Development and Characterization of Mixed Niosomes for Oral Delivery Using Candesartan Cilexetil as a Model Poorly Water-Soluble Drug

The aim of this study was to prepare candesartan cilexetil-loaded niosomes and mixed niosomes to enhance the aqueous solubility of the drug, thus improving its oral bioavailability. The formulations were prepared using various types and combinations of surfactants, copolymers, and charge-inducing agents. The candesartan cilexetil entrapment efficiency, particle size, and zeta potential of these niosomes varied within the range of 99.06 ± 1.74 to 36.26 ± 2.78, 157.3 ± 3.3 to 658.3 ± 12.7 nm, and −14.7 ± 2.8 to −44.5 ± 1.5 mV, respectively. The in vitro drug release from niosomes was improved after niosomal entrapment compared to pure candesartan cilexetil. The sedimentation behavior study and formulation stability tests against bile salt revealed that mixed niosomes prepared by combining Span 60 and Pluronic P85 demonstrated better stability. The differential scanning calorimetry analysis showed the conversion of crystal structure of candesartan cilexetil to the soluble amorphous form after niosomal encapsulation which induced the drug release. Consequently, oral drug delivery by Span 60/Pluronic P85-mixed niosomes seems feasible due to enhanced drug release and stability.KEY WORDS: in vitro drug release, niosomes, oral drug delivery, stability, surfactants     

Preparation and Characterization of Stable pH-Sensitive Vesicles Composed of α-Tocopherol Hemisuccinate

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t_1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.Key words: cleavage, long circulation, PEG-α-tocopherol hemisuccinate, pH-sensitive, vesicles     

Designing anti‐diabetic β‐cells microcapsules using polystyrenic sulfonate,polyallylamine, and a tertiary bile acid: Morphology,bioenergetics, and cytokine analysis

Purpose: Recently sodium alginate (SA)‐poly‐l‐ornithine (PLO) microcapsules containing pancreatic β‐cells that showed good morphology but low cell viability (<27%) was designed. In this study, two new polyelectrolytes, polystyrenic sulfonate (PSS; at 1%) and polyallylamine (PAA; at 2%) were incorporated into a microencapsulated‐formulation, with the aim of enhancing the physical properties of the microcapsules. Following incorporation, the structural characteristics and cell viability were investigated. The effects of the anti‐inflammatory bile acid, ursodeoxycholic acid (UDCA), on microcapsule morphology, size, and stability as well as β‐cell biological functionality was also examined. Methods: Microcapsules were prepared using PLO‐PSS‐PAA‐SA mixture and two types of microcapsules were produced: without UDCA (control) and with UDCA (test). Microcapsule morphology, stability, and size were examined. Cell count, microencapsulation efficiency, cell bioenergetics, and activity were also examined. Results: The new microcapsules showed good morphology but cell viability remained low (29% ± 3%). UDCA addition improved cell viability post‐microencapsulation (42 ± 5, P < 0.01), reduced swelling (P < 0.01), improved mechanical strength (P < 0.01), increased Zeta‐potential (P < 0.01), and improved stability. UDCA addition also increased insulin production (P < 0.01), bioenergetics (P < 0.01), and decreased β‐cell TNF‐α (P < 0.01), IFN‐gamma (P < 0.01), and IL‐6 (P < 0.01) secretions. Conclusions: Addition of 4% UDCA to a formulation system consisting of 1.8% SA, 1% PLO, 1% PSS, and 2% PAA enhanced cell viability post‐microencapsulation and resulted in a more stable formulation with enhanced encapsulated β‐cell metabolism, bioenergetics, and biological activity with reduced inflammation. This suggests potential application of UDCA, when combined with SA, PLO, PSS, and PAA, in β‐cell microencapsulation and diabetes treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:501–509, 2016     

Modified Cellulose Nanocrystal for Vitamin C Delivery

Cellulose nanocrystal grafted with chitosan oligosaccharide (CNC-CS_OS) was used to encapsulate vitamin C and prepare CNCS/VC complexes using tripolyphosphte via ionic complexation. The stability of vitamin C and the antioxidant activity of the CNCS/VC complexes were elucidated. The formation of the complex was confirmed using DSC and UV–vis spectrophotometry, and TEM was used to study the morphology of the complexes. The encapsulation efficiency of vitamin C at pH 3 and 5 was 71.6% ± 6.8 and 91.0 ± 1.0, respectively. Strong exothermic peaks observed in isothermal titration calorimetric (ITC) studies at pH 5 could be attributed to additional electrostatic interactions between CNC-CS_OS and vitamin C at pH 5. The in vitro release of vitamin C from CNCS/VC complexes showed a sustained release of up to 20 days. The vitamin C released from CNCS/VC complex displayed higher stability compared with the control vitamin C solution, and this was also confirmed from the ITC thermograms. CNC-CS_OS possessed a higher scavenging activity and faster antioxidant activity compared with its precursors, i.e., oxidized CNC and CS_OS and their physical mixtures. Complexing vitamin C into CNC-CS_OS particles yielded a dynamic antioxidant agent, where the vitamin C is released over time and displayed sustained antioxidant properties. Therefore, CNCS/VC can potentially be used in cosmeceutical applications as topical formulations.KEY WORDS: cellulose nanocrystals, chitosan oligosaccharide, controlled release, vitamin C antioxidant activity     

Development of Novel Chitosan Microcapsules for Pulmonary Delivery of Dapsone: Characterization,Aerosol Performance,and In Vivo Toxicity Evaluation

Pneumocystis carinii pneumonia (PCP) is a major opportunistic infection that affects patients with human immunodeficiency virus. Although orally administered dapsone leads to high hepatic metabolism, decreasing the therapeutic index and causing severe side effects, this drug is an effective alternative for the treatment of PCP. In this context, microencapsulation for pulmonary administration can offer an alternative to increase the bioavailability of dapsone, reducing its adverse effects. The aim of this work was to develop novel dapsone-loaded chitosan microcapsules intended for deep-lung aerosolized drug delivery. The geometric particle size (D_4,3) was approximately 7 μm, the calculated aerodynamic diameter (d_aero) was approximately 4.5 μm, and the mass median aerodynamic diameter from an Andersen cascade impactor was 4.7 μm. The in vitro dissolution profile showed an efficient dapsone encapsulation, demonstrating the sustained release of the drug. The in vitro deposition (measured by the Andersen cascade impactor) showed an adequate distribution and a high fine particles fraction (FPF = 50%). Scanning electron microscopy of the pulmonary tissues demonstrated an adequate deposition of these particles in the deepest part of the lung. An in vivo toxicity experiment showed the low toxicity of the drug-loaded microcapsules, indicating a protective effect of the microencapsulation process when the particles are microencapsulated. In conclusion, the pulmonary administration of the novel dapsone-loaded microcapsules could be a promising alternative for PCP treatment.KEY WORDS: dapsone, dry powders inhalers, in vivo toxicity, microparticles, pulmonary drug delivery     

Evaluation of Matrix Tablets Based on Eudragit?E100/Carbopol?971P Combinations for Controlled Release and Improved Compaction Properties of Water Soluble Model Drug Paracetamol

The purpose of this work was to investigate the influence of Eudragit®E100 polymer in modifying the release rates and compaction properties of water soluble model drug paracetamol from Carbopol®971P NF polymer matrix tablets prepared by direct compression. The effects of the ratio of the two polymers, the total polymeric content, and the tablets mechanical strength on paracetamol release rates were investigated. Dissolution studies were conducted using USP XX Π rotating paddle apparatus at 50 rpm and 37°C at three different stages (pH 1.2, 4.8, and 6.8). Results showed that the polymers combination improved significantly the compaction properties of paracetamol tablets as evident by the higher crushing strengths (8.3 ± 0.4 Kp) compared to polymer-free tablets (3.4 ± 0.2 Kp) at intermediate compression pressure of 490 MPa. When combined with Carbopol®971P NF, Eudragit®E100 was found to be capable of extending paracetamol release for more than 12 h compared to 1 h for polymers-free tablets. The combined polymers were able to control paracetamol release in a pH independent pattern. The f2 (similarity factor) analysis showed that the ratio between the polymers and the total polymer concentration exhibited significant impact on drug release rates. In conclusion, Eudragit®E100 when combined with Carbopol®971P NF was capable of improving the compaction and sustained release properties of paracetamol. Korsmeyer–Peppas model was found to be the most suitable for fitting drug release data. The polymer combinations can potentially be used to control the release rates of highly water soluble drugs.KEY WORDS: Carbopol®971P NF, Eudragit®E100, matrix tablet, pH-independent release, sustained     

Masking the Bitter Taste of Injectable Lidocaine HCl Formulation for Dental Procedures

Several attempts have been made to mask the bitter taste of oral formulations, but none have been made for injectable formulations. This study aims to mask the bitter taste of dental lidocaine HCl (LID) injection using hydroxypropyl-β-cyclodextrin (HP-β-CD) and sodium saccharin. Inclusion complexes of LID and HP-β-CD were prepared by the solution method in 1:1 and 1:2 M ratios. Inclusion complexes in solution were studied using phase solubility in phosphate buffer solutions (pH 8, 9, and 10). Freeze-dried inclusion complexes were characterized using differential scanning calorimetry (DSC), X-ray, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), and in vitro release. Injectable formulations were prepared using inclusion complexes and characterized for stability and for taste using an Alpha MOS ASTREE electronic tongue (ETongue). The association constants of HP-β-CD with lidocaine-free base and its ionized form were found to be 26.23 ± 0.00025 and 0.8694 ± 0.00045 M⁻¹, respectively. Characterization studies confirmed the formation of stable inclusion complexes of LID and HP-β-CD. Injectable formulations were found to be stable for up to 6 months at 4°C, 25°C, and 40°C. The taste evaluation study indicated that HP-β-CD (1:1 and 1:2 M ratios) significantly improved the bitter taste of LID injectable formulation. In conclusion, inclusion complex in the 1:1 M ratio with 0.09% sodium saccharin was considered to be optimum in masking the bitter taste of LID.KEY WORDS: bitter taste, HP-β-CD, inclusion complex, injectable, lidocaine HCl, taste masking     

Freeze-Dried Targeted Mannosylated Selenium-Loaded Nanoliposomes: Development and Evaluation

The aim of this investigation was to develop and evaluate freeze-dried mannosylated liposomes for the targeted delivery of selenium. Dipalmitoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol were dissolved in a chloroform and methanol mixture and allowed to form a thin film within a rotatory evaporator. This thin film was hydrated with a sodium selenite (5.8 μM) solution to form multilamellar vesicles and homogenized under high pressure to yield unilamellar nanoliposomes. Se-loaded nanoliposomes were mannosylated by 0.1% w/v mannosamine (Man-Lip-Se) prior to being lyophilized. Mannosamine concentration was optimized with cellular uptake studies in M receptor expressing cells. Non-lyophilized and lyophilized Man-Lip-Se were characterized for size, zeta potential, and entrapment efficiency. The influence of liposomal composition on the characteristics of Man-Lip-Se were evaluated using acidic and basic medium for 24 h. Thermal analysis and powder X-ray diffraction were used to determine the interaction of components within the Man-Lip-Se. The size, zeta potential and entrapment efficiency of the optimum Man-Lip-Se were observed to be 158 ± 28.9 nm, 33.21 ± 0.89 mV, and 77.27 ± 2.34%, respectively. An in vitro Se release of 70–75% up to 24 h in PBS pH 6.8 and <8% Se release in acidic media (0.1 N HCl) in 1 h was observed. The Man-Lip-Se were found to withstand gastric-like environments and showed sustained release. Stable freeze-dried Man-Lip-Se were successfully formulated with a size of <200 nm, ∼75% entrapment, and achieved controlled release of Se with stability under acidic media, which may be of importance in the targeted delivery of Se to the immune system.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-013-9988-3) contains supplementary material, which is available to authorized users.Key words: mannosylation, nanoliposome, selenium, thermal properties     

Preparation and Characterization of Fenofibrate-Loaded Nanostructured Lipid Carriers for Oral Bioavailability Enhancement

The aim of this study is to investigate the potential of nanostructured lipid carriers (NLCs) in improving the oral bioavailability of a lipid lowering agent, fenofibrate (FEN). FEN-loaded NLCs (FEN-NLCs) were prepared by hot homogenization followed by an ultrasonication method using Compritol 888 ATO as a solid lipid, Labrafil M 1944CS as a liquid lipid, and soya lecithin and Tween 80 as emulsifiers. NLCs were characterized in terms of particle size and zeta pote\ntial, surface morphology, encapsulation efficiency, and physical state properties. Bioavailability studies were carried out in rats by oral administration of FEN-NLC. NLCs exhibited a spherical shape with a small particle size (84.9 ± 4.9 nm). The drug entrapment efficiency was 99% with a loading capacity of 9.93 ± 0.01% (w/w). Biphasic drug release manner with a burst release initially, followed by prolonged release was depicted for in vitro drug release studies. After oral administration of the FEN-NLC, drug concentration in plasma and AUC_t-∞ was fourfold higher, respectively, compared to the free FEN suspension. According to these results, FEN-NLC could be a potential delivery system for improvement of loading capacity and control of drug release, thus prolonging drug action time in the body and enhancing the bioavailability.KEY WORDS: bioavailability, fenofibrate, nanoparticles, nanostructured lipid carriers     

The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells

Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO₂max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells.     

The effects of salbutamol on epithelial ion channels depend on the etiology of acute respiratory distress syndrome but not the route of administration

Introduction

We investigated the effects of intravenous and intratracheal administration of salbutamol on lung morphology and function, expression of ion channels, aquaporin, and markers of inflammation, apoptosis, and alveolar epithelial/endothelial cell damage in experimental pulmonary (p) and extrapulmonary (exp) mild acute respiratory distress syndrome (ARDS).

Methods

In this prospective randomized controlled experimental study, 56 male Wistar rats were randomly assigned to mild ARDS induced by either intratracheal (n = 28, ARDSp) or intraperitoneal (n = 28, ARDSexp) administration of E. coli lipopolysaccharide. Four animals with no lung injury served as controls (NI). After 24 hours, animals were anesthetized, mechanically ventilated in pressure-controlled mode with low tidal volume (6 mL/kg), and randomly assigned to receive salbutamol (SALB) or saline 0.9% (CTRL), intravenously (i.v., 10 μg/kg/h) or intratracheally (bolus, 25 μg). Salbutamol doses were targeted at an increase of ≈ 20% in heart rate. Hemodynamics, lung mechanics, and arterial blood gases were measured before and after (at 30 and 60 min) salbutamol administration. At the end of the experiment, lungs were extracted for analysis of lung histology and molecular biology analysis. Values are expressed as mean ± standard deviation, and fold changes relative to NI, CTRL vs. SALB.

Results

The gene expression of ion channels and aquaporin was increased in mild ARDSp, but not ARDSexp. In ARDSp, intravenous salbutamol resulted in higher gene expression of alveolar epithelial sodium channel (0.20 ± 0.07 vs. 0.68 ± 0.24, p < 0.001), aquaporin-1 (0.44 ± 0.09 vs. 0.96 ± 0.12, p < 0.001) aquaporin-3 (0.31 ± 0.12 vs. 0.93 ± 0.20, p < 0.001), and Na-K-ATPase-α (0.39 ± 0.08 vs. 0.92 ± 0.12, p < 0.001), whereas intratracheal salbutamol increased the gene expression of aquaporin-1 (0.46 ± 0.11 vs. 0.92 ± 0.06, p < 0.001) and Na-K-ATPase-α (0.32 ± 0.07 vs. 0.58 ± 0.15, p < 0.001). In ARDSexp, the gene expression of ion channels and aquaporin was not influenced by salbutamol. Morphological and functional variables and edema formation were not affected by salbutamol in any of the ARDS groups, regardless of the route of administration.

Conclusion

Salbutamol administration increased the expression of alveolar epithelial ion channels and aquaporin in mild ARDSp, but not ARDSexp, with no effects on lung morphology and function or edema formation. These results may contribute to explain the negative effects of β2-agonists on clinical outcome in ARDS.     

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β

Introduction

Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.

Methods

Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO₂) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).

Results

Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.

Conclusions

Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.     

Development of Acid-Resistant Alginate/Trimethyl Chitosan Nanoparticles Containing Cationic β-Cyclodextrin Polymers for Insulin Oral Delivery

In this study, the use of trimethylchitosan (TMC), by higher solubility in comparison with chitosan, in alginate/chitosan nanoparticles containing cationic β-cyclodextrin polymers (CPβCDs) has been studied, with the aim of increasing insulin uptake by nanoparticles. Firstly, TMCs were synthesized by iodomethane, and CPβCDs were synthesized within a one-step polycondensation reaction using choline chloride (CC) and epichlorohydrine (EP). Insulin–CβCDPs complex was prepared by mixing 1:1 portion of insulin and CPβCDs solutions. Then, nanoparticles prepared in a three-step procedure based on the iono-tropic pregelation method. Nanoparticles screened using experimental design and Placket Burman methodology to obtain minimum size and polydispercity index (pdI) and the highest entrapment efficiency (EE). CPβCDs and TMC solution concentration and pH and alginate and calcium chloride solution concentrations are found as the significant parameters on size, PdI, and EE. The nanoparticles with proper physicochemical properties were obtained; the size, PdI, and EE% of optimized nanoparticles were reported as 150.82 ± 21 nm, 0.362 ± 0.036, and 93.2% ± 4.1, respectively. The cumulative insulin release in intestinal condition achieved was 50.2% during 6 h. By SEM imaging, separate, spherical, and nonaggregated nanoparticles were found. In the cytotoxicity studies on Caco-2 cell culture, no significant cytotoxicity was observed in 5 h of incubation, but after 24 h of incubation, viability was decreased to 50% in 0.5 mμ of TMC concentration. Permeability studies across Caco-2 cells had been carried out, and permeability achieved in 240 min was 8.41 ± 0.39%, which shows noticeable increase in comparison with chitosan nanoparticles. Thus, according to the results, the optimized nanoparticles can be used as a new insulin oral delivery system.KEY WORDS: alginate, cationic β-cyclodextrin, insulin nanoparticle, oral delivery, trimethyl chitosan     

Design and Optimization of PLGA-Based Diclofenac Loaded Nanoparticles

Drug based nanoparticle (NP) formulations have gained considerable attention over the past decade for their use in various drug formulations. NPs have been shown to increase bioavailability, decrease side effects of highly toxic drugs, and prolong drug release. Nonsteroidal anti-inflammatory drugs such as diclofenac block cyclooxygenase expression and reduce prostaglandin synthesis, which can lead to several side effects such as gastrointestinal bleeding and renal insufficiency. The aim of this study was to formulate and characterize diclofenac entrapped poly(lactide-co-glycolide) (PLGA) based nanoparticles. Nanoparticles were formulated using an emulsion-diffusion-evaporation technique with varying concentrations of poly vinyl alcohol (PVA) (0.1, 0.25, 0.5, or 1%) or didodecyldimethylammonium bromide (DMAB) (0.1, 0.25, 0.5, 0.75, or 1%) stabilizers centrifuged at 8,800 rpm or 12,000 rpm. The resultant nanoparticles were evaluated based on particle size, zeta potential, and entrapment efficacy. DMAB formulated NPs showed the lowest particle size (108±2.1 nm) and highest zeta potential (−27.71±0.6 mV) at 0.1 and 0.25% respectively, after centrifugation at 12,000 rpm. Results of the PVA based NP formulation showed the smallest particle size (92.4±7.6 nm) and highest zeta potential (−11.14±0.5 mV) at 0.25% and 1% w/v, respectively, after centrifugation at 12,000 rpm. Drug entrapment reached 77.3±3.5% and 80.2±1.2% efficiency with DMAB and PVA formulations, respectively. The results of our study indicate the use of DMAB for increased nanoparticle stability during formulation. Our study supports the effective utilization of PLGA based nanoparticle formulation for diclofenac.     

Caprylate-Conjugated Cisplatin for the Development of Novel Liposomal Formulation

Cisplatin, first (platinum) compound to be evolved as an anticancer agent, has found its important place in cancer chemotherapy. However, the dose-dependent toxicities of cisplatin, namely nephrotoxicity, ototoxicity, peripheral neuropathy, and gastrointestinal toxicity hinder its widespread use. Liposomes can reduce the toxicity of cisplatin and provide a better therapeutic action, but the low lipid solubility of cisplatin hinders its high entrapment in such lipid carrier. In the present investigation, positively charged reactive aquated species of cisplatin were complexed with negatively charged caprylate ligands, resulting in enhanced interaction of cisplatin with lipid bilayer of liposomes and increase in its encapsulation in liposomal carrier. Prepared cisplatin liposomes were found to have a vesicular size of 107.9 ± 6.2 nm and zeta potential of −3.99 ± 3.45 mV. The optimized liposomal formulation had an encapsulation efficiency of 96.03 ± 1.24% with unprecedented drug loading (0.21 mg cisplatin / mg of lipids). The in vitro release studies exhibited a pH-dependent release of cisplatin from liposomes with highest release (67.55 ± 3.65%) at pH 5.5 indicating that a maximum release would occur inside cancer cells at endolysosomal pH. The prepared liposomes were found to be stable in the serum and showed a low hemolytic potential. In vitro cytotoxicity of cisplatin liposomes on A549 lung cancer cell line was comparable to that of cisplatin solution. The developed formulation also had a significantly higher median lethal dose (LD₅₀) of 23.79 mg/kg than that of the cisplatin solution (12 mg/kg). A promising liposomal formulation of cisplatin has been proposed that can overcome the disadvantages associated with conventional cisplatin therapy and provide a higher safety profile.Key Words: cisplatin, complexation, cytotoxicity, LD₅₀, liposome     

Healthy birth weight results in higher vitamin A storage in neonate piglets administered high-dose supplements

A proposed intervention for newborn infants in countries with suspected vitamin A (VA) deficiency is to administer 50,000 IU retinyl palmitate at birth to reduce mortality risk. However, no studies have investigated birth weight effects. In this study, low birth weight (LBW; <1 kg, n = 18) and healthy birth weight (HBW) piglets (>1.5 kg, n = 18) from VA-depleted sows were dosed with 25,000 or 50,000 IU retinyl palmitate (26.2 or 52.4 µmol retinol equivalents) at birth to compare VA reserves. Blood was collected at varying times (n = 3–5/time/dose), and piglets were killed at 12 or 24 h for blood, liver, kidneys, spleen, lungs, adrenal gland, and intestinal contents. HBW piglets had significantly higher birth, death, and organ weights than LBW (P < 0.0001 for all). HBW and LBW piglets, which received VA, had higher liver and kidney VA concentrations (0.18 ± 0.09, 0.24 ± 0.10 µmol/g liver and 13.4 ± 4.1, 14.2 ± 4.5 nmol/g kidney, respectively) than controls (n = 10) (0.051 ± 0.01 µmol/g liver and 1.01 ± 0.43 nmol/g kidney) (P = 0.0061 and < 0.0001, respectively). Total liver (9.75 ± 5.16 µmol) and kidney retinol (204 ± 79.1 nmol) were higher in HBW than LBW piglets (P < 0.0001). Extrahepatic tissues, except lung, had higher VA concentration than controls (P < 0.0001). Serum retinol and ester concentrations were higher in treated than control piglets (P = 0.0028, P < 0.0001, respectively), and significantly changed during the times sampled (P = 0.022, P = 0.011, respectively). Peak serum retinyl ester concentrations, which occurred at 3 h, were higher in piglets that received 50,000 IU (4.2 ± 4.4 µmol/L) than 25,000 IU (2.7 ± 2.3 µmol/L) (P = 0.031). Regardless of dose amount, HBW piglets stored more supplemental VA than LBW piglets when administered at birth.     

Severe and uncontrolled adult asthma is associated with vitamin D insufficiency and deficiency

Background

Vitamin D has effects on the innate and adaptive immune system. In asthmatic children low vitamin D levels are associated with poor asthma control, reduced lung function, increased medication intake, and exacerbations. Little is known about vitamin D in adult asthma patients or its association with asthma severity and control.

Methods

Clinical parameters of asthma control and 25-hydroxyvitamin D (25(OH)D) serum concentrations were evaluated in 280 adult asthma patients (mean ± SD: 45.0 ± 13.8 yrs., 40% male, FEV1 74.9 ± 23.4%, 55% severe, 51% uncontrolled).

Results

25(OH)D concentrations in adult asthmatics were low (25.6 ±11.8 ng/ml) and vitamin D insufficiency or deficiency (vitamin D <30 ng/ml) was common (67%). 25(OH)D levels were related to asthma severity (intermittent: 31.1 ± 13.0 ng/ml, mild: 27.3 ± 11.9 ng/ml, moderate: 26.5 ± 12.0 ng/ml, severe: 24.0 ± 11.8 ng/ml, p = 0.046) and control (controlled: 29.5 ± 12.5 ng/ml, partly controlled 25.9 ± 10.8 ng/ml, uncontrolled: 24.2 ± 11.8 ng/ml, p = 0.030). The frequency of vitamin D insufficiency or deficiency was significantly higher in patients with severe or uncontrolled asthma and was associated with a lower FEV1 (vitamin D <30 vs. ≥30 ng/ml 2.3 ± 0.9 L vs. 2.7 ± 1.0 L, p = 0.006), higher levels of exhaled NO (45 ± 46 ppb vs. 31 ± 37 ppb, p = 0.023), a higher BMI (28.3 ± 6.2 vs. 25.1 ± 3.9, p < 0.001), and sputum eosinophilia (5.1 ± 11.8% vs. 0.5 ± 1.0%, p = 0.005). The use of oral corticosteroids or sputum eosinophilia was associated with a 20% or 40% higher risk of vitamin D insufficiency or deficiency.

Conclusions

25(OH)D levels below 30 ng/ml are common in adult asthma and most pronounced in patients with severe and/or uncontrolled asthma, supporting the hypothesis that improving suboptimal vitamin D status might be effective in prevention and treatment of asthma.     

Left ventricular assist device for end-stage heart failure: results of the first LVAD destination program in the Netherlands

PurposeMechanical circulatory support with a continuous-flow left ventricular assist device (LVAD) may be a valuable treatment in end-stage heart failure patients for an extended period of time. The purpose of this study was to evaluate the safety and efficacy of implantation of a continuous-flow LVAD in end-stage heart failure patients within the first destination program in the Netherlands.MethodsA third-generation LVAD was implanted in 16 heart failure patients (age 61 ± 8; 81 % male; left ventricular ejection fraction 20 ± 6 %) as destination therapy. All patients were ineligible for heart transplant. At baseline, 3 and 6 months, New York Heart Association (NYHA) functional class, quality-of-life and exercise capacity were assessed. Clinical adverse events were registered.ResultsSurvival at 30 days and 6 months was 88 and 75 %, respectively. In the postoperative phase, 6 (38 %) patients required continuous veno-venous haemofiltration for renal failure and 2 (13 %) patients required extracorporeal membrane oxygenation because of severe right ventricular failure. During follow-up, NYHA functional class and quality-of-life improved from 3.7 ± 0.1 to 2.3 ± 0.1 and 57 ± 5 to 23 ± 3 at 6 months (P < 0.001), respectively. The 6 min walking distance improved from 168 ± 42 m to 291 ± 29 m at 6 months (P = 0.001).ConclusionContinuous-flow LVAD therapy is a promising treatment for patients with end-stage heart failure ineligible for heart transplant.     

Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes

Introduction

Our objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli.

Methods

Cartilage explants from FrzB^−/− and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1β (IL-1β) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB^−/− and wild-type mice. The expression and release of MMP-3 and −13 were determined by RT-PCR, western blot and ELISA. The accumulation of β-catenin was assessed by RT-PCR and western blot.

Results

Cartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB^−/− cartilage explants compared to wild-type (P = 0.17). IL-1β dose-dependently induced Mmp-13 and −3 gene expression and protein release by cultured chondrocytes. IL-1β-mediated increase in MMP-13 and −3 was slightly enhanced in FrzB^−/− chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and β-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB^−/− chondrocytes to IL-1β and load may be associated with an over-stimulation of the canonical Wnt/β-catenin pathway.

Conclusions

Our results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/β-catenin pathway.