Initial Development and Characterization of PLGA Nanospheres Containing Ropivacaine

Local anesthetics are able to induce pain relief by binding to the sodium channels of excitable membranes, blocking the influx of sodium ions and the propagation of the nervous impulse. Ropivacaine (RVC) is an amino amide, enantiomerically pure, local anesthetic largely used in surgical procedures, which present physico-chemical and therapeutic properties similar to those of bupivacaine but decreased toxicity and motor blockade. The present work focuses on the preparation and characterization of nanospheres containing RVC; 0.25% and 0.50% RVC were incorporated in poly(d,l-lactide-co-glycolide (PLGA) 50:50) nanospheres (PLGA-NS), prepared by the nanoprecipitation method. Characterization of the nanospheres was conducted through the measurement of pH, particle size, and zeta potential. The pH of the nanoparticle system with RVC was 6.58. The average diameters of the RVC-containing nanospheres was 162.7 ± 1.5 nm, and their zeta potentials were negative, with values of about ?10.81 ± 1.16 mV, which promoted good stabilization of the particles in solution. The cytotoxicity experiments show that RVC-loaded PLGA-NS generate a less toxic formulation as compared with plain RVC. Since this polymer drug-delivery system can effectively generate an even less toxic RVC formulation, this study is fundamental due to its characterization of a potentially novel pharmaceutical form for the treatment of pain with RVC.     

Effect of heating temperature and time on pharmaceutical characteristics of albumin microspheres containing 5-fluorouracil

Conclusion  In this study, we found that both heating temperature and heating time affect mean particle size, particle size distribution, and drug entrapment efficiency of albumin microspheres. The change in heating temperature may affect the particle size of the product, especially when heating is carried out at a lower temperature (90°C–120°C). Hence the temperature should be selected on the basis of desired size range. Given that it is desirable for a maximum amount of the drug used in the preparation to become entrapped in microspheres, heating temperature and heating time for denaturation of albumin should be selected cautiously, as both have a significant effect on drug entrapment efficiency. In the present case, the highest entrapment was found in batches prepared by heating at 90°C for 5 minutes. However, the extent of stabilization at the selected temperature and the time of heating should also be taken into consideration, as they may affect the release of drugs to target tissue.     

Effect of storage time and temperature on steroid and protein hormone concentrations in porcine plasma and serum

The effect of storage time and temperature of porcine blood prior to quantitation of hormone concentrations by radioimmunoassay (RIA) was evaluated. Blood from each of four luteal phase gilts was used to determine cortisol (CS) and progesterone (P) concentrations, while blood from each of four ovariectomized gilts and each of four lactating sows was used to determine luteinizing hormore (LH) and prolactin (PRL) concentrations, respectively. Blood was collected via jugular puncture from each animal within a 30-sec time period and placed into 18 heparinized and 18 nonheparinized tubes. One sample with and without heparin was stored in ice water (4°C) or at 28°C for 0.25, 2, 4, 6, 8, 12, 24, 36 or 48 hours. After storage, blood was centrifuged at 4°C and plasma or serum was collected and stored at ?20°C until quantitated by RIA. There were no differences (P>0.05) between plasma and serum concentrations (X ± SE, ng/ml) of CS (26.9 ± 0.8 vs 28.5 ± 0.8), P (24.7 ± 0.7 vs 24.8 ± 0.8), LH (2.1 ± 0.1 vs 2.2 ± 0.1) or PRL (53.2 ± 2.3 vs 52.6 ± 2.1). Similarly, storage temperature (4 vs 28°C) did not affect the concentrations of P (25.7 ± 0.8 vs 23.9 ± 0.7), LH (2.2 ± 0.1 vs 2.2 ± 0.1) or PRL (53.7 ± 2.1 vs 53.2 ± 2.3). Howver, CS concentrations decreased (P<0.05) from 28.5 ± 0.5 (4°C) to 26.9 ± 0.8 ng/ml (28°C). There was an animla x time interaction for CS concentration when plasma and serum were stored at both 4°C (P<0.001) and 28°C (P<0.003). There was also and animal x time interaction (P<0.03) for LH concentrations. The P and PRL concentrations decreased linearly by 0.0615 ng/hr (P<0.001) and 0.0625 ng/hr (P<0.004), respectively, with increased storage time.     

Synthesis and Characterization of PLGA Shell Microcapsules Containing Aqueous Cores Prepared by Internal Phase Separation

The preparation of microcapsules consisting of poly(d,l-lactide-co-glycolide) (PLGA) polymer shell and aqueous core is a clear challenge and hence has been rarely addressed in literature. Herein, aqueous core-PLGA shell microcapsules have been prepared by internal phase separation from acetone-water in oil emulsion. The resulting microcapsules exhibited mean particle size of 1.1?±?0.39 μm (PDI?=?0.35) with spherical surface morphology and internal poly-nuclear core morphology as indicated by scanning electron microscopy (SEM). The incorporation of water molecules into PLGA microcapsules was confirmed by differential scanning calorimetry (DSC). Aqueous core-PLGA shell microcapsules and the corresponding conventional PLGA microspheres were prepared and loaded with risedronate sodium as a model drug. Interestingly, aqueous core-PLGA shell microcapsules illustrated 2.5-fold increase in drug encapsulation in comparison to the classical PLGA microspheres (i.e., 31.6 vs. 12.7%), while exhibiting sustained release behavior following diffusion-controlled Higuchi model. The reported method could be extrapolated to encapsulate other water soluble drugs and hydrophilic macromolecules into PLGA microcapsules, which should overcome various drawbacks correlated with conventional PLGA microspheres in terms of drug loading and release.     

Stability of niosomes with encapsulated vitamin D3 and ferrous sulfate generated using a novel supercritical carbon dioxide method

Niosomes were prepared using a novel supercritical carbon dioxide based method to simultaneously encapsulate ferrous sulfate and vitamin D₃ as hydrophilic and hydrophobic cargo, respectively. Vesicle particle size was determined to be bimodal with peak diameters of 1.44?±?0.16?μm and 7.21?±?0.64?μm, with the smaller peak comprising 98.8% of the total niosomal volume. Encapsulation efficiency of ferrous sulfate was 25.1?±?0.2% and encapsulation efficiency of vitamin D₃ was 95.9?±?1.47%. Physical stability of the produced niosomes was assessed throughout a storage period of 21 days. Niosomes showed good physical stability at 20?°C, but storage at 4?°C showed an initial burst release, indicating possible rupture of the niosomal membrane. The Korsmeyer–Peppas equation was used to model the release of ferrous sulfate over time at both storage temperatures.     

The effect of temperature on the rate of sprout growth and development within stored onion bulbs

In order to understand better the effects of storage temperature on the time to visible sprouting in stored onions, sprout growth was measured by regularly dissecting samples from bulbs stored at 1, 10, 15 or 25°C for 243 days. The dry-weight of the shoot or sprout within stored onion bulbs increased exponentially with time. The rate of increase of sprout dry weight, as well as the rate of leaf initiation by the shoot apex was faster at 17° than at 10 or 25°C, and almost zero at 1°C. The rate of loss of dry weight from storage tissue was similar at 17°C and 25°C but slower at 10°C and slower still at 1°C.     

Dependence of the viability and virulence of siberian isolates of the entomopathogenic fungus Beauveria bassiana (Bals-Griv.) Vuill. on the period of their storage at positive temperatures

A study of Siberian isolates of Beauveria bassiana (Bals-Griv.) Vuill. has shown the dependence of their viability and virulence on the storage period and temperature. The isolates remain viable and virulent for a longer period (up to 3 years) when they are stored at a low positive temperature (+8°C) than when stored at room temperature (+18°C). According to our correlation analysis, when stored at room temperature, the virulence of the studied isolates towards the test insects depends on the storage period (57–72%) and, to a lesser extent (28–43%), on other unaccounted factors.     

Post-harvest Changes in the Respiratory Activity of and Ethanol Accumulation in Fresh Rice Grains

Fresh rice grains stored under anaerobic conditions at 4°C showed a strong activity of anaerobic respiration at 30°C. When stored at 30°C, the rates of both oxygen consumption and carbon dioxide evolution declined rapidly. The ethanol content in paddy at the post-harvest stage was increased at 4°C, whereas no significant accumulation of ethanol was observed at 30°C. The accumulated ethanol in paddy was depleted as the storage temperature was raised from 4 to 30°C. In contrast, a temperature-dependent accumulation was observed with a lowering from 30 to 4°C. On the other hand, ethanol content in brown rice changed little with storage temperature. On the basis of these results, it is assumed that ethanol is more easily accumulated in the rice grains against diffusion to the atmosphere at the lower temperature.     

Effect of temperature and ripening stages on membrane integrity of fresh-cut tomatoes

The shelf-life of fresh-cut tomatoes mainly depends on loss of tissue integrity and firmness that occurs also in intact fruits after long-term cold storage due to chilling injury. Round-fruit tomatoes (Solanum lycopersicum L.) cv. Jama were stored in 1.1-L plastic (polyethylene) fresh-cut produce containers as 10.0-mm-thick tomato slices and as intact tomatoes at 4 ± 0.5 °C. The aim of this work was to study the loss of membrane integrity and biochemical processes involved in membrane disruption. Electrolyte leakage and lipid peroxidation were studied at different stages of maturity: mature green, pink (PK), fully ripe and two different storage temperatures: 4 and 15 °C. The tomato slices of PK stage stored at 4 °C did not show changes for both parameters, while significant increase in membrane leakage and lipid peroxidation was observed at 15 °C, especially after 24 h of storage. The enzymes showed a simultaneous increase in their activities with a rise in electrolyte leakage and lipid peroxidation after 7 days of storage. Finally, phospholipase C (PLC) and phospholipase D (PLD) were investigated for intact fruit and tomato slices stored at 4 °C. The PLC had higher activity compared with PLD. In conclusion, the loss of membrane integrity in fresh-cut tomatoes is mainly affected by ripening stages, storage temperature and duration. The wounds enhance the PLC and PLD activities and they play a role late during storage.     

Overexpression of GRF Encapsulated in PLGA Microspheres in Animal Skeletal Muscle Induces Body Weight Gain

Biodegradable nanospheres or microspheres have been widely used as a sustained release system for the delivery of bioagents. In the present study, injectable sustained-release growth hormone-releasing factor (GRF) (1–32) microspheres were prepared by a double emulsion-in liquid evaporation process using biodegradable polylactic-co-glycolic acid (PLGA) as the carrier. The entrapment efficiency was 89.79% and the mean particle size was 4.41 μm. The microspheres were injected into mouse tibialis muscle. After 30 days, mice injected with GRF (1–32) microspheres (group I) gained significantly more weight than any other treatment group, including mice injected with the naked plasmid (group II) (10.26 ± 0.13 vs. 9.09 ± 0.56; P < 0.05), a mixture of microspheres and plasmid (group III) (10.26 ± 0.13 vs. 8.57 ± 0.02; P < 0.05), or saline (IV) (10.26 ± 0.13 vs. 6.47 ± 0.26; P < 0.05). In addition, mice treated with the GRF (1–32) microspheres exhibited the highest expression levels of GRF as detected by PCR, RT-PCR, and ELISA (mean 2.56 ± 0.40, P < 0.05, overall comparison of treatment with groups II, III, and IV). Additionally, rabbits were injected in the tibialis muscle with the same treatments described above. After 30 days, the group treated with GRF (1–32) microspheres gained the most weight. At day 30 postinjection, weight gain in group I was 63.93% higher than group II (plasmid) (877.10 ± 24.42 vs. 535.05 ± 26.38; P < 0.05), 108.59% higher than group III (blank MS) (877.10 ± 24.42 vs. 420.50 ± 19.39; P < 0.05), and 93.94% higher than group IV (saline) (877.10 ± 24.42 vs. 452.25 ± 27.38; P < 0.05). Furthermore, IGF-1 levels in the serum from GRF microsphere-treated group were elevated relative to all other groups. The present results suggest that encapsulation of GRF with PLGA increases GRF gene expression in muscle after local plasmid delivery, and stimulates significantly more weight gain than delivery of the naked plasmid alone.     

Stored xylem sap from wheat and barley in drying soil contains a transpiration inhibitor with a large molecular size

Xylem sap was collected from wheat and barley growing in a drying soil, and the effect of the sap on transpiration was detected by a bioassay with detached wheat leaves. The inhibitory activity of fresh sap was small, and could be largely accounted for by the abscisic acid content (about 2×10^-5mol m^-3). When fresh sap was stored at -20°C for several days, the activity increased. Maximum activity developed after a week. This increase in activity was due to a compound that increased in size with storage at -20°C. When fresh sap was fractionated with filters of different molecular size exclusion characteristics, and the separated fractions stored at -20°C for a week, activity developed only in the fraction containing compounds smaller than 0·3 kDa. However, when sap already stored at -20°C was fractionated, activity was only in fractions containing compounds larger than 0·3 kDa. The increase in activity and in size did not occur with storage in liquid nitrogen (-196°C) or at -80°C. These results suggest that storage at -20°C causes the aggregation or polymerization of a small compound with low activity to form a large compound with high activity. This change is not catalysed by an enzyme because it can occur in a fraction from which molecules larger than 0·3 kDa are removed. It is probably promoted by high solute concentrations when ice crystals form. Sap collected from plants in soils of high water potential had little or no activity after storage at -20°C.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Effects of hot water treatments and storage temperatures on the ripening and the use of electrical impedance as an index for assessing post-harvest changes in mango fruits

The effects of hot water treatment and storage temperature (4°C, 13°C or 22°C) on the quality and impedance of outer and inner mesocarp of mango were assessed in two experiments during storage, impedance being a potential non‐destructive measure of tissue damage following heat treatment. Fruits were subjected to equivalent heat units at 36.5°C for 60 min plus 46.5°C for 43 min or 46.5°C for 90 min by hot water treatments (hwt) on the assumption of cumulative heat effects and a base temperature of 12–13°C. Fruit reflectance decreased whereas chroma and hue angle increased over storage time and also with increase in storage temperature. The yellow colour increased with a rise in storage temperature in hot water treated mangoes. Soluble solids content of mangoes held at 22°C was highest at 5 days of storage but decreased subsequently over storage time. Impedance of all fruits decreased with increase in frequency, storage temperature and time in store. The impedance of hwt mangoes was lower than that of non‐hwt fruits 8 h after immersion, but recovered almost to control levels on day 5 at 4°C or 13°C, but decreased gradually after 5 days at 13°C. Impedance of all mangoes stored at 22°C decreased continuously during storage. Impedance was higher in the inner mesocarp than outer pulp. Impedance of hwt fruits was poorly correlated with soluble solid content and chroma but well correlated with reflectance of fruit pulp at 22°C. Changes in impedance of mangoes are discussed in relation to physiological and biochemical changes that occur during heat treatment and storage.     

The influence of temperature on weight loss from stored onion bulbs due to desiccation, respiration and sprouting

When onion bulbs were stored for 9 months at 2, 7.5, 15 or 25 °C and 70% r.h., the losses due to desiccation increased with temperature but less than 20 % was due to respiration at any of the storage temperatures. Respiration rates of onion bulbs transferred from 2 to 25 °C were higher from February onwards than those of bulbs stored continuously at 25 °C. Conversely, bulbs transferred from 25 to 2 °C respired less from February onwards than those kept at 2 °C. Sprouting, at the final assessment in June, was highest at 15 and 7.5 °C and lowest at 2 °C. Total weight loss was above 45 % in all the storage treatments except at 2 °C (12%). Storage at 7.5 °C is suitable until March but long-term storage until June requires low temperatures.     

Changes in carotenoid and chlorophyll content of black tomatoes (Lycopersicone sculentum L.) during storage at various temperatures

Black tomatoes have a unique color and higher lycopene content than typical red tomatoes. Here, black tomatoes were investigated how maturation stage and storage temperature affected carotenoid and chlorophyll accumulation. Immature fruits were firmer than mature fruits, but failed to develop their distinctive color and contained less lycopene when stored at 8 °C. Hunter a^* values of black tomatoes increased with storage temperature and duration; storage of immature fruits at high temperature favored lycopene accumulation. Chlorophyll levels of black tomatoes declined during storage, but differences between mature and immature tomatoes stored at 12 °C were minimal. β-Carotene levels of black tomatoes increased during early storage, but rapidly declined beginning 13 d post-harvest. The highest lycopene and chlorophyll levels were observed in mature black tomatoes stored at 12 °C for 13 d; these conditions also yielded the best quality fruit. Thus, the unique pigmentation properties of black tomatoes can be precisely controlled by standardizing storage conditions.     

Effect of storage time and pretreatment on seed germination of the threatened coniferous species Fokienia hodginsii

We report the effects of storage time and pretreatment on seed germination of Fokienia hodginsii. Lower mean germination was observed in seeds stored for 2 years (6.41 ± 1.23 seeds/replicate) compared with those stored for 1 year (8.52 ± 1.06 seeds/replicate). Seeds collected from a southern location had statistically higher mean germination (9.67 ± 1.28 seeds/replicate) than those collected from a northern location (7.99 ± 1.36 seeds/replicate). Higher mean T₅₀ was observed in seeds stored for 2 years (37.02 ± 4.43 days) compared with those stored for 1 year (30.69 ± 5.06 days). Mean germination of untreated fresh seeds was 9.97 ± 1.34 seeds/replicate and that of treated fresh seeds in 60°C water was 12.95 ± 1.24 seeds/replicate. Fresh seeds treated with 50°C and 70°C water had a significantly lower mean germination compared with untreated seeds and seeds treated in 60°C water. Mean T₅₀ was lowest in seeds treated with 60°C water.     

Changes in germinability,lipid peroxidation,and antioxidant enzyme activities in pear stock (<Emphasis Type="Italic">Pyrus betulaefolia</Emphasis> Bge.) seeds during room- and low-temperature storage

The aim of this study was to determine if loss of germinability in Pyrus betulaefolia seeds stored at 4°C and at room temperature is associated with a loss of membrane lipid peroxidation or changes in antioxidant enzyme activities. The results indicated that germination percentage clearly decreased when seeds were stored at room temperature rather than at 4°C from 6 to 12 months. Room-temperature storage of the pear stock seed for 12 months decreased germination to 15.52%, but germination percentage was not changed when seed was stored at 4°C for 12 months. MDA, a marker for membrane lipid peroxidation, increased significantly under room-temperature storage conditions. Antioxidant enzyme (SOD, POD, and CAT) activities were a good indicator of germination percentage in pear stock seeds. Antioxidant enzyme activities of pear stock seeds at 4°C were higher than antioxidant enzyme activities in seeds stored at room temperature from 6 to 12 months. Antioxidant enzyme activities of the pear stock seed decreased markedly under conditions of room-temperature storage from 6 to 12 months. The results of this study showed that long-term room-temperature storage was detrimental for maintaining the vigor of P. betulaefolia seeds. The mechanisms responsible for this outcome are a higher level of membrane lipid peroxidation and a lower level of activity of antioxidant enzymes.     

Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

Biology of Lysiphlebus fabarum following cold storage of larvae and pupae

Cold storage is one means of preserving parasitoids prior to release in augmentation biological control programs. This study examined the feasibility of storing larval and pupal stages of a sexual population of Lysiphlebus fabarum Marshall (Hymenoptera: Braconidae: Aphidiinae) at 6 ± 1 and 8 ± 1 °C, 50–60% r.h., and L14:D10 photoperiod. These life stages were stored for periods of 1, 2, and 3 weeks under fluctuating thermal regimes (2 h daily at 21 ± 1 °C). Generally, pupae gave better results than larvae, and 6 °C was better than 8 °C, considering wasp survival, wasp size (tibial and antennal lengths), egg load, and egg size. The best results were obtained with pupae stored for 2 weeks under a fluctuating temperature regime at 6 °C. Females emerging from this treatment did not differ from controls (developing directly at 21 °C) in body size, egg size, or progeny sex ratio, and suffered less than 20% mortality. Egg loads were reduced in these wasps, but the reductions were substantially less than occurred in other 2‐week‐storage treatments. Wasps stored in this manner successfully parasitized similar numbers of aphids as controls and produced similar progeny sex ratios. These results reveal a suitable set of low‐temperature conditions that can be used to delay the development of L. fabarum for 2 weeks with minimal impact on wasp fitness.     

Cold tolerance of coccons ofAllorhogas pyralophagus [Hym.: Braconidae]

Cold tolerance studies were conducted under laboratory conditions for coccons ofAllorhogas pyralophagus Marsh, a Mexican parasitoid of graminaceous borers. By storage, developmental time (from cocoon to adult emergence) could be extended by 2 to 6 times. However, cocoons stored for more than 14 days at 2°C failed to survive, while at 5 and 10°C, about 50% emergence was recorded for upto 21 days of storage. With respect to survival and adult longevity, 10°C seemed to be the most suitable storage temperature. Pre-emergence period was also significantly increased by storing cocoons for 21 to 35 days at this temperature. Sex-ratio of emerging adults was not significantly affected by storage. Fecundity was adversely affected in all the treatments except in the case of females emerging from cocoons stored at 5°C for 7 days. The progeny of parasitoids which emerged from cocoons stored at 5 and 10°C for 35 days consisted of only males. It is clear from the present study thatA. pyralophagus cocoons are more amenable to short-term storage. Contribution No. 285/87 of the Indian Institute of Horticultural Research, Bangalore-560 089.