmacêutica/métodos , Proteínas/química , Composição de Medicamentos/métodos , Congelamento
6.
Mol Pharm ; 21(6): 3017-3026, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38758116

RESUMO

Sucrose and trehalose pharmaceutical excipients are employed to stabilize protein therapeutics in a dried state. The mechanism of therapeutic protein stabilization is dependent on the sugars being present in an amorphous solid-state. Colyophilization of sugars with high glass transition polymers, polyvinylpyrrolidone (PVP), and poly(vinylpyrrolidone vinyl acetate) (PVPVA), enhances amorphous sugar stability. This study investigates the stability of colyophilized sugar-polymer systems in the frozen solution state, dried state postlyophilization, and upon exposure to elevated humidity. Binary systems of sucrose or trehalose with PVP or PVPVA were lyophilized with sugar/polymer ratios ranging from 2:8 to 8:2. Frozen sugar-PVPVA solutions exhibited a higher glass transition temperature of the maximally freeze-concentrated amorphous phase (Tg') compared to sugar-PVP solutions, despite the glass transition temperature (Tg) of PVPVA being lower than PVP. Tg values of all colyophilized systems were in a similar temperature range irrespective of polymer type. Greater hydrogen bonding between sugars and PVP and the lower hygroscopicity of PVPVA influenced polymer antiplasticization effects and the plasticization effects of residual water. Plasticization due to water sorption was investigated in a dynamic vapor sorption humidity ramping experiment. Lyophilized sucrose systems exhibited increased amorphous stability compared to trehalose upon exposure to the humidity. Recrystallization of trehalose was observed and stabilized by polymer addition. Lower concentrations of PVP inhibited trehalose recrystallization compared to PVPVA. These stabilizing effects were attributed to the increased hydrogen bonding between trehalose and PVP compared to trehalose and PVPVA. Overall, the study demonstrated how differences in polymer hygroscopicity and hydrogen bonding with sugars influence the stability of colyophilized amorphous dispersions. These insights into excipient solid-state stability are relevant to the development of stabilized biopharmaceutical solid-state formulations.


Assuntos
Estabilidade de Medicamentos , Excipientes , Liofilização , Polímeros , Povidona , Temperatura de Transição , Trealose , Liofilização/métodos , Povidona/química , Trealose/química , Excipientes/química , Polímeros/química , Sacarose/química , Açúcares/química , Ligação de Hidrogênio , Armazenamento de Medicamentos , Química Farmacêutica/métodos , Varredura Diferencial de Calorimetria , Umidade , Pirrolidinas/química , Compostos de Vinila/química
7.
Biomacromolecules ; 25(5): 3190-3199, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38693753

RESUMO

Intracellular bacteria in dormant states can escape the immune response and tolerate high-dose antibiotic treatment, leading to severe infections. To overcome this challenge, cascade-targeted nanoplatforms that can target macrophages and intracellular bacteria, exhibiting synergetic antibiotic/reactive oxygen species (ROS)/nitric oxide (NO)/immunotherapy, were developed. These nanoplatforms were fabricated by encapsulating trehalose (Tr) and vancomycin (Van) into phosphatidylserine (PS)-coated poly[(4-allylcarbamoylphenylboric acid)-ran-(arginine-methacrylamide)-ran-(N,N'-bisacryloylcystamine)] nanoparticles (PABS), denoted as PTVP. PS on PTVP simulates a signal of "eat me" to macrophages to promote cell uptake (the first-step targeting). After the uptake, the nanoplatform in the acidic phagolysosomes could release Tr, and the exposed phenylboronic acid on the nanoplatform could target bacteria (the second-step targeting). Nanoplatforms can release Van in response to infected intracellular overexpressed glutathione (GSH) and weak acid microenvironment. l-arginine (Arg) on the nanoplatforms could be catalyzed by upregulated inducible nitric oxide synthase (iNOS) in the infected macrophages to generate nitric oxide (NO). N,N'-Bisacryloylcystamine (BAC) on nanoplatforms could deplete GSH, allow the generation of ROS in macrophages, and then upregulate proinflammatory activity, leading to the reinforced antibacterial capacity. This nanoplatform possesses macrophage and bacteria-targeting antibiotic delivery, intracellular ROS, and NO generation, and pro-inflammatory activities (immunotherapy) provides a new strategy for eradicating intracellular bacterial infections.


Assuntos
Antibacterianos , Nanopartículas , Óxido Nítrico , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Óxido Nítrico/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Camundongos , Animais , Células RAW 264.7 , Nanopartículas/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/imunologia , Imunoterapia/métodos , Vancomicina/farmacologia , Vancomicina/química , Vancomicina/administração & dosagem , Infecções Bacterianas/tratamento farmacológico , Trealose/química , Trealose/farmacologia
8.
J Chem Phys ; 160(24)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38912631

RESUMO

We study, through molecular dynamics simulations, three aqueous solutions with one lysozyme protein and three different concentrations of trehalose and dimethyl sulfoxide (DMSO). We analyze the structural and dynamical properties of the protein hydration water upon cooling. We find that trehalose plays a major role in modifying the structure of the network of HBs between water molecules in the hydration layer of the protein. The dynamics of hydration water presents, in addition to the α-relaxation, typical of glass formers, a slower long-time relaxation process, which greatly slows down the dynamics of water, particularly in the systems with trehalose, where it becomes dominant at low temperatures. In all the solutions, we observe, from the behavior of the α-relaxation times, a shift of the Mode Coupling Theory crossover temperature and the fragile-to-strong crossover temperature toward higher values with respect to bulk water. We also observe a strong-to-strong crossover from the temperature behavior of the long-relaxation times. In the aqueous solution with only DMSO, the transition shifts to a lower temperature than in the case with only lysozyme reported in the literature. We observe that the addition of trehalose to the mixture has the opposite effect of restoring the original location of the strong-to-strong crossover. In all the solutions analyzed in this work, the observed temperature of the protein dynamical transition is slightly shifted at lower temperatures than that of the strong-to-strong crossover, but their relative order is the same, showing a correlation between the motion of the protein and that of the hydration water.


Assuntos
Dimetil Sulfóxido , Simulação de Dinâmica Molecular , Muramidase , Trealose , Água , Trealose/química , Dimetil Sulfóxido/química , Muramidase/química , Água/química , Crioprotetores/química , Criopreservação/métodos , Temperatura Baixa
9.
Cryobiology ; 115: 104886, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38555011

RESUMO

Nowadays, the physical nature of supersaturated binary aqueous sugar solutions in the vicinity of the glass transition represents a very important issue due to their biological applications in cryopreservation of cells and tissues, food science and stabilization and storage of nano genetic drugs. We present the construction of the Supplemented Phase Diagram and the non-equilibrium nature of the undersaturated-supersaturated kinetic transition. The description of its thermodynamic nature is achieved through the study of behavior of their viscosity as temperature is lowered and concentration increased. In this work, we find a universal character for the viscosities of several sugar water solutions.


Assuntos
Criopreservação , Vitrificação , Água , Viscosidade , Criopreservação/métodos , Água/química , Açúcares/química , Crioprotetores/química , Crioprotetores/farmacologia , Termodinâmica , Transição de Fase , Soluções , Sacarose/química , Trealose/química , Temperatura
10.
Cryobiology ; 115: 104898, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38663665

RESUMO

Trehalose is widely acknowledged for its ability to stabilize plasma membranes during dehydration. However, the exact mechanism by which trehalose interacts with lipid bilayers remains presently unclear. In this study, we conducted atomistic molecular dynamic simulations on asymmetric model bilayers that mimic the membrane of human red blood cells at various trehalose and water contents. We considered three different hydration levels mimicking the full hydration to desiccation scenarios. Results indicate that the asymmetric distribution of lipids did not significantly influence the computed structural characteristics at full and low hydration. At dehydration, however, the order parameter obtained from the symmetric bilayer is significantly higher compared to those obtained from asymmetric ones. Analysis of hydrogen bonds revealed that the protective ability of trehalose is well described by the water replacement hypothesis at full and low hydration, while at dehydration other interaction mechanisms associated with trehalose exclusion from the bilayer may involve. In addition, we found that trehalose exclusion is not attributed to sugar saturation but rather to the reduction in hydration levels. It can be concluded that the protective effect of trehalose is not only related to the hydration level of the bilayer, but also closely tied to the asymmetric distribution of lipids within each leaflet.


Assuntos
Membrana Eritrocítica , Ligação de Hidrogênio , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Trealose , Trealose/metabolismo , Trealose/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Humanos , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/química , Água/química , Água/metabolismo , Eritrócitos/metabolismo , Eritrócitos/química , Dessecação
11.
Int J Mol Sci ; 25(15)2024 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-39126073

RESUMO

The spread of multidrug-resistant mycobacterium strains requires the development of new approaches to combat diseases caused by these pathogens. For that, photodynamic inactivation (PDI) is a promising approach. In this study, a tricarbocyanine (TCC) is used for the first time as a near-infrared (740 nm) activatable PDI photosensitizer to kill mycobacteria with deep light penetration. For better targeting, a novel tricarbocyanine dye functionalized with two trehalose units (TCC2Tre) is developed. The photodynamic effect of the conjugates against mycobacteria, including Mycobacterium tuberculosis, is evaluated. Under irradiation, TCC2Tre causes more effective killing of mycobacteria compared to the photosensitizer without trehalose conjugation, with 99.99% dead vegetative cells of M. tuberculosis and M. smegmatis. In addition, effective photoinactivation of dormant forms of M. smegmatis is observed after incubation with TCC2Tre. Mycobacteria treated with TCC2Tre are more sensitive to 740 nm light than the Gram-positive Micrococcus luteus and the Gram-negative Escherichia coli. For the first time, this study demonstrates the proof of principle of in vitro PDI of mycobacteria including the fast-growing M. smegmatis and the slow-growing M. tuberculosis using near-infrared activatable photosensitizers conjugated with trehalose. These findings are useful for the development of new efficient alternatives to antibiotic therapy.


Assuntos
Raios Infravermelhos , Mycobacterium smegmatis , Mycobacterium tuberculosis , Fármacos Fotossensibilizantes , Trealose , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/efeitos da radiação , Trealose/farmacologia , Trealose/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Carbocianinas/química , Carbocianinas/farmacologia , Fotoquimioterapia/métodos
12.
Int J Mol Sci ; 25(18)2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39337517

RESUMO

The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand-receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound 4d, tri-substituted and five carbons long, induced significantly greater levels of interleukin-1ß (IL-1ß), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of 4d highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure-activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.


Assuntos
Lectinas Tipo C , Leucócitos Mononucleares , Trealose , Humanos , Lectinas Tipo C/metabolismo , Lectinas Tipo C/agonistas , Trealose/farmacologia , Trealose/química , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Citocinas/metabolismo , Interleucina-6/metabolismo , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Receptores Imunológicos/agonistas , Receptores Imunológicos/metabolismo
13.
Molecules ; 29(15)2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39124891

RESUMO

Desert strains of the genus Chroococcidiopsis are among the most desiccation-resistant cyanobacteria capable of anhydrobiosis. The accumulation of two sugars, sucrose and trehalose, facilitates the entrance of anhydrobiotes into a reversible state of dormancy by stabilizing cellular components upon water removal. This study aimed to evaluate, at the atomistic level, the role of trehalose in desiccation resistance by using as a model system the 30S ribosomal subunit of the desert cyanobacterium Chroococcidiopsis sp. 029. Molecular dynamic simulations provided atomistic evidence regarding its protective role on the 30S molecular structure. Trehalose forms an enveloping shell around the ribosomal subunit and stabilizes the structures through a network of direct interactions. The simulation confirmed that trehalose actively interacts with the 30S ribosomal subunit and that, by replacing water molecules, it ensures ribosomal structural integrity during desiccation, thus enabling protein synthesis to be carried out upon rehydration.


Assuntos
Cianobactérias , Simulação de Dinâmica Molecular , Trealose , Trealose/metabolismo , Trealose/química , Cianobactérias/metabolismo , Cianobactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Dessecação , Modelos Moleculares
14.
AAPS PharmSciTech ; 25(7): 220, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39313719

RESUMO

Ketoconazole (K) is a poorly water-soluble drug that faces significant challenges in achieving therapeutic efficacy. This study aimed to enhance the dissolution rate of ketoconazole by depositing spray-dried ketoconazole (SK) onto the surface of ground trehalose dihydrate (T) using spray drying. Ketoconazole-trehalose surface solid dispersions (SKTs) were prepared in ratios of 1:1 (SK1T1), 1:4 (SK1T4), and 1:10 (SK1T10), and characterized them using particle size analysis, scanning electron microscopy, powder X-ray diffraction, and in vitro dissolution studies. Results showed that the dissolution rates of the dispersions were significantly higher than those of pure ketoconazole, with the 1:10 ratio showing the highest dissolution rate. The improved dissolution was attributed to the formation of a new crystalline phase and better dispersion of ketoconazole particles. These findings suggest that the surface solid dispersion approach could be a valuable method for enhancing the bioavailability of poorly water-soluble drugs.


Assuntos
Cetoconazol , Tamanho da Partícula , Solubilidade , Trealose , Difração de Raios X , Cetoconazol/química , Cetoconazol/administração & dosagem , Trealose/química , Difração de Raios X/métodos , Microscopia Eletrônica de Varredura/métodos , Secagem por Atomização , Química Farmacêutica/métodos , Pós/química , Disponibilidade Biológica , Composição de Medicamentos/métodos , Antifúngicos/química , Antifúngicos/administração & dosagem
15.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 41(2): 368-375, 2024 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-38686419

RESUMO

The freeze-drying is a technology that preserves biological samples in a dry state, which is beneficial for storage, transportation, and cost saving. In this study, the bovine pericardium was treated with a freeze-drying protectant composed of polyethylene glycol (PEG) and trehalose (Tre), and then freeze-dried. The results demonstrated that the mechanical properties of the pericardium treated with PEG + 10% w/v Tre were superior to those of the pericardium fixed with glutaraldehyde (GA). The wet state water content of the rehydrated pericardium, determined using the Karl Fischer method, was (74.81 ± 1.44)%, which was comparable to that of the GA-fixed pericardium. The dry state water content was significantly reduced to (8.64 ± 1.52)%, indicating effective dehydration during the freeze-drying process. Differential scanning calorimetry (DSC) testing revealed that the thermal shrinkage temperature of the pericardium was (84.96 ± 0.49) ℃, higher than that of the GA-fixed pericardium (83.14 ± 0.11) ℃, indicating greater thermal stability. Fourier transform infrared spectroscopy (FTIR) results showed no damage to the protein structure during freeze-drying. Hematoxylin and eosin (HE) staining demonstrated that the freeze-drying process reduced pore formation, prevented ice crystal growth, and resulted in a tighter arrangement of tissue fibers. The frozen-dried bovine pericardium was subjected to tests for cell viability and hemolysis rate. The results revealed a cell proliferation rate of (77.87 ± 0.49)%, corresponding to a toxicity grade of 1. Additionally, the hemolysis rate was (0.17 ± 0.02)%, which is below the standard of 5%. These findings indicated that the frozen-dried bovine pericardium exhibited satisfactory performance in terms of cytotoxicity and hemolysis, thus meeting the relevant standards. In summary, the performance of the bovine pericardium treated with PEG + 10% w/v Tre and subjected to freeze-drying could meet the required standards.


Assuntos
Liofilização , Pericárdio , Polietilenoglicóis , Trealose , Animais , Pericárdio/química , Trealose/química , Trealose/farmacologia , Bovinos , Polietilenoglicóis/química , Glutaral/química , Varredura Diferencial de Calorimetria
16.
Biochemistry ; 62(5): 1044-1052, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36802580

RESUMO

Extremotolerant organisms and industry exploit sugars as desiccation protectants, with trehalose being widely used by both. How sugars, in general, and the hydrolytically stable sugar trehalose, in particular, protect proteins is poorly understood, which hinders the rational design of new excipients and implementation of novel formulations for preserving lifesaving protein drugs and industrial enzymes. We employed liquid-observed vapor exchange nuclear magnetic resonance (LOVE NMR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA) to show how trehalose and other sugars protect two model proteins: the B1 domain of streptococcal protein G (GB1) and truncated barley chymotrypsin inhibitor 2 (CI2). Residues with intramolecular H-bonds are most protected. The LOVE NMR and DSC data indicate that vitrification may be protective. Combining LOVE NMR and TGA data shows that water retention is not important. Our data suggest that sugars protect protein structure as they dry by strengthening intraprotein H-bonds and water replacement and that trehalose is the stress-tolerance sugar of choice because of its covalent stability.


Assuntos
Açúcares , Trealose , Trealose/química , Proteínas/química , Carboidratos/química , Água , Varredura Diferencial de Calorimetria
17.
Arch Biochem Biophys ; 740: 109584, 2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37001749

RESUMO

The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K0.5 and Vmax when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of Vmax when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features.


Assuntos
Maltose , Trealose , Animais , Coelhos , Trealose/química , Maltose/química , Maltose/metabolismo , Celobiose , L-Lactato Desidrogenase/metabolismo , Dissacarídeos/farmacologia , Dissacarídeos/metabolismo , Concentração de Íons de Hidrogênio
18.
Mol Pharm ; 20(9): 4587-4596, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37535010

RESUMO

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze-thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003-0.800% w/v, to prevent LDH (10 µg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.


Assuntos
Gelo , Poloxâmero , Congelamento , Trealose/química , Proteínas , L-Lactato Desidrogenase/química , Tensoativos , Sacarose/química , Liofilização , Varredura Diferencial de Calorimetria
19.
Mol Pharm ; 20(11): 5682-5689, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37782000

RESUMO

Protein-based drugs are becoming increasingly important, but there are challenges associated with their formulation (for example, formulating stable inhalable aerosols while maintaining the proper long-term stability of the protein). Determining the morphology of multicomponent, protein-based drug formulations is particularly challenging. Here, we use dynamic nuclear polarization (DNP) solid-state NMR spectroscopy to determine the hierarchy of components within spray-dried particles containing protein, trehalose, leucine, and trileucine. DNP NMR was applied to these formulations to assess the localization of the components within the particles. We found a consistent scheme, where trehalose and the protein are co-located within the same phase in the core of the particles and leucine and trileucine are distributed in separate phases at the surface of the particles. The description of the hierarchy of the organic components determined by DNP NMR enables the rationalization of the performance of the formulation.


Assuntos
Excipientes , Trealose , Leucina/química , Trealose/química , Excipientes/química , Aerossóis/química , Espectroscopia de Ressonância Magnética , Pós/química , Administração por Inalação , Tamanho da Partícula
20.
Pharm Res ; 40(5): 1283-1298, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37012535

RESUMO

OBJECTIVES: Solid biopharmaceutical products can circumvent lower temperature storage and transport and increase remote access with lower carbon emissions and energy consumption. Saccharides are known stabilizers in a solid protein produced via lyophilization and spray drying (SD). Thus, it is essential to understand the interactions between saccharides and proteins and the stabilization mechanism. METHODS: A miniaturized single droplet drying (MD) method was developed to understand how different saccharides stabilize proteins during drying. We applied our MD to different aqueous saccharide-protein systems and transferred our findings to SD. RESULTS: The poly- and oligosaccharides tend to destabilize the protein during drying. The oligosaccharide, Hydroxypropyl ß-cyclodextrin (HPßCD) shows high aggregation at a high saccharide-to-protein molar ratio (S/P ratio) during MD, and the finding is supported by nanoDSF results. The polysaccharide, Dextran (DEX) leads to larger particles, whereas HPBCD leads to smaller particles. Furthermore, DEX is not able to stabilize the protein at higher S/P ratios either. In contrast, the disaccharide Trehalose Dihydrate (TD) does not increase or induce protein aggregation during the drying of the formulation. It can preserve the protein's secondary structure during drying, already at low concentrations. CONCLUSION: During the drying of S/P formulations containing the saccharides TD and DEX, the MD approach could anticipate the in-process (in) stability of protein X at laboratory-scale SD. In contrast, for the systems with HPßCD, the results obtained by SD were contradictory to MD. This underlines that depending on the drying operation, careful consideration needs to be applied to the selection of saccharides and their ratios.


Assuntos
Dessecação , Secagem por Atomização , 2-Hidroxipropil-beta-Ciclodextrina , Liofilização , Proteínas , Oligossacarídeos , Estabilidade de Medicamentos , Trealose/química
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