Lyophilization
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Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- MeSH
- fosfáty * chemie MeSH
- histidin * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová chemie MeSH
- lyofilizace * metody MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- pufry MeSH
- spektrofotometrie ultrafialová metody MeSH
- trehalosa * chemie MeSH
- Publikační typ
- časopisecké články MeSH
Nanomedicine for treating post-viral infectious disease syndrome is at an emerging stage. Despite promising results from preclinical studies on conventional antioxidants, their clinical translation as a therapy for treating post-COVID conditions remains challenging. The limitations are due to their low bioavailability, instability, limited transport to the target tissues, and short half-life, requiring frequent and high doses. Activating the immune system during coronavirus (SARS-CoV-2) infection can lead to increased production of reactive oxygen species (ROS), depleted antioxidant reserve, and finally, oxidative stress and neuroinflammation. To tackle this problem, we developed an antioxidant nanotherapy based on lipid (vesicular and cubosomal types) nanoparticles (LNPs) co-encapsulating ginkgolide B and quercetin. The antioxidant-loaded nanocarriers were prepared by a self-assembly method via hydration of a lyophilized mixed thin lipid film. We evaluated the LNPs in a new in vitro model for studying neuronal dysfunction caused by oxidative stress in coronavirus infection. We examined the key downstream signaling pathways that are triggered in response to potassium persulfate (KPS) causing oxidative stress-mediated neurotoxicity. Treatment of neuronally-derived cells (SH-SY5Y) with KPS (50 mM) for 30 min markedly increased mitochondrial dysfunction while depleting the levels of both glutathione peroxidase (GSH-Px) and tyrosine hydroxylase (TH). This led to the sequential activation of apoptotic and necrotic cell death processes, which corroborates with the crucial implication of the two proteins (GSH-Px and TH) in the long-COVID syndrome. Nanomedicine-mediated treatment with ginkgolide B-loaded cubosomes and vesicular LNPs showed minimal cytotoxicity and completely attenuated the KPS-induced cell death process, decreasing apoptosis from 32.6% (KPS) to 19.0% (MO-GB), 12.8% (MO-GB-Quer), 14.8% (DMPC-PEG-GB), and 23.6% (DMPC-PEG-GB-Quer) via free radical scavenging and replenished GSH-Px levels. These findings indicated that GB-LNPs-based nanomedicines may protect against KPS-induced apoptosis by regulating intracellular redox homeostasis.
- MeSH
- antioxidancia * farmakologie MeSH
- COVID-19 metabolismus MeSH
- farmakoterapie COVID-19 * MeSH
- ginkgolidy * farmakologie MeSH
- glutathionperoxidasa * metabolismus MeSH
- laktony farmakologie MeSH
- lidé MeSH
- nanočástice * MeSH
- nanomedicína * metody MeSH
- neurony účinky léků virologie MeSH
- oxidační stres * účinky léků MeSH
- quercetin farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- SARS-CoV-2 účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The purpose of the current study was to evaluate the functional activity and storage viability (at 4 °C and 35 °C) of an immobilized as well as lyophilized multienzyme, viz., pectinase, cellulase, and amylase (PCA) that was produced by Bacillus subtilis NG105 under solid state fermentation (SSF) at 35 °C for 10 days using mosambi peel as a substrate. After SSF, the culture media was divided into two aliquots. From the first aliquot, the produced ME was extracted, precipitated, and further immobilized on calcium alginate beads (MEICA). In order to immobilize on mosambi peel matrix, the second aliquot was mixed with acetone and subsequently lyophilized (MELMP). Thus, ready MEICA and MELMP extracted 87.5 and 91.5% juice from mango pulp, respectively. In the reusability study, after 5 cycles, MEICA exhibited 23.8%, 24.4%, and 36.5% PCA activity, respectively. The PCA activity of MEICA and MELMP was examined after 60 days of storage at 4 °C. The result revealed that the PCA for MEICA declined from 100 to 66%, 58.2%, and 64.5%, respectively, while for MELMP, it dropped from 100 to 84.2%, 82.1%, and 69.7%, respectively. Further, after 60 days of storage, the reduction of total protein content (TPC) in free multienzyme (FME), MEICA, and MELMP was 92.2%, 91.5%, and 36.3% observed, respectively. In the localization study, the maximum levels of multienzyme activity were found in cell exudates. This study demonstrated that immobilizing of multienzyme through lyophilization on waste substrates like mosambi peel boosted its stability and shelf-life along with greatly reducing the cost of products.
BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.
- MeSH
- antibakteriální látky farmakologie aplikace a dávkování MeSH
- endopeptidasy * farmakologie aplikace a dávkování MeSH
- gramnegativní bakteriální infekce * farmakoterapie MeSH
- gramnegativní bakterie * účinky léků MeSH
- krysa rodu rattus MeSH
- myši inbrední BALB C * MeSH
- myši MeSH
- proteinové inženýrství metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Inhalation drug administration is increasingly used for local pharmacotherapy of lung disorders and as an alternative route for systemic drug delivery. Modern inhalation powder systems aim to target drug deposition in the required site of action. Large porous particles (LPP), characterized by an aerodynamic diameter over 5 μm, density below 0.4 g/cm3, and the ability to avoid protective lung mechanisms, come to the forefront of the research. They are mostly prepared by spray techniques such as spray drying or lyophilization using pore-forming substances (porogens). These substances could be gaseous, solid, or liquid, and their selection depends on their polarity, solubility, and mutual compatibility with the carrier material and the drug. According to the pores-forming mechanism, porogens can be divided into groups, such as osmogens, extractable porogens, and porogens developing gases during decomposition. This review characterizes modern trends in the formulation of solid microparticles for lung delivery; describes the mechanisms of action of the most often used porogens, discusses their applicability in various formulation methods, emphasizes spray techniques; and documents discussed topics by examples from experimental studies.
- MeSH
- aplikace inhalační MeSH
- farmaceutická chemie metody MeSH
- lékové transportní systémy * metody MeSH
- lidé MeSH
- plíce * metabolismus účinky léků MeSH
- poréznost MeSH
- prášky, zásypy, pudry MeSH
- příprava léků metody MeSH
- velikost částic * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.
- MeSH
- cytokiny metabolismus MeSH
- kryoprezervace metody MeSH
- kultivační média speciální chemie MeSH
- kultivované buňky MeSH
- lidé MeSH
- lyofilizace * MeSH
- mezenchymální kmenové buňky * metabolismus cytologie MeSH
- sekretom metabolismus MeSH
- teplota MeSH
- trehalosa metabolismus farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Freezing and lyophilization have been utilized for decades to stabilize pharmaceutical and food products. Freezing a solution that contains dissolved salt and/or organic matter produces pure primary ice crystal grains separated by freeze-concentrated solutions (FCS). The microscopic size of the primary ice crystals depends on the cooling conditions and the concentration of the solutes. It is generally accepted that primary ice crystals size influences the rate of sublimation and also can impact physico-chemical behaviour of the species in the FCS. This article, however, presents a case where the secondary ice formed inside the FCS plays a critical role. We microscoped the structures of ice-cast FCS with an environmental scanning electron microscope and applied the aggregation-sensitive spectroscopic probe methylene blue to determine how the microstructure affects the molecular arrangement. We show that slow cooling at -50 °C produces large salt crystals with a small specific surface, resulting in a high degree of molecular aggregation within the FCS. In contrast, fast liquid nitrogen cooling yields an ultrafine structure of salt crystals having a large specific surface area and, therefore, inducing smaller aggregation. The study highlights a critical role of secondary ice in solute aggregation and introduces methylene blue as a molecular probe to investigate freezing behaviour of aqueous systems with crystalline solute.
- MeSH
- led * MeSH
- lyofilizace MeSH
- methylenová modř * MeSH
- roztoky MeSH
- voda chemie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.
Oxidative stress is an important toxicity and genotoxicity mechanism of many chronic adverse health outcomes. This study developed a sensitive extraction method for urine matrix (based on lyophilization, without the need for pre-cleaning by solid phase extraction), coupled to LC-MS/MS analysis of the biomarker 8-hydroxy-2'-deoxyguanosine (8-OHdG). The methodology was validated in urine samples from a cohort of Spanish pregnant women collected during the first, second and third trimester of pregnancy, and urine samples collected within 24 h after delivery (n = 85). A detection and quantification limit of 0.01 and 0.05 μg/L, respectively, were established. The median 8-OHdG concentration was 2.18 μg/L (range 0.33-7.79); and the corresponding creatinine-adjusted concentrations ranged from 1.04 to 13.12 with median of 4.48 μg 8-OHdG/g creatinine. The concentrations of non-adjusted 8-OHdG significantly decreased (p < 0.05) in the 3rd trimester and post-delivery urine samples when compared to the 1st trimester levels. 8-OHdG concentrations were further studied in placenta samples matching the same urine samples (n = 26), with a median value of 1.3 ng 8-OHdG/g of tissue. Placental 8-OHdG concentrations were correlated with urinary levels of non-adjusted 8-OHdG in the 3rd trimester. Considering the small cohort size, results must be interpreted with caution, however statistical analyses revealed elevated urinary non-adjusted 8-OHdG levels in the 1st trimester of mothers that delivered boys compared to those who delivered girls (p < 0.01). Increased urinary non-adjusted 8-OHdG concentrations at the time of delivery were significantly associated with clinical records (any type of clinical record during pregnancy; p < 0.05). The novel extraction and analytical method for the assessment of 8-OHdG is applicable for sensitive analysis of multiple analytes or biomarkers in urine matrix. This method could also be applied for other matrices such as blood or tissues. Our findings show that 8-OHdG in urine of pregnant women could predict oxidative stress in placenta and can be related to characteristics such as maternal obesity, mode of delivery and newborn sex.
- MeSH
- 8-hydroxy-2'-deoxyguanosin MeSH
- biologické markery moč MeSH
- chromatografie kapalinová metody MeSH
- deoxyguanosin * moč MeSH
- kreatinin moč MeSH
- lidé MeSH
- novorozenec MeSH
- oxidační stres MeSH
- placenta MeSH
- poškození DNA MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- těhotenství MeSH
- těhotné ženy * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The QuantiFERON®-Monitor (QFM) is an assay that measures interferon-γ production and was developed to provide an objective marker of complex immune response. In this study, we evaluated the use of the QFM test in patients with two forms of multiple sclerosis (MS), relapsing-remitting form treated with fingolimod (fMS) and secondarily progressive form not treated pharmacologically (pMS), and in healthy controls (HC). We hypothesized that IFN-γ levels would be lower in those subjects who are relatively more immunosuppressed and higher in those with normal or activated immune function. METHODS: This single-center observational study was conducted from November 2020 to October 2021 and compared results in three groups of patients: 86 healthy controls, 96 patients with pMS, and 78 fMS. Combination of lyophilized stimulants was added to 1 ml heparinized whole blood within 8 hr of collection. Plasmatic IFN-γ was measured using the ELISA kit for the QFM and data were obtained in IU/ml. RESULTS: The results showed that controls had nearly 2-fold higher levels of IFN-γ (QFM score) in median (q25, q75) 228.00 (112.20, 358.67) than the MS patient groups: pMS 144.80 (31.23, 302.00); fMS 130.50 (39.95, 217.07) which is statistically significant difference P-value: HC vs. pMS = 0.0071; HC vs. fMS = 0.0468. This result was also confirmed by a validation analysis to exclude impact of variable factors, such as disease duration and Expanded Disability Status Scale scores. CONCLUSIONS: Results showed that controls had higher levels of IFN-γ production than the MS patient groups and suggest that MS patients included in this study have a lower ability of immune system activation than HC. Results confirm that fingolimod is able to suppress production of IFN-γ. The fact that the QFM score of MS patients is significantly lower than that of HC may indicate a dysfunctional state of the immune system in baseline conditions.
