Thermosensitive Poloxamer 407/Poly(Acrylic Acid) Hydrogels with Potential Application as Injectable Drug Delivery System

Thermosensitive hydrogels are of great interest for in situ gelling drug delivery. The thermosensitive vehicle with a gelation temperature in a range of 30–36°C would be convenient to be injected as liquid and transform into gel after injection. To prepare novel hydrogels gelling near body temperature, the gelation temperature of poloxamer 407 (PX) were tailored by mixing PX with poly(acrylic acid) (PAA). The gelation behaviors of PX/PAA systems as well as the interaction mechanism were investigated by tube inversion, viscoelastic, shear viscosity, DSC, SEM, and FTIR studies. The gelation temperature of the plain PX solutions at high concentration of 18, 20, and 22% (w/w) gelled at temperature below 28°C, which is out of the suitable temperature range. Mixing PX with PAA to obtain 18 and 20% (w/w) PX with 1% (w/w) PAA increased the gelation temperature to the desired temperature range of 30–36°C. The intermolecular entanglements and hydrogen bonds between PX and PAA may be responsible for the modulation of the gelation features of PX. The mixtures behaved low viscosity liquid at room temperature with shear thinning behavior enabling their injectability and rapidly gelled at body temperature. The gel strength increased, while the pore size decreased with increasing PX concentration. Metronidazole, an antibiotic used for periodontitis, was incorporated into the matrices, and the drug did not hinder their gelling ability. The gels showed the sustained drug release characteristic. The thermosensitive PX/PAA hydrogel could be a promising injectable in situ gelling system for periodontal drug delivery.     

Liquid Crystalline Systems for Transdermal Delivery of Celecoxib: In Vitro Drug Release and Skin Permeation Studies

Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.KEY WORDS: celecoxib, drug delivery systems, liquid crystalline system, monoolein, skin permeation     

Water potential — water saturation deficit relationship during dehydration and resaturation of leaves

The relationship between the water potential (Ψ_w) and the water saturation deficit (Δ W _sat) in kale and maize leaf tissue was measured during dehydration and resaturation either of leavesin situ or of cut leaves. The curves relating Ψ_w toΔW _sat were similar in all variants, but at the same values ofΔ W _sat corresponding values of Ψ_w were always lower in leavesin situ than in cut leaves and during dehydration than during resaturation.     

Optimizing Exchange Transfusion for Severe Unconjugated Hyperbilirubinemia: Studies in the Gunn Rat

Background

Severe unconjugated hyperbilirubinemia carries the risk of neurotoxicity. Phototherapy (PT) and exchange transfusion (ET) are cornerstones in the treatment of unconjugated hyperbilirubinemia. Studies to improve ET efficacy have been hampered by the low application of ET in humans and by the lack of an in vivo model. The absence of an appropriate animal model has also prevented to determine the efficacy of adjunct or alternative treatment options such as albumin (Alb) administration.

Aim

To establish an in vivo model for ET and to determine the most effective treatment (combination) of ET, PT and Alb administration.

Methods

Gunn rats received either PT, PT+Alb, ET, ET+PT, ET+PT+Alb or sham operation (each n = 7). ET was performed via the right jugular vein in ∼20 min. PT (18 µW/cm²/nm) was started after ET or at T₀. Albumin i.p. injections (2.5 g/kg) were given after ET or before starting PT. Plasma unconjugated bilirubin (UCB), plasma free bilirubin (Bf), and brain bilirubin concentrations were determined.

Results

We performed ET in 21 Gunn rats with 100% survival. At T₁, ET was profoundly more effective in decreasing both UCB −44%, p<0.01) and Bf −81%, p<0.05) than either PT or PT+Alb. After 48 h, the combination of ET+PT+Alb showed the strongest hypobilirubinemic effect (−54% compared to ET).

Conclusions

We optimized ET for severe unconjugated hyperbilirubinemia in the Gunn rat model. Our data indicate that ET is the most effective treatment option, in the acute as well as the follow-up situation.     

Water stress development in kale leavesin situ and in water supplied cut leaves

The development of temporary water stress during the day-light hours, characterized by a decrease of the values of the water potential (?_w) and increase of the values of water saturation deficit (ΔW _sat) was found not only in the leaves of intact kale plants but also in cut leaves with their petioles immersed in water. These results indicate that the leaf resistance to water transport could not be supposed as negligible. The same decrease of ? _w was accompanied with the higher increase of ΔW _sat in cut leaves than in leavesin situ.     

Preparation of <Emphasis Type="Italic">in situ</Emphasis> Injectable Chitosan/Gelatin Hydrogel Using an Acid-tolerant Tyrosinase

An in situ injectable chitosan/gelatin hydrogel was formed under slightly acidic conditions (pH 4.0 ~ 4.5) using an acid-tolerant tyrosinase, tyrosinase-CNK. A homogeneous chitosan/tyrosinase-CNK solution was prepared in one part of a dual-barrel syringe, and highly soluble gelatin in distilled water was prepared in the other part of the syringe without any additional crosslinking materials. Chitosan/gelatin hydrogel was formed in situ by simple injection of the solutions at room temperature followed by curing at 37°C. However, conventional mushroom tyrosinase did not catalyze this permanent gel formation. Tyrosinase- CNK-catalyzed glycol chitosan/gelatin hydrogel was similarly formed by this in situ injection approach. The hydrogels exhibited a high swelling ratio of 20-fold their own weight, interconnected micropores with an average diameter of approximately 260 μm and in vitro biodegradability suitable for tissue engineering and drug delivery applications. These results showed that tyrosinase-CNK-mediated chitosan/gelatin hydrogel formation has remarkable potential for the development of novel formulations for in situ injectable gel-forming systems.     

Chloroplast osmotic adjustment and water stress effects on photosynthesis

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψ_w) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψ_w reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψ_w may involve stromal volume maintenance.     

Exploring Microstructural Changes in Structural Analogues of Ibuprofen-Hosted In Situ Gelling System and Its Influence on Pharmaceutical Performance

The present work explores inner structuration of in situ gelling system consisting of glyceryl monooleate (GMO) and oleic acid (OA). The system under study involves investigation of microstructural changes which are believed to govern the pharmaceutical performance of final formulation. The changes which are often termed mesophasic transformation were analysed by small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), rheology and plane polarised light (PPL) microscopy. The current work revealed transformation of blank system from W/O emulsion to reverse hexagonal structure upon addition of structural analogues of ibuprofen. Such transformations are believed to occur due to increased hydrophobic volume within system as probed by SAXS analysis. The findings of SAXS studies were well supported by DSC, rheology and PPL microscopy. The study established inverse relationship between log P value of structural analogues of ibuprofen and the degree of binding of water molecules to surfactant chains. Such relationship had pronounced effect on sol–gel transformation process. The prepared in situ gelling system showed sustained drug release which followed Higuchi model.KEY WORDS: flurbiprofen, hexagonal phase, ibuprofen, ketoprofen, liquid crystal, sustained drug release     

Glass transition behavior of octyl β-D-glucoside and octyl β-D-thioglucoside/water binary mixtures

The lyotropic behavior and glass-forming properties of octyl β-d-glucoside (C8Glu) and octyl β-d-thioglucoside (C8SGlu)/water binary mixtures were evaluated using differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). The results clearly indicate that the mixture forms a glass in the supercooling state of liquid crystalline phases such as cubic, lamellar, and smectic. The glass transition temperature (T_g) of the mixture was strongly dependent on solute concentration, with a higher concentration correlating with a higher T_g. The experimental T_g was consistent with the predicted value calculated using the Couchman-Karasz equation in both the C8Glu and C8SGlu/water mixtures. The change of heat capacity at T_g showed the two bending points under variation of concentrations. And the highest temperature of phase transition from lamellar to isotropic solution was observed at around 50% molar concentration. It was expected that non-percolated state of water existed in extremely higher concentration ranges.     

In Vitro Insulin Release from Thermosensitive Chitosan Hydrogel

Recently, great attention has been paid to in situ gel-forming chitosan/glycerol-phosphate (chitosan/Gp) solution due to their good biodegradability and thermosensitivity. This in situ gel-forming system is injectable fluid that can be introduced into the body in a minimally invasive manner prior to solidifying within the desired tissue. At the present study, insulin release from chitosan/Gp solution has been investigated. Insulin in different concentrations was loaded in two formulations of chitosan/Gp solution and in vitro drug release was studied over a period of 3 weeks. Results indicated that the release of insulin from chitosan/Gp gel decreases by increasing in Gp salt and initial insulin concentration. Stability of released insulin was investigated by 8-anilino-1-naphthalenesulfonate probe. Results proved that insulin have been released in its native form. Because of simple preparation and administration, prolonged release of insulin and stability of released insulin, this in situ gel-forming system could be used as a controlled release delivery system for insulin.KEY WORDS: biodegradable, chitosan, controlled release, in situ forming, insulin     

A Modified <Emphasis Type="Italic">In Situ</Emphasis> Method to Determine Release from a Complex Drug Carrier in Particle-Rich Suspensions

Effective and compound-sparing methods to evaluate promising drug delivery systems are a prerequisite for successful selection of formulations in early development stages. The aim of the study was to develop a small-scale in situ method to determine drug release and supersaturation in highly concentrated suspensions of enabling formulations. Mesoporous magnesium carbonate (MMC), which delivers the drug in an amorphous form, was selected as a drug carrier. Five model compounds were loaded into the MMC at a 1:10 ratio using a solvent evaporation technique. The μDiss Profiler was used to study the drug release from MMC in fasted-state simulated intestinal fluid. To avoid extensive light scattering previously seen in particle-rich suspensions in the μDiss Profiler, an in-house-designed protective nylon filter was placed on the in situ UV probes. Three types of release experiments were conducted for each compound: micronized crystalline drug with MMC present, drug-loaded MMC, and drug-loaded MMC with 0.01% w/w hydroxypropyl methyl cellulose. The nylon filters effectively diminished interference with the UV absorption; however, the release profiles obtained were heavily compound dependent. For one of the compounds, changes in the UV spectra were detected during the release from the MMC, and these were consistent with degradation of the compound. To conclude, the addition of protective nylon filters to the probes of the μDiss Profiler is a useful contribution to the method, making evaluations of particle-rich suspensions feasible. The method is a valuable addition to the current ones, allowing for fast and effective evaluation of advanced drug delivery systems.     

Further evidence for closed, nonspherical aggregates in the cubic I1 phase of lysolecithin and water

Measurements of time-resolved fluorescence quenching have been performed in the binary lauroyllysophosphatidylcholine (LaLPC)/water system. The aggregation numbers, N, are determined for the micellar solution phase (N_micelle ≈ 80) and the cubic liquid crystalline I₁ phase (N_cub ≈ 90) at 298-303 K. When a quencher is present, the fluorescence decays for the hexagonal phase of the LaLPC/water system and for the bicontinuous cubic phase of monooleoylglycerol/water system are nonexponential, as expected for phase structures having long-range continuous apolar regions. Nuclear magnetic resonance (NMR) measurements of the lipid translational diffusion conclusively show that the cubic I₁ phase consists of closed micelles. NMR spectra of ³¹P obtained at 202.4 MHz of this cubic phase exhibit a characteristic line shape, which is compatible with a phase structure containing short nonspherical micelles. A comparison between electron spin resonance (ESR) spin-label spectra recorded for a micellar solution and the cubic phases of the LaLPC and monooleoylglycerol systems are also shown to support a structure of closed micelles in the cubic I₁ phase of the lysolecithin system.     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Application of a multi-cylinder evapotranspirometer method for evapotranspiration measurements in wetlands

This paper introduces a multi-cylinder evapotranspirometer method, which can directly measure evapotranspiration (ET) from emergent plants in different species and states as well as simultaneously measure evaporation (E_W) from an open water surface. Values of daily ET from three contrasting reed (Phragmites australis) stands, with different leaf area indexes (LAI), were obtained through in situ measurements of the Baiyangdian wetland using this method during the growing seasons in 2008 and 2009. The results showed that the ET rate of the reed belt was very high, even exceeding 20 mm d⁻¹ under extreme weather conditions. Depending on the LAI change, the annual ET from the different reed canopies ranged from 970 to 2035 mm, whereas the ET/E_W ratios ranged from 2.05 to 3.98. Accuracy analysis results showed that the errors of the measurement from this method were no more than 2 mm. The relative errors of the measurement were correspondingly from 0.04% to 0.33%. It is indicated that the accuracy of our measurement is good enough for the requirements of the ET measurement.     

Nanosuspension Based In Situ Gelling Nasal Spray of Carvedilol: Development, In Vitro and In Vivo Characterization

The objective of the present investigation was to develop in situ gelling nasal spray formulation of carvedilol (CRV) nanosuspension to improve the bioavailability and therapeutic efficiency. Solvent precipitation–ultrasonication method was opted for the preparation of CRV nanosuspension which further incorporated into the in situ gelling polymer phase. Optimized formulation was extensively characterized for various physical parameters like in situ gelation, rheological properties and in vitro drug release. Formation of in situ gel upon contact with nasal fluid was conferred via the use of ion-activated gellan gum as carrier. In vivo studies in rabbits were performed comparing the nasal bioavailability of CRV after oral, nasal, and intravenous administration. Optimized CRV nanosuspension prepared by combination of poloxamer 407 and oleic acid showed good particle size [d (0.9); 0.19 μm], zeta potential (+10.2 mV) and polydispersity (span; 0.63). The formulation containing 0.5% w/v gellan gum demonstrated good gelation ability and desired sustained drug release over period of 12 h. In vivo pharmacokinetic study revealed that the absolute bioavailability of in situ nasal spray formulation (69.38%) was significantly increased as compared to orally administered CRV (25.96%) with mean residence time 8.65 h. Hence, such in situ gel system containing drug nanosuspension is a promising approach for the intranasal delivery in order to increase nasal mucosal permeability and in vivo residence time which altogether improves drug bioavailability.KEY WORDS: bioavailability, Carvedilol, in situ gel, intranasal, nanosuspension     

Model Drug as Pore Former for Controlled Release of Water-Soluble Metoprolol Succinate from Ethylcellulose-Coated Pellets Without Lag Phase: Opportunities and Challenges

The objective of the present study was to evaluate the feasibility of using model drug metoprolol succinate (MS) as a pore former to modify the initial lag phase (i.e., a slow or non-release phase in the first 1–2 h) associated with the drug release from coated pellets. MS-layered cores with high drug-layering efficiency (97% w/w) were first prepared by spraying a highly concentrated drug aqueous solution (60% w/w, 70°C) on non-pareils without using other binders. The presence of MS in ethylcellulose (EC) coating solution significantly improved the coating process by reducing pellets sticking, which often occurs during organic coating. There may be a maximum physical compatibility of MS with EC, and the physical state of the drug in the functional coating layer of EC/MS (80:20) was simultaneously crystalline and non-crystalline (amorphous or solid molecule solution). The lag phase associated with hydroxypropylcellulose (HPC) as a pore former was not observed when MS was used as a pore former. The drug release from EC/MS-coated pellets was pH independent, inversely proportional to the coating levels, and directly related to the pore former levels. The functional coating layer with MS as a pore former was not completely stabilized without curing. Curing at 60°C for 1 day could substantially improve the stability of EC/MS-coated pellets. The physical state of the drug in the free film of EC/MS (85:15) changed partially from amorphous to crystal when cured at 60°C for 1 day, which should be attributed to the incompatibility of the drug with EC.KEY WORDS: coated pellets, curing treatment, lag phase, metoprolol succinate, pore former     

Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L_o) and liquid-disordered (L_d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L_o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In ¹³C-¹³C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.     

Nodule Activity and Allocation of Photosynthate of Soybean during Recovery from Water Stress

Nodulated soybean plants (Glycine max [L.] Merr. cv Ransom) in a growth-chamber study were subjected to a leaf water potential (Ψ_w) of −2.0 megapascal during vegetative growth. Changes in nonstructural carbohydrate contents of leaves, stems, roots, and nodules, allocation of dry matter among plant parts, in situ specific nodule activity, and in situ canopy apparent photosynthetic rate were measured in stressed and nonstressed plants during a 7-day period following rewatering. Leaf and nodule Ψ_w also were determined. At the time of maximum stress, concentration of nonstructural carbohydrates had declined in leaves of stressed, relative to nonstressed, plants, and the concentration of nonstructural carbohydrates had increased in stems, roots, and nodules. Sucrose concentrations in roots and nodules of stressed plants were 1.5 and 3 times greater, respectively, than those of nonstressed plants. Within 12 hours after rewatering, leaf and nodule Ψ_w of stressed plants had returned to values of nonstressed plants. Canopy apparent photosynthesis and specific nodule activity of stressed plants recovered to levels for nonstressed plants within 2 days after rewatering. The elevated sucrose concentrations in roots and nodules of stressed plants also declined rapidly upon rehydration. The increase in sucrose concentration in nodules, as well as the increase of carbohydrates in roots and stems, during water stress and the rapid disappearance upon rewatering indicates that inhibition of carbohydrate utilization within the nodule may be associated with loss of nodule activity. Availability of carbohydrates within the nodules and from photosynthetic activity following rehydration of nodules may mediate the rate of recovery of N₂-fixation activity.     

Polymorphism of phosphatidylethanolamines from two natural sources

Two pure phosphatidylethanolamines, isolated from porcine erythrocytes (PPE) and from larvae of Phormia regina (FPE), were analysed and found to differ only in their fatty-acyl composition. An X-ray diffraction study was made of the equilibrium structures formed by these two lipids, in water, as a function of lipid concentration and of temperature. They behave identically and the structural parameters of a widely occurring lamellar phase were determined and found to be similar to phosphatidylcholine. The lamellar phase, composed of alternating layers of lipid, of thickness d₁, and water, of thickness d_w, swells only to a limited extent (d_w maximum is 13 Å) and d₁ varies from 38 Å to 43 Å as the lipid concentration varies from 70% to 90% dry weight. The equilibrium structures formed by FPE and PPE in low pH buffers are quite different, PPE being identical to that in water but FPE forming a cubic phase. The reason for the difference is unclear, but the results suggest the importance of fatty-acyl composition and of the effect of pH on the polymorphism of these systems.     

Application of a liquid chromatography/tandem mass spectrometry method for the pharmacokinetic study of dihydroartemisinin injectable powder in healthy Chinese subjects

A simple, rapid and accurate liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of dihydroartemisinin (DHA) in human plasma. Following a simple single-step liquid–liquid extraction with ethyl acetate, the analyte was separated on a C₁₈ column by isocratic elution with methanol–water–10 mM ammonium acetate (80:10:10, v/v/v), and analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 1.01–2020 ng/mL. Intra- and inter-day precisions were less than 9.0%, and accuracy ranged from 93.0 to 98.2%. The pharmacokinetics of DHA injectable powder was studied for the first time in healthy subjects by this method. After single intravenous infusion of DHA injectable powder 40, 80 and 160 mg, the elimination half-life (t_1/2λZ) was 1.69, 1.88 and 1.92 h, respectively; mean C_max and AUC increased in proportion to the doses. The pharmacokinetics of DHA fit the linear dynamic feature over the DHA dose range studied.