Acute and subacute toxicity studies on triptolide and triptolide-loaded polymeric micelles following intravenous administration in rodents

Except its anti-tumour effects, triptolide (TP) also shows multiple pharmacological side activities, such as immune-suppressive and male anti-fertility. To increase the therapeutic index of TP, a novel polymeric micelle system containing TP (TP-PM) has been developed to treat tumour. Our previous studies have demonstrated the good anti-tumour efficacy of TP-PM. This paper investigated the acute toxicity in mice and subacute toxicity in rats of TP-PM and TP. Results demonstrated that the LD₅₀ for TP-PM and TP administered intravenously were 1.06 mg/kg and 0.83 mg/kg in mice, respectively. In subacute toxicity study, TP-PM and TP were administered intravenously at the dose levels of 0.1 mg/kg and 0.3 mg/kg for 14 d. Compared to the control, there was significant decrease in the serum AST activities, the testis ACP activities, thymus index, testis index, and significant increase in spleen index, and obvious histopathological changes in rats treated with TP, however, the toxicities of TP-PM on liver, kidney, testis and spleen are slighter than TP. Compared to TP, TP-PM significantly increased the ACP activity of the testis and decreased the MDA level in serum. So, the polymeric micelles may be a novel drug delivery carrier of TP for reducing the toxicities of TP.     

Cytotoxicity of Triptolide and Triptolide loaded polymeric micelles in vitro

Triptolide (TP), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F is characterized by strong anti-tumor effects on various cancer cells. Except its anti-tumor effects, TP also shows multiple pharmacological side activities, such as anti-inflammatory, immune-suppressive and male anti-fertility. In order to reduce these side effects, especially the immuno-suppressive activity when used to cure cancer, a novel polymeric micelle system containing TP (TP-PM) was constructed. The immune-modulation effects of TP-PM have been evaluated by previous studies. In this study, we compared the cytotoxicity of TP and TP-PM on Jurkat and HT29 cells. Therefore, we determined the cell viability, membrane integrity, cell proliferation, apoptosis, and caspase 3/7 activity after exposure to TP and TP-PM. The results demonstrated that actually low concentrated TP and TP-PM could induce an inhibition of cell growth and proliferation as well as membrane damage in both tumor cell lines. TP and TP-PM induced apoptosis and caused activation of caspase 3/7 even at low concentrations. Both formulations destroyed the membrane of Jurkat cells, nevertheless, TP-PM showed stronger pernicious effects. In general, TP-PM induced in both tested cell lines stronger effects than TP. Therefore, polymeric micelles can be considered as promising carriers for TP in cancer therapy.     

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(d,l-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents—stearylamine and polyethylenimine—in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague–Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (−6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28–42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels—interferon γ and interleukin-2—were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(d,l-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.     

Localization and quantification of biodegradable particles in an intestinal cell model: The influence of particle size

The physicochemical parameters influencing particle crossing through the epithelial barrier have been largely studied and size has been shown as a crucial factor. Most part of this work has been done with polystyrene (PS) particles; however, few studies were conducted with well characterized biodegradable polymeric nanoparticles, more suitable for drug delivery. The aim of the present work was to study the influence of the size of well characterized biodegradable polymeric particles on cellular uptake by Caco-2 cells. Poly (d,l-lactide-co-glycolide acid) (PLGA) particles loaded with a fluorescent dye, 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiO), were prepared by the emulsion evaporation process. Five batches of particles with narrow size distribution (100, 300, 600, 1000, and 2000 nm) were produced using selective centrifugation. Surface properties (zeta potential, hydrophobicity and residual surfactant rate) were similar among all batches. Preliminary study showed that after growth and differentiation, cell counting differed from plate to plate. Therefore, a quantitative method using fluorescence spectroscopy was developed to estimate the number of particles interacting with a single cell. The results were compared to the interaction obtained with polystyrene particles, commonly used as particle model. The interaction was clearly dependant on particle size and concentration. Particles in the range of 100 nm presented a higher interaction when compared to larger particles. More than 6000 (d,l-lactide-co-glycolide acid) particles and 200,000 polystyrene particles were quantified per cell. Cellular localisation of particles by confocal microscopy showed the association of the poly (d,l-lactide-co-glycolide acid) particles with the cells. Small particles were observed intracellularly, whereas particles larger than 300 nm were associated with the apical membranes. Interestingly, some of the 100 nm PLGA particles were localized in the nuclei of the cells.     

Preventive effect of L-carnosine on changes in the thermal nociceptive threshold in streptozotocin-induced diabetic mice

Sensory abnormality is one of the serious complications in diabetes. Since the effective therapeutic regimen to ameliorate the diabetic sensory abnormality is very few, the present study was then designed to investigate the effect of zinc l-carnosine on the changes of nociceptive threshold in diabetic mice. Zinc l-carnosine (75–300 mg/kg, p.o.) was administered once daily from 1 day after streptozotocin treatment. Diabetic mice showed shorter tail-flick latency at 1–4 weeks after streptozotocin treatment and longer tail-flick latency at 6–9 weeks after its treatment. The shortened tail-flick latency in early stage of diabetic mice was ameliorated by treatment with zinc l-carnosine. Moreover, zinc l-carnosine also slowed the onset of hypoalgesia in diabetic mice. Tail-flick latency in non-diabetic mice was not affected by the zinc l-carnosine treatment, indicating that zinc l-carnosine did not affect normal nociceptive transmission. Moreover, l-carnosine, but not zinc sulfate, ameliorated the abnormal sensory perception in diabetic mice. Interestingly, the ameliorative effect of zinc l-carnosine on the abnormal sensory perception in diabetic mice is much stronger than that of l-carnosine. These results provide the evidence of the ameliorative potential of zinc l-carnosine on the progressive diabetic neuropathy. Moreover, l-carnosine combined with zinc shows more potent amelioration of abnormal sensory perception in diabetic mice than by itself.     

Arginine homeostasis in allergic asthma

Allergic asthma is a chronic disease characterized by early and late asthmatic reactions, airway hyperresponsiveness, airway inflammation and airway remodelling. Changes in l-arginine homeostasis may contribute to all these features of asthma by decreased nitric oxide (NO) production and increased formation of peroxynitrite, polyamines and l-proline. Intracellular l-arginine levels are regulated by at least three distinct mechanisms: (i) cellular uptake by cationic amino acid (CAT) transporters, (ii) metabolism by NO-synthase (NOS) and arginase, and (iii) recycling from l-citrulline. Ex vivo studies using animal models of allergic asthma have indicated that attenuated l-arginine bioavailability to NOS causes deficiency of bronchodilating NO and increased production of procontractile peroxynitrite, which importantly contribute to allergen-induced airway hyperresponsiveness after the early and late asthmatic reaction, respectively. Decreased cellular uptake of l-arginine, due to (eosinophil-derived) polycations inhibiting CATs, as well as increased consumption by increased arginase activity are major causes of substrate limitation to NOS. Increasing substrate availability to NOS by administration of l-arginine, l-citrulline, the polycation scavenger heparin, or an arginase inhibitor alleviates allergen-induced airway hyperresponsiveness by restoring the production of bronchodilating NO. In addition, reduced l-arginine levels may contribute to the airway inflammation associated with the development of airway hyperresponsiveness, which similarly may involve decreased NO synthesis and increased peroxynitrite formation. Increased arginase activity could also contribute to airway remodelling and persistent airway hyperresponsiveness in chronic asthma via increased synthesis of l-ornithine, the precursor of polyamines and l-proline. Drugs that increase the bioavailability of l-arginine in the airways – particularly arginase inhibitors – may have therapeutic potential in allergic asthma.     

A 13-week subchronic oral toxicity study of l-serine in rats

A subchronic oral toxicity study was conducted to evaluate the safety of l-serine in Sprague–Dawley rats. The test article was administered once daily by gavage in male and female rats at dose levels of 0, 500, 1500, and 3000 mg/kg body weight/day for 13 weeks. Daily clinical signs, body weight, and food consumption were not affected by ingestion of the test article. There were no treatment-related adverse effects on urinalysis, hematology, serum biochemistry, organ weights, gross and histopathological examination. It was concluded that the no-observed-effect level (NOEL) for l-serine was 3000 mg/kg bw/day for both genders.     

Simultaneous determination of nine major active compounds in Dracocephalum rupestre by HPLC

A new method was developed for the simultaneous determination of nine major constituents in Dracocephalum rupestre, including 5,7-dihydroxychromone (1), eriodictyol-7-O-β-d-glucoside (2), luteolin-7-O-β-d-glucoside (3), naringenin-7-O-β-d-glucoside (4), apigenin-7-O-β-d-glucoside (5), eriodictyol (6), luteolin (7), naringenin (8) and apigenin (9). The quantitative determination was conducted by reversed phase high-performance liquid chromatography with photodiode array detector (LC–PDA). Separation was performed on an Agilent Eclipse XDB-C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.5% aqueous acetic acid. The components were identified by retention time, ultraviolet (UV) spectra and quantified by LC–PDA at 260 nm. All calibration curves showed good linearity (r² > 0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.0%. The recoveries were between 95.15 and 104.45%. The limits of detection (LOD) ranged from 0.002 to 0.422 μg/ml and limits of quantification (LOQ) ranged from 0.005 to 1.208 μg/ml, respectively. The identity of the peaks was further confirmed by high-performance liquid chromatography with triple-quadrupole mass spectrometry system coupled with electrospray ionization (ESI) interface. The developed method was applied to the determination of nine constituents in 14 samples of D. rupestre collected at various harvesting times. Most compounds accumulated at much higher amounts in about June–July. The satisfactory results indicated that the developed method was readily utilized as a quality control method for D. rupestre.     

Uptake and efflux of methylmercury in vitro: Comparison of transport mechanisms in C6, B35 and RBE4 cells

Methylmercury (MeHg) is a neurotoxicant which enters the brain and may cause permanent change. Thus, the properties of MeHg transport across cell membranes are a key factor in designing an appropriate model for MeHg neurotoxicity. This study uses cell cultures to examine the uptake and efflux mechanisms of methylmercury in C6 glioma, B35 neuroblastoma and rat brain endothelial (RBE4) cells. The cellular uptake and efflux of MeHg was investigated using ¹⁴C-labeled MeHg. The uptake of MeHg-chloride was temperature-independent while the uptake of MeHg-l-cysteine was temperature-dependent in all the three cell types. This indicates that uptake of MeHg-chloride is due to passive diffusion and uptake of MeHg-l-cysteine is due to a protein carrier. Substrates of the amino acid transport system L inhibited uptake of MeHg-l-cysteine in C6 and RBE4 cells, but not B35 cells, indicating a role for system L in MeHg-uptake in the former two. Probenecid, Na⁺-free medium, MeHg and several l-amino acids did not alter the efflux of MeHg from C6 and RBE4 cells. The amino acids l-cysteine and cystine however, increased the efflux. Both cysteine and cystine are important in the generation of glutathione (GSH), suggesting the involvement of GSH in MeHg efflux. HgCl₂ at low concentrations (0.5 and 1.0 μM) decreased the MeHg efflux and at high concentrations (5.0 and 10.0 μM) increased the efflux. This inhibiting effect of HgCl₂ at low concentrations is possibly due to binding to GSH while the effect of high HgCl₂ concentrations is attributed to disrupted membrane integrity, as measured by Trypan blue. This study demonstrates differing transport mechanisms of MeHg in the cell lines C6, B35 and RBE4.     

Stabilized polymeric micelles by electrostatic interactions for drug delivery system

Methoxy poly(ethylene glycol)-block-oligo(l-aspartic acid)-block-poly(-caprolactone) with four aspartic acid groups was synthesized with a molecular weight and M_w/M_n of 8930 and 1.22. Polymeric micelles were formed by dialysis and stabilized by electrostatic interactions between the carboxylic acid groups and calcium cations. The critical micelle concentration of mPEG–Asp–PCL was determined to be 0.078 mg/mL. At 0.02 mg/mL, the dissociation of micelles without ionic stabilization formed an opaque, phase-separated solution, while the stabilized micelles under the same conditions showed structural stability through ionic stabilization. The paclitaxel-loading and efficiency were 8.7% and 47.6%, respectively, and the drug loading increased the mean diameter from 73.0 nm to 87 nm, which was increased further to 96 nm after ionic fixation. Rapid releases of approximately 65% of the encapsulated paclitaxel from a non-stabilized micelle and 45% from a stabilized micelle were observed in the first 24 h at pH 7.4 in a PBS solution containing 0.1 wt% Tween 80. The stabilized micelles then showed a sustained, slow release pattern over a couple of weeks, while the profile from the non-stabilized micelles reached a plateau at approximately 75% after 50 h. The enhanced micelle stability independent of concentration through ionic stabilization opens a way for preparing long circulating delivery systems encapsulating water-insoluble drugs.     

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

This study investigates the participation of the endothelial factors in the α-adrenoceptor contractile responses in mesenteric resistance arteries from 15 days ouabain-treated (25 µg/kg/day) and untreated rats. Ouabain treatment increased blood pressure and heart rate without changing the contractile response to phenylephrine (3 nM–30 µM). Endothelium removal or N^G-nitro-l-arginine methyl ester (l-NAME, 100 µM), increased the responses to phenylephrine. The endothelial modulation was similar in both rat groups, but the l-NAME effects were bigger in arteries from ouabain-treated rats. However, the endothelial NOS expression and the relaxation to acetylcholine (0.1 nM–10 µM) remained unaltered after ouabain treatment. The coincubation with l-NAME and indomethacin (100 µM) leftward shifted the concentration–response curves to phenylephrine in arteries from untreated rats similarly to the displacement after incubation only with l-NAME. However, in mesenteric arteries from treated rats, the co-incubation with indomethacin and l-NAME did not alter the response to phenylephrine. The addition of the inhibitor of calcium activated potassium channels tetraethylammonium (2 mM) further leftward shifted the phenylephrine curves only in arteries from untreated rats. Cyclooxygenase-2 (COX-2) expression was greater in vessels from ouabain-treated rats. In conclusion, the chronic ouabain treatment for 15 days modified the participation of endothelial factors in response to phenylephrine in mesenteric resistance arteries, by increasing the release of NO and prostanoids and impairment the endothelium-derived hyperpolarizing factor (EDHF) release. This was accompanied by an increased COX-2 expression. Although this balance avoids changes in the phenylephrine concentration–response curves, these vascular changes might contribute to maintain the ouabain-induced hypertension.     

Ionically Fixed Polymeric Nanoparticles as a Novel Drug Carrier

Purpose

In this study, we have prepared a novel polymeric drug delivery system comprised of ionically fixed polymeric nanoparticles (IFPN) and investigated their potential as a drug carrier for the passive targeting of water-insoluble anticancer drugs.

Materials and Methods

For this purpose, the physicochemical characteristics of the IFPN were investigated by comparing them with conventional polymeric micelles. IFPN containing paclitaxel were prepared and evaluated for in vitro stability and in vivo pharmacokinetics.

Results

The IFPN were successfully fabricated using a monomethoxypolyethylene glycol-polylactide (mPEG-PLA) diblock copolymer and a sodium salt of d,l-poly(lactic acid) (d,l-PLACOONa) upon the addition of CaCl₂. The transmittance of the IFPN solution was much lower than that of a polymeric micelle solution at the same polymer concentration implicating an increase in the number of appreciable particles. The particle size of the IFPN was approximately 20～30 nm which is in the range of particle sizes that facilitate sterile filtration using a membrane filter. The IFPN also have a regular spherical shape with a narrow size distribution. The zeta potential of the IFPN was almost neutral, similar to that of the polymeric micelles. In contrast, mixed micelles with a combination of mPEG-PLA and d,l-PLACOONa prior to the addition of Ca²⁺ showed a negative charge (?17 mV), possibly due to the carboxyl anion of polylactic acid exposed on the surface of the micelles. The IFPN formulation was highly kinetically stable in aqueous medium compared to the polymeric micelle formulation. The molecular weight of d,l-PLACOONa in the IFPN and the mPEG-PLA/d,l-PLACOONa molar ratio had a great influence upon the kinetic stability of the IFPN. Pharmacokinetic studies showed that the area under the concentration vs time curve (AUC) of IFPN in blood was statistically higher (about two times) when compared with that of Cremophor EL-based formulation (Taxol^® equivalent) or polymeric micelle formulation.

Conclusions

The results suggests that the IFPN were retained in the circulation long enough to play a significant role as a drug carrier in the bloodstream, possibly resulting in improved therapeutic efficiency. Therefore, the IFPN are expected to be a promising novel polymeric nanoparticulate system for passive tumor targeting of water-insoluble anticancer drugs including paclitaxel.     

Development and validation of a HPLC–ES-MS/MS method for the determination of glucosamine in human synovial fluid

A new HPLC method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in human synovial fluid was developed and validated. Synovial fluid samples were analyzed after a simple protein precipitation step with trichloroacetic acid using a polymer-based amino column with a mobile phase composed of 10 mM ammonium acetate (pH 7.5)–acetonitrile (20:80, v/v) at 0.3 mL/min flow rate. d-[1-¹³C]glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition (m/z 180 → 72 and 181 → 73 for glucosamine and internal standard, respectively). The limit of quantification (injected volume = 3 μL) was 0.02 ng, corresponding to 10 ng/mL in synovial fluid. Calibration curves obtained using matrix-matched calibration standards and internal standard at 600 ng/mL were linear up to 2000 ng/mL. Precision values (%R.S.D.) were ≤14% in the entire analytical range. Accuracy (%bias) ranged from −11% to 10%. The recoveries measured at three concentration levels (50, 800, and 1500 ng/mL) were higher than 89%. The method was successfully applied to measure endogenous glucosamine levels in synovial fluid samples collected from patients with knee osteoarthritis and glucosamine levels after oral administration of glucosamine sulfate (DONA^®) at the dose of 1500 mg/day for 14 consecutive days (steady-state).     

Enantiomer separation of α-hydroxy acids in high-performance immunoaffinity chromatography

In this study, a monoclonal anti-d-hydroxy acid antibody was immobilized onto a synthetic high-flow-through chromatographic support material to produce a chiral stationary phase suitable for enantiomer separation of free α-hydroxy acids. Chiral separation of several aliphatic and aromatic members of this class of compounds was achieved in HPLC under mild isocratic buffer conditions using phosphate buffered saline, pH 7.4, as mobile phase. Due to the high degree of stereoselectivity exhibited by the immobilized antibody, in all cases the l-enantiomer eluted with the void volume, while the d-enantiomer was retained and eluted second. The effect of the mobile phase parameters flow rate, temperature, pH, and ionic strength on the enantiomer separation of the model analyte mandelic acid was investigated. While it was found that variations in the flow rate did not change the retention factor k₂, dramatic effects on the interaction between the immobilized antibody and d-mandelic acid were observed when any of the other mobile phase parameters were modulated.     

A highly branched 1,3-β-d-glucan extracted from Aureobasidium pullulans induces cytokine production in DBA/2 mouse-derived splenocytes

We recently elucidated the structure of a highly branched 1,3-β-d-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of β-d-glucans are known to depend on their structures, the effects of a highly branched 1,3-β-d-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-γ and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-α in DBA/2 mouse-derived splenocytes in vitro.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Treatment of Venezuelan equine encephalitis virus infection with (-)-carbodine

Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(−) enantiomer of carbodine [(−)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC₅₀) of 0.2 μg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (−)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kg d) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (−)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (−)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (−)-carbodine is effective when initiated after the establishment of brain infection.     

pH-sensitive double-hydrophilic block copolymer micelles for biological applications

In the recent years, double-hydrophilic block copolymer (DHBC) micelles have appeared as potential vectors for pharmaceutical applications due to their simple preparation method in aqueous solvent. The present study aims at underscoring the strategy for the choice of the partners in the formulation of DHBC micelles presenting a good stability in physiological conditions (pH 7.4, 0.15 mol/L NaCl) and a pH-sensitivity allowing their disassembly at pH 5. Using light scattering and Laser-Doppler electrophoresis, micelles of polymethacrylic acid-b-polyethylene oxide complexing either poly-l-lysine (PLL) or an oligochitosan were characterised. Whatever the polyamine counter-polyion considered, the micelles were perfectly formed for an amine/methacrylic acid molar charge ratio of one. They were characterised by a hydrodynamic diameter of 28 nm for PLL and 60 nm for oligochitosan and by a neutral zeta potential. The stability study as a function of the pH and of the ionic strength revealed different behaviours. Oligochitosan micelles were stable until pH 7 and unstable at 0.15 mol/L NaCl. On the contrary, PLL micelles were stable in physiological conditions and disassembled at pH 5. As a conclusion, the choice of the partners to formulate double-hydrophilic block copolymer based-micelles is strategic in order to obtain well-adapted vectors applied to the pharmaceutical field.     

Specific and non-specific phagocytosis of ligand-grafted PLGA microspheres by macrophages

We evaluated the influence of ligand grafting on the rate and intensity of uptake of poly(d,l-lactide-co-glycolide) microparticles by alveolar macrophages. Microspheres with a mean diameter of 2.5 μm were obtained by spray drying. Three ligands (WGA, an RGD containing peptide and mannose-PEG₃-NH₂) and a cationic molecule (PLL) were covalently grafted on the particle surface using the carbodiimide method. Their grafting efficiency was quantified, and WGA grafting was characterized by confocal laser scanning microscopy (CLSM) and by atomic force microscopy (AFM). The uptake by macrophages of surface-modified microspheres was quantified by CLSM. This work showed that the uptake of negatively charged ligand-grafted microspheres (−26 to −51 mV) was increased up to two to four times according to the ligand compared to ungrafted microspheres (−81 mV) and displayed saturation as opposed to the cationic PLL-grafted microspheres. Moreover, a specific receptor-mediated phagocytosis mechanism was suggested based on free ligand, cytochalasin D and +4 °C incubation that decreased the microparticle uptake. Furthermore, this work clearly showed that the relative contribution of specific and non-specific processes to the overall uptake varied greatly according to the ligands, and was dependent on the particle-to-cell ratio. In conclusion, this work showed that ligand grafting can enhance the uptake of microparticles, with a variable relative contribution of specific and non-specific uptake mechanism.     

Prolonged analgesic effect of PLGA-encapsulated bee venom on formalin-induced pain in rats

To enhance the medicinal activity of bee venom (BV) acupuncture, bee venom was loaded into biodegradable poly(d,l-lactide-co-glycolide) nanoparticles (BV-PLGA-NPs) by a water-in-oil-in-water-emulsion/solvent-evaporation technique. Rat formalin tests were performed after subcutaneous injection of BV-PLGA-NPs to the Zusanli acupuncture point (ST36) at 0.5, 1, 2, 6, 12, 24, and 48 h before plantar injection of 2% formalin. BV-PLGA-NPs treatment showed comparable analgesic activity to typical BV acupuncture during the late phase, compared with saline-treated controls, and the analgesic effect lasted for 12 h. PLGA-encapsulation was also effective in alleviating the edema induced by allergens in bee venom. These results indicate that PLGA-encapsulation provided a more prolonged effect of BV acupuncture treatment, while maintaining a comparable therapeutic effect.