Storage In Dry State Research Articles

Microneedle-based intradermal delivery of stabilized dengue virus.

Current live‐attenuated dengue vaccines require strict cold chain storage. Methods to preserve dengue virus (DENV) viability, which enable vaccines to be transported and administered at ambient temperatures, will be decisive towards the implementation of affordable global vaccination schemes with broad immunization coverage in resource‐limited areas. We have developed a microneedle (MN)‐based vaccine platform for the stabilization and intradermal delivery of live DENV from minimally invasive skin patches. Dengue virus‐stabilized microneedle arrays (VSMN) were fabricated using saccharide‐based formulation of virus and could be stored dry at ambient temperature up to 3 weeks with maintained virus viability. Following intradermal vaccination, VSMN‐delivered DENV was shown to elicit strong neutralizing antibody responses and protection from viral challenge, comparable to that of the conventional liquid vaccine administered subcutaneously. This work supports the potential for MN‐based dengue vaccine technology and the progression towards cold chain‐independence.Dengue virus can be stabilized using saccharide‐based formulations and coated on microneedle array vaccine patches for storage in dry state with preserved viability at ambient temperature (VSMN; virus‐stabilized microneedle arrays).

The possible causal relationship between fragmentation of genomic DNA and formation of viable, but non-culturable probiotic bacteria upon storage in dry state.

In this study, the aim was to establish if loss of DNA integrity is a cause of loss of culturability for probiotic bacteria during storage in dry state. The number of colony forming units (CFU), number of metabolically active cells, and DNA integrity during dry storage of probiotic strains, B. animalis subsp. lactis BB-12 and L. acidophilus LA-5, were investigated. The probiotic strains were freeze-dried and stored at 20°C, with and without oxygen present, and at water activity levels 0.22 or 0.32. Dry storage resulted in a decrease in CFU during the entire storage period. The number of metabolically active cells was unchanged during storage of B. animalis subsp. lactis BB-12, but did decrease during the first week of storage of L. acidophilus LA-5. Loss of DNA integrity was evident for both strains during storage and correlated well with the loss of CFU. Both loss of CFU and loss of DNA integrity were significantly greater for both strains when oxygen was present and when aw was increased. Statistical analysis indicates a possible causal relationship between DNA degradation and loss of culturability and this idea is consistent with the function of DNA at cell division. The study contributes with new knowledge of the cause for loss of CFU during dry storage of probiotic bacteria, which possibly can aid in the improvement of preservation techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:231-242, 2018.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.

Resistance of various yeast ecological groups to prolonged storage in dry state

Resistance of 14 yeast species belonging to different ecological groups to extensive storage in a dried state was investigated. Pedobiotic yeasts isolated mainly from the soils of humid areas (Cryptococcus podzolicus, Cr. terricola, and Lipomyces starkeyi) were the least resistant. The yeasts associated with the nectar of entomophilous plants (Metschnikowia reukaufii and Candida bombi) also exhibited low resistance to drying. Complete death of these species occurred during the first month of storage. Eurybiotic species from various environments (Cryptococcus magnus, Cryptococcus victoriae, Debaryomyces hansenii, and Cryptococcus wieringae) were somewhat more resistant. Pigmented plant-associated yeasts (Rhodotorula mucilaginosa and Sporobolomyces roseus), as well as the pathogenic or opportunistic Candida strains (C. albicans and C. parapsilosis), were the most resistant to drying. Thus, occurrence of yeasts in natural habitats is closely associated with their ability to survive prolonged drying.

Secondary Structure of rhBMP-2 in a Protective Biopolymeric Carrier Material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.

Cryptic anhydrobiotic potential in man: Implications in medicine

Anhydrobiosis ("life without water") is a state of suspended animation in a dry, ametabolic condition which some species enter before desiccation stress. This state confers resistance to other abiotic stresses such as radiation and heat. Once specimens are rehydrated life resumes. Application of such long-term storage in dry state without loss of viability would bring a considerable advance in organ transplant, maintenance of cell/tissue banks, vaccine storage and transport and many other medical applications. Since anhydrobiosis brings biological advantage to organisms, one would expect it to be positively selected during evolution. Moreover, our still limited understanding of the human genome, together with occasional discoveries of hidden endogenous processes, as well as reports of suspended animation in higher animals leads us to hypothesize the existence of cryptic anhydrobiosis in man. This hypothesis could be tested in laboratory animals and human-derived biological samples using several physical-chemical pre-conditioning/desiccation/rehydration parameters. Finding cryptic anhydrobiosis in human tissues would be a breakthrough in medicine providing means for long-term storage of biological samples in several stressful conditions.

Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance

Liquid and gas chromatography are commonly used to measure organophosphorus pesticides. However, these methods are relatively time consuming and require a tedious sample pretreatment. Here, we applied the localized surface plasmon resonance (LSPR) of gold nanoparticles covalently coupled with acetylcholinesterase (AChE) to create a biosensor for detecting an example of serial signals responding to paraoxon in the range of 1-100 ppb by an AChE modified LSPR sensor immersing in a 0.05 mM ACh solution. The underlying mechanism is that paraoxon prevents acetylcholine chloride (ACh) reacting with AChE by destroying the OH bond of serine in AChE. We found that the AChE modified LSPR sensors prepared by incubation with 12.5 mU/mL of AChE in phosphate buffer solution at pH 8.5 room temperature for 14 h have the best linear inhibition response with a 0.234 ppb limit of paraoxon detection. A 14% of inhibition on the sensor corresponds to the change of paraoxon concentration from 1 to 100 ppb. The sensor remained 94% of its original activity after six cycles of inhibition with 500 ppb paraoxon followed with reactivation of AChE by 0.5 mM 2-pyriding-aldoxime methoiodide (2-PAM). In addition, the sensor retains activity and gives reproducible results after storage in dry state at 4 degrees C for 60 days. In conclusion, we demonstrated that the AChE modified LSPR sensors can be used to determine the concentration of paraoxon biosensor with high sensitive and stable characteristics.

Glutamate Biosensor Using a DNA-Cu(II)/polyamine Membrane as a Novel Electrocataytic Layer for Cathodic Determination of Hydrogen Peroxide

Novel glutamate biosensor was fabricated by coupling of a glutamate oxidase (GlOx) to a copper ion embedded polyion complex membrane composed of double-stranded DNA and polyallylamine (PAA), which prepared on a surface of the glassy carbon (GC) electrode. A hydrogen peroxide (H2O2) produced during the GlOx reaction was cathodically detected, based on an electrocatalytic activity of a DNA-Cu(II) complex for a reduction of H2O2. The cathodic current responses to L-glutamate obtained at −0.2 V (vs. Ag/AgCl) and at pH 6.0 linearly increased up to 100 µM with a detection limit of 1 µM (S/N=3). The sensitivity (slope of the linear region) was 0.80 nA/µM, and the response time was less than 20∼30 s. The sensor retained its response characteristics for at least five days by the storage in dry state at 4°C.

Composite Biosensor for Sulfite Assay: Use of Water-Insoluble Hexacyanoferrate(III) Salts as Electron-Transfer Mediators

Water-insoluble salts of hexacyanoferrate(III) and cationic surface active agents were synthetized and used as electron-mediators for sulfite oxidase. The biosensor was prepared from a composite consisting of modified graphite (50 % w/w) and n-eicosane (50 % w/w). Graphite was modified with mediators or with both mediator and sulfite oxidase for surface- and bulk-modified electrode, respectively. The main advantage of biosensors with insoluble mediators is the possibility to operate at a potential of 0 mV (vs. SCE), thus less interferences are expected, in comparison to soluble hexacyanoferrate(III) where a potential of +300 mV must be used. The maximum sensitivity 7.8 × 10−4 μA/μM was obtained for bulk-modified biosensor, prepared from graphite modified with 5 % w/w of hexadecyltrimethylammonium hexacyanoferrate(III) and 1.25 units/mg (of graphite) of sulfite oxidase. The sensitivity of the biosensor decreased to 24 % of the initial sensitivity after one month storage in dry state at ambient temperature. The use of trehalose as an enzyme stabilization agent has led to the improved stability: 40 % of the initial stability was retained after one month.