Wendell Q. Sun

STABILITY OF DRY LIPOSOMES IN SUGAR GLASSES

Sugars, particularly trehalose and sucrose, are used to stabilize liposomes during hydration (freeze-drying and air-drying). As a result, dry liposomes are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution. We investigated the effects of the glassy state on liposome fusion and solute retention in the dry state. Solute leakage from dry liposomes was extremely slow at temperatures below the glass transition temperature (Tg); however, it increased exponentially as temperature increased to near or above the Tg, indicating that the glassy state had to be maintained for dry liposomes to retain trapped solutes. The leakage of solutes from dry liposomes followed the law of first-order kinetics and was correlated linearly with liposome fusion. The kinetics of solute leakage showed an excellent fit with the Arrhenius equation at temperatures both above and below the Tg, with a transitional break near the Tg. The activation energy of solute leakage was 1320 kJ/mol at temperatures above the Tg, but increased to 1991 kJ/mol at temperatures below the Tg. The stabilization effect of sugar glass on dry liposomes may be associated with the elevated energy barrier for liposome fusion and the physical separation of dry liposomes in the glassy state. The half-life of solute retention in dry liposomes may be prolonged by storing dry liposomes at temperatures below the Tg and by increasing the Tg of the dry liposome preparation.

Cytoplasmic Vitrification and Survival of Anhydrobiotic Organisms

Abstract We examine the relationship between cytoplasmic vitrification and survival of anhydrobiotic organisms under extreme desiccation condition. The ability of anhydrobiotic organisms to survive desiccation is associated with the accumulation of carbohydrates. Spores, yeasts and microscopic animals accumulate trehalose, whereas pollen, plant seeds and resurrection plants contain sucrose and oligosaccharides such as raffinose and stachyose. During dehydration, these carbohydrates and other components help the organisms enter into the vitreous state (cytoplasmic vitrification). The immobilization by vitrification may minimize stress damages on the cellular structures and protect their biological capabilities during dehydration and rehydration; however, cytoplasmic vitrification alone is found to be insufficient for anhydrobiotic organisms to survive extreme dehydration. The survival of dry organisms in the desiccated state requires the maintenance of the vitreous state. When the vitreous state is lost, free radical oxidation, phase separation and cytoplasmic crystallization would occur and impose real threat to the survival of dry organisms.

A Porcine-Derived Acellular Dermal Scaffold That Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure

Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes. The ability to produce a porcine-derived graft that retains the structural integrity of the extracellular matrix and minimizes potential antigenic response to galactose-alpha-(1,3)-galactose terminal disaccharide (alpha-Gal) may allow the scaffold to support regeneration of native tissue. Dermal tissue from wild-type (WT-porcine-derived acellular dermal matrix [PADM]) or Gal-deficient (Gal(-/-) PADM) pigs was processed to remove cells and DNA while preserving the structural integrity of the extracellular matrix. In addition, the WT tissue was subjected to an enzymatic treatment to minimize the presence of alpha-Gal (Gal-reduced PADM). Extracellular matrix composition and integrity was assessed by histological, immunohistochemical (IHC), and ultrastructural analysis. In vivo performance was evaluated by implantation into the abdominal wall of Old World primates in an exisional repair model. Anti-alpha-Gal activity in the serum of monkeys implanted subcutaneously was assessed by ELISA. Minimal modification to the extracellular matrix was assessed by evaluation of intact structure as demonstrated by staining patterns for type I and type VII collagens, laminin, and fibronectin similar to native porcine skin tissues. Explants from the abdominal wall showed evidence of remodeling, notably fibroblast cell repopulation and revascularization, as early as 1 month. Serum ELISA revealed an initial anti-alpha-Gal induction that decreased to baseline levels over time in the primates implanted with WT-PADM, whereas no or minimal anti-Gal activity was detected in the primates implanted with Gal(-/-) PADM or Gal-reduced PADM. The combination of a nondamaging process, successful removal of cells, and reduction of xenogeneic alpha-Gal antigens from the porcine dermal matrix are critical for producing a material with the ability to remodel and integrate into host tissue and ultimately support soft tissue regeneration.

Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization.

Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (Tg). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the Tg. Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage.

Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis.

The formation of intracellular glass is proposed to be relevant to protein stabilization and survival of anhydrobiotic organisms in the dry state. The stability of proteins in the amorphous carbohydrate matrix and its relevance to seed survival have been investigated in the present study. Glucose-6-phosphate dehydrogenase (G6PDH) was preserved in the amorphous glucose/sucrose (1:10, w/w) matrix by freeze-drying. The stability of freeze-dried G6PDH was examined at temperatures above and below the glass transition temperature (Tg). The rate of G6PDH inactivation in the amorphous carbohydrate matrix deviated significantly from the Arrhenius kinetics, and conformed to the Williams-Landel-Ferry (WLF) relationship. The temperature dependence of G6PDH inactivation in two sets of samples with different Tg values was compared. Identical temperature dependence of G6PDH inactivation was observed after temperature normalization by (T-Tg). Seed survival of Vigna radiata Wilczek (mung bean) showed a similar WLF kinetics at storage temperatures T > or = Tg. In situ protein stability in mung bean embryonic axes was studied using differential scanning calorimetry (DSC). Thermal stability of seed proteins exhibited a strong dependence on the Tg of intracellular glass. These results indicate an important role of the glassy state in protein stabilization. Our data suggest an association between protein stability in intracellular glass and seed survival during storage.

Desiccation sensitivity and activities of free radical-scavenging enzymes in recalcitrant Theobroma cacao seeds

Mature and immature axes of Theobroma cacao (cocoa) seeds tolerated desiccation under a rapid-drying regime to critical water contents of 1.0 and 1.7 g g -1 dw, respectively. These critical water contents corresponded to water contents below which activities of free radical-scavenging enzymes (ascorbate peroxidase, peroxidase and superoxide dismutase) decreased rapidly during desiccation. The decline in axis viability below the critical water content was correlated with sharp increases in lipid peroxidation and cellular leakage. Cotyledon tissues were more desiccation-tolerant than axes, with a low critical water content of 0.24 g g –1 dw. Desiccation sensitivity in cotyledon tissues was also correlated with the decrease in superoxide dismutase activity and increased lipid peroxidation products. However, in the cotyledons, no ascorbate peroxidase activity was detected at any water content, and peroxidase activity was gradually reduced as desiccation proceeded. Cocoa embryonic axes contained large amounts of sucrose, raffinose and stachyose but only traces of reducing monosaccharides. Desiccation sensitivity of recalcitrant cocoa axes did not appear to be due to the lack of sugar-related protective mechanisms during desiccation, and it was more likely related to the decrease of enzymic protection against desiccation-induced oxidative stresses.

Effect of Sucrose/Raffinose Mass Ratios on the Stability of Co-Lyophilized Protein During Storage Above the Tg

AbstractPurpose. To examine the potential of raffinose as an excipient in stabilizing protein and to study the effect of sucrose/raffinose mass ratios on the stability of co-lyophilized protein and amorphous solids during storage at an elevated temperature. Methods. Glucose-6-phosphate dehydrogenase (G6PDH) was co-lyophilized with sucrose and raffinose mixed at different mass ratios. The activity of dried G6PDH was monitored during storage at 44°C. Thermal properties of sucrose/raffinose matrices were determined by differential scanning calorimetry (DSC). Results. Mass ratios of sucrose to raffinose did not affect the recovery of G6PDH activity after freeze-drying, but significantly affected the stability of freeze-dried G6PDH during storage. The sucrose-alone formulation offered the best enzyme stabilization during storage. With increasing fraction of raffinose, the G6PDH stability decreased, sugar crystallization inhibited, and crystal-melting temperature increased. Conclusions. Despite the higher Tg of the formulations with higher fraction of raffinose, they provided less protection for G6PDH than did sucrose alone during storage. Our data do not support the prediction from recent thermophysical studies that raffinose should be superior to sucrose and trehalose as a potential excipient or stabilizer.

Rate of Dehydration and Cumulative Desiccation Stress Interacted to Modulate Desiccation Tolerance of Recalcitrant Cocoa and Ginkgo Embryonic Tissues

Rate of dehydration greatly affects desiccation tolerance of recalcitrant seeds. This effect is presumably related to two different stress vectors: direct mechanical or physical stress because of the loss of water and physicochemical damage of tissues as a result of metabolic alterations during drying. The present study proposed a new theoretic approach to represent these two types of stresses and investigated how seed tissues responded differently to two stress vectors, using the models of isolated cocoa (Theobroma cacao) and ginkgo (Ginkgo biloba) embryonic tissues dehydrated under various drying conditions. This approach used the differential change in axis water potential (ΔΨ/Δt) to quantify rate of dehydration and the intensity of direct physical stress experienced by embryonic tissues during desiccation. Physicochemical effect of drying was expressed by cumulative desiccation stress [∫ o t f(ψ,t)], a function of both the rate and time of dehydration. Rapid dehydration increased the sensitivity of embryonic tissues to desiccation as indicated by high critical water contents, below which desiccation damage occurred. Cumulative desiccation stress increased sharply under slow drying conditions, which was also detrimental to embryonic tissues. This quantitative analysis of the stress-time-response relationship helps to understand the physiological basis for the existence of an optimal dehydration rate, with which maximum desiccation tolerance could be achieved. The established numerical analysis model will prove valuable for the design of experiments that aim to elucidate biochemical and physiological mechanisms of desiccation tolerance.

Discrete levels of desiccation sensitivity in various seeds as determined by the equilibrium dehydration method

This study examined the hypothesis that desiccation sensitivities of recalcitrant and intermediate seeds can be categorized into discrete levels of critical water potential. The equilibrium dehydration method was used to determine the critical water potential (CWP) below which desiccation damage started to occur. The CWP values of Bruguiera cylindrica, Lansium domesticum, Litchi chinensis and Lumitzera racemosa are approximately ‐4 MPa. The CWP values of Andira inermis, Avicennia alba, Castanea sinensis (from New Zealand), Citrus aurantifolia, Ginkgo biloba, Nephelium lappaceum and Theobroma cacao (immature axis) are approximately ‐8 MPa. The CWP values of Acer pseudoplatanus, Castanea sinensis (from China), Quercus rubra and Theobroma cacao (mature axis) are approximately ‐12 MPa. The CWP values of Artocarpus heterophyllus and Hevea brasiliensis are approximately � 23 MPa, while the CWP values of Acer platanoides, Azadirachta indica, Carica papaya and Coffea arabica are approximately ‐73 MPa. Together with data available in earlier literature, these CWP values suggest that there are five discrete levels of critical water potential among desiccation-sensitive seed tissues. These data support the hypothesis that discrete levels of desiccation sensitivity occur among recalcitrant and intermediate seeds, and suggest that specific damaging and protective mechanisms exist at certain hydration levels.

Correlation of modified water sorption properties with the decline of storage stability of osmotically-primed seeds of Vigna radiata (L.) Wilczek

Osmotic priming in a polyethylene glycol solution (300 g/kg water) for 48 h resulted in a partial loss of desiccation tolerance for seeds of Vigna radiata (L.) Wilczek (mung bean). The percentage of germination began to decrease after primed seeds were dried down to water contents less than 0.06 g/g DW. As compared with control seeds, primed mung bean seeds also had poorer storage stability. The decline of storage stability after osmotic priming was correlated with the modifications of seed water sorption properties. Priming significantly increased the amount of water associated with the weak water-binding sites, and reduced the amount of water associated with the strong binding sites and multi-molecular binding sites in seed tissues. The enhancement of molecular mobility in seeds, as a result of such water redistribution, probably accelerates seed deterioration and decreases storage stability.