Surface functionalization of doxorubicin-loaded liposomes with octa-arginine for enhanced anticancer activity

Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL® or Lipodox®) were surface functionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide was conjugated to the polyethylene glycol–dioleoyl phosphatidylethanolamine (PEG–DOPE) amphiphilic co-polymer. The resultant R8–PEG–PE conjugate was introduced into the lipid bilayer of liposomes at 2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modification did not alter the particle size distribution, as measured by Particle Size Analyzer and transmission electron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of the modified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular and intratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laser scanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes (Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes at higher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomes at tested time point of 1 h. The significantly higher accumulation of loaded drug to its site of action for R8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 ± 7% for R8-Dox-L compared to 90.6 ± 2% for Dox-L at Dox dose of 50 μg/mL for 4 h followed by 24 h incubation) and enhanced suppression of tumor growth (348 ± 53 mm³ for R8-Dox-L, compared to 504 ± 54 mm³ for Dox-L treatment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeutic window of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.     

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W₁/O/W₂ solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA–PEG–P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA–PEG–P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA–PEG–P(HB-HO) NPs (IC₅₀ = 0.87 μM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA–PEG–P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91 ± 17.43 mm³, significantly smaller than normal saline control group (542.58 ± 45.19 mm³). All these results have illustrated that our techniques for the preparing of DOX/FA–PEG–P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.     

Effects of liposomes with polyisoprenoids,potential drug carriers,on the cardiovascular and excretory system in rats

BackgroundThe unpredictable side effects of a majority currently used drugs are the substantial issue, in which patients and physicians are forced to deal with. Augmenting the therapeutic efficacy of drugs may prove more fruitful than searching for the new ones. Since recent studies show that new cationic derivatives of polyisoprenoid alcohols (APrens) might exhibit augmenting properties, we intend to use them as a component of liposomal drug carriers. In this study we investigate if these compounds do not per se cause untoward effects on the living organism.MethodsMale Sprague–Dawley rats received for four weeks daily injections (0.5 ml sc) of liposomes built of dioleoyl phosphatidylethanolamine (DOPE), liposomes built of DOPE and APren-7 (ratio 10:1) or water solvent. Weekly, rats were observed in metabolic cages (24 h); blood and urine were sampled for analysis; body weight (BW) and systolic blood pressure (SBP) were determined. After chronic experiment, kidneys and heart were harvested for histological and morphometric analysis.ResultsThe 4-week BW increments were in the range of 97 ± 4 to 102 ± 4%, intergroup differences were not significant. Microalbuminuria was the lowest in the group receiving liposomes with APren-7 (0.22 ± 0.03 mg/day). Water and food intake, plasma and urine parameters were similar in all groups.ConclusionsNewly designed liposomes containing APren-7 did not affect functions of the excretory and cardiovascular systems, and renal morphology; therefore we find them suitable as a component of liposomal drug carriers.     

Principles of rational design of thermally targeted liposomes for local drug delivery

Drug release from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes occurs close to the main transition temperature T_m = 41 °C. The exact release temperature can be adjusted by additional lipids, which shift T_m. A major issue is drug leakage at 37 °C. We here describe a novel approach with improved drug retention yet rapid release. To obtain spherical, smooth liposomes we included: i) 2 mol% cholesterol, to soften bilayers (Lemmich et al 1997), ii) lipids, which due to their spontaneous curvature stabilize the negative and positive curvatures of the inner and outer leaflets of unilamellar liposomes. In addition to differential scanning calorimetry (DSC) and fluorescence spectroscopy, the lipid mixtures were analyzed by a Langmuir balance for their elastic properties and lipid packing, aiming at high elasticity modulus C_S^− 1. Maxima in C_S^− 1 coincided with minima in the free energy of lateral mixing. These liposomes have reduced drug leakage, yet retain rapid release.From the Clinical EditorThis paper reports the development of optimized DPPC liposomes for drug delivery, with reduced drug leakage but maintained rapid release.     

Polyamine Aza-Cyclic Compounds Demonstrate Anti-Proliferative Activity In Vitro But Fail to Control Tumour Growth In Vivo

Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079–8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity. To explore this polyamine pharmacophore we cross-linked the aza-cyclic compound, hexacyclen, with 1,4-dibromobutane or 1,8-dibromooctane to yield the polyamines [poly(butylhexacyclen)—CL4] or [poly(octylhexacyclen)—CL8] respectively, both free of primary amines. We characterised the compounds and their respective nanoparticles and examined their interaction with the putative targets of the cationic polyamines: the cell membrane and DNA. Like DAB 16, CL4 and CL8 bind plasmid DNA and all three compounds interrupted the cell cycle of A431 epidermoid carcinoma cells in the S-phase. Additionally all three compounds disrupted erythrocyte membranes, with CL8 and DAB 16 being more active, in this respect, than CL4. CL4 (IC₅₀ = 775.1 μg mL^? 1) and CL8 (IC₅₀ = 8.4 μg mL^? 1), in a similar manner to DAB 16, were anti-proliferative against A431 cells; although unlike DAB 16, CL4 and CL8 were not tumouricidal against A431 xenografts in mice, indicating that primary amines may play an important role in the in vivo activity of DAB 16.     

In Vitro Characterization of a Liposomal Formulation of Celecoxib Containing 1,2-Distearoyl-sn-Glycero-3-Phosphocholine,Cholesterol, and Polyethylene Glycol and its Functional Effects Against Colorectal Cancer Cell Lines

Nanosized liposomal drug delivery systems are well suited for selective drug delivery at tumor sites. Celecoxib (CLX) is a highly hydrophobic cyclooxygenase-2 inhibitor that can reduce the incidence of colorectal polyps; however, the adverse cardiovascular effects limit its applicability. Here, we report a liposomal formulation of CLX using 1,2-Distearoyl-sn-glycero-3-phosphocholine, cholesterol, and polyethylene glycol. Encapsulation efficiency of the drug was greater than 70%; the release was slow and sustained with only 12%–20% of CLX released in the first 12 h. Flow cytometry and confocal microscopy studies using the colon cancer cell lines HCT-116 and SW620 showed significantly higher cellular association and internalization of the liposomes after incubation for 6 h when compared with 30 min. The liposomes did not colocalize with transferrin, but had a punctuate appearance, indicating vesicular localization. Cell proliferation was inhibited by 95% and 78%, respectively, in SW620 and HT29 cells after incubation with 600 μM liposomal CLX for 72 h. Moreover, cellular motility, as shown by a scratch wound healing assay, was also significantly (p = 0.006) inhibited when SW620 cells were incubated with 400 μM liposomal CLX. This is the first report of the successful encapsulation of CLX in a long-circulating liposomal formulation that could be effective against colorectal cancer.     

PEGylated chitosan-based polymer micelle as an intracellular delivery carrier for anti-tumor targeting therapy

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles presented a potential candidate for intracellular drug delivery carrier due to its special spatial structure. In this article, CSO-SA was further modified by polyethylene glycol (PEG). The physicochemical properties of PEGylated CSO-SA (PEG-CSO-SA) micelles were characterized. After PEGylation, the critical micelle concentration (CMC) of PEG-CSO-SA had no significant change; the micelle size increased; and the zeta potential decreased. The cellular uptake of CSO-SA micelles before and after PEGylation in macrophage RAW264.7, immortalized rat liver cells BRL-3A and human liver tumor cells HepG2 was studied. About 58.4 ± 0.63% of CSO-SA micelles were uptaked by RAW264.7 in 24 h, however, only 17.7 ± 0.94% of PEG-CSO-SA micelles were internalized into RAW264.7 after the CSO-SA was modified with PEG in five molar times. Meanwhile, there were no changes in the uptake after PEGylation of CSO-SA in BRL-3A and HepG2. Using mitomycin C as a model drug, the in vitro anti-tumor activities of the drug loaded in the micelles were investigated. The 50% cellular growth inhibition (IC₅₀) of the drug decreased from 1.97 ± 0.2 to 0.13 ± 0.02 μg/mL after mitomycin C was loaded into CSO-SA micelles, and the IC₅₀ value of the drug had no obvious change when the CSO-SA was modified by PEG.     

Development of PVP/PEG mixtures as appropriate carriers for the preparation of drug solid dispersions by melt mixing technique and optimization of dissolution using artificial neural networks

The effect of plasticizer’s (PEG) molecular weight (MW) on PVP based solid dispersions (SDs), prepared by melt mixing, was evaluated in the present study using Tibolone as a poorly water soluble model drug. PEGs with MW of 400, 600, and 2000 g/mol were tested, and the effect of drug content, time and temperature of melt mixing on the physical state of Tibolone, and the dissolution characteristics from SDs was investigated. PVP blends with PEG400 and PEG600 were completely miscible, while blends were heterogeneous. Furthermore, a single T_g recorded in all samples, indicating that Tibolone was dispersed in a molecular lever (or in the form of nanodispersions), varied with varying PEG’s molecular weight, melt mixing temperature, and drug content, while FTIR analysis indicated significant interactions between Tibolone and PVP/PEG matrices. All prepared solid dispersion showed long-term physical stability (18 months in room temperature). The extent of interaction between mixture components was verified using Fox and Gordon–Taylor equations. Artificial neural networks, used to correlate the studied factors with selected dissolution characteristics, showed good prediction ability.     

Neuroprotective effects of neolignans isolated from Magnoliae Cortex against glutamate-induced apoptotic stimuli in HT22 cells

In the course of screening for neuroprotective natural products, Magnoliae Cortex showed potent inhibition of hippocampal neuronal HT22 cell death. Obovatol, honokiol, and magnolol were isolated from the ethanolic extract of Magnoliae Cortex. Isolated compounds obovatol, honokiol, and magnolol were protective against 5 mM glutamate-induced cell death. When cells were stressed using glutamate, cell viability decreased to 16.98 ± 4.58% over the control (100.00 ± 10.15%). In contrast, 10 μM obovatol, 10 μM honokiol, and 50 μM magnolol increased cell viability to 91.80 ± 1.70%, 93.59 ± 1.93%, and 85.36 ± 7.40%, respectively. The neuroprotective effects of obovatol and honokiol were attributable to the inhibition of intracellular reactive oxygen species production, followed by protection of the mitochondrial membrane potential (ΔΨ_m), recovery of Bcl-2 and Bid levels, inhibition of apoptosis-inducing factor expression, and phosphorylation of mitogen-activated protein kinases such as p38 kinases, extracellular signal-regulated kinases, and c-Jun N-terminal kinases. On the contrary, magnolol did not show any significant effect on the ΔΨ_m and apoptotic factors. Among three compounds, obovatol most strongly scavenged 2,2-diphenyl-1-picrylhydrazyl radicals and inhibited the elevation of intracellular reactive oxygen species levels in glutamate-stressed HT22 cells. These data suggest that obovatol and honokiol may have clinical applications for preventing neurodegenerative disorders.     

Development of a binary lipid nanoparticles formulation of itraconazole for parenteral administration and controlled release

The principal aim of this study was to develop an intravenous formulation of itraconazole (ITZ) using lipid nanoparticles based on binary mixture of liquid and solid lipids. Lipid nanoparticles were developed to provide the controlled release of ITZ as well as to improve the solubility of ITZ. Lipid nanoparticles were prepared with tristearin as a solid lipid, triolein as a liquid lipid, and a surfactant mixture of eggPC, Tween 80 and DSPE-PEG₂₀₀₀. ITZ was incorporated at the concentration of 20 mg/g. Lipid nanoparticles were manufactured by high-pressure homogenization method. The particle size and polydispersity index (PI) of lipid nanoparticles were below 280 nm and 0.2, respectively. Zeta potentials and incorporation efficiencies of lipid nanoparticles were around ?30 mV and above 80%, respectively. Lipid nanoparticles containing 1% of liquid lipid showed the smallest particles size and the highest incorporation efficiency. Results from SEM, DSC and PXRD revealed that ITZ in lipid nanoparticles exists in an amorphous state. Release rates were increased as the amount of liquid lipid in lipid core increased, demonstrating that the release of ITZ from lipid nanoparticles could be controlled by modulation of the amount of liquid lipid in lipid core. Pharmacokinetic studies were performed after intravenous administration of lipid nanoparticles in rats at the dose of 5 mg/kg. The plasma concentration of ITZ was prolonged after intravenous administration of lipid nanoparticles. It is concluded that binary lipid nanoparticles could control the release and pharmacokinetic parameters of ITZ.     

Investigation of PEG Crystallization in Frozen PEG-Sucrose-Water Solutions. I. Characterization of the Nonequilibrium Behavior during Freeze-Thawing

Our objective was to characterize the nonequilibrium thermal behavior of frozen aqueous solutions containing PEG and sucrose. Aqueous solutions of (i) sucrose (10%, w/v) with different concentrations of PEG (1–20%, w/v), and (ii) PEG (10%, w/v) with different concentrations of sucrose (2–20%, w/v), were cooled to ? 70°C at 5°C/min and heated to 25°C at 2°C/min in a differential scanning calorimeter. Annealing was performed at temperatures ranging from ? 50 to ? 20°C for 2 or 6 h. Similar experiments were also performed in the low-temperature stage of a powder X-ray diffractometer. A limited number of additional DSC experiments were performed wherein the samples were cooled to ? 100°C. In unannealed systems with a fixed sucrose concentration (10%, w/v), the T′_g decreased from ? 35 to ? 48°C when PEG concentration was increased from 1% to 20% (w/v). On annealing at ? 25°C, PEG crystallized. This was evident from the increase in T′_g and the appearance of a secondary melting endotherm in the DSC. Low- temperature XRD provided direct evidence of PEG crystallization. Annealing at temperatures ≤?40°C did not result in crystallization and a devitrification event was observed above the T′_g. In unannealed systems with a fixed PEG concentration (10%, w/v), the T′_g increased from ? 50 to ? 40°C when sucrose concentration was increased from 5% to 50%, w/v. As the annealing time increased (at ? 25°C), the T′_g approached that of a sucrose-water system, reflecting progressive PEG crystallization. A second glass transition at ~? 65°C was evident in unannealed systems [10%, w/v sucrose and 10 (or 20%), w/v PEG] cooled to ? 100°C. Investigation of the nonequilibrium behavior of frozen PEG-sucrose-water ternary system revealed phase separation in the freeze-concentrate. Annealing facilitated PEG crystallization.     

Effects of Bacillus subtilis ANSB060 on growth performance,meat quality and aflatoxin residues in broilers fed moldy peanut meal naturally contaminated with aflatoxins

This study was conducted to investigate the toxic effects of aflatoxins and the efficacy of Bacillus subtilis ANSB060 for the amelioration of aflatoxicosis in broiler chickens. Six replicates of ten broilers each were assigned to one of seven dietary treatments, which were labeled C0 (basal diet); M0 (basal diet containing moldy peanut meal); C500 and C1000 (C0 + 500 or 1000 g/t aflatoxin biodegradation preparations, composed mainly of ANSB060); and M500, M1000 and M2000 (M0 + 500, 1000 or 2000 g/t aflatoxin biodegradation preparations). The concentrations of aflatoxin B₁, B₂, G₁ and G₂ in the moldy diets (M0, M500, M100 and M2000) fluctuated around 70.7 ± 1.3, 11.0 ± 1.5, 6.5 ± 0.8 and 2.0 ± 0.3 μg/kg, respectively. The results showed that the M0 diet caused a significant decrease in average daily weight gain and increased feed requirements, with a gain ratio increasing from d 8 to 42, deterioration in meat quality and aflatoxin residues in broilers’ livers as compared with the C0 diet. The addition of ANSB060 to the aflatoxin-contaminated diets offset these negative effects, leading to the conclusion that ANSB060 has a protective effect on growth performance and meat quality while reducing the amount of aflatoxin residues in the livers of broilers fed naturally moldy peanut meal.     

Inhalable Liposomes of Low Molecular Weight Heparin for the Treatment of Venous Thromboembolism

This study tests the feasibility of inhalable pegylated liposomal formulations of low molecular weight heparin (LMWH) for treatment of two clinical manifestations of vascular thromboembolism: deep vein thrombosis (DVT) and pulmonary embolism (PE). Conventional distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and long-circulating pegylated (DSPE–PEG-2000 and DSPE-PEG-5000) liposomes were prepared by hydration method. Formulations were evaluated for particle size, entrapment efficiency, stability, pulmonary absorption, anticoagulant, and thrombolytic effects in rats. Pulmonary absorption was monitored by measuring plasma antifactor Xa activity; anticoagulant and thrombolytic effects were studied by measuring reduction in thrombus weight and amount of dissolved radioactive clot in the blood, respectively. Pegylated liposomal were smaller and showed greater drug entrapment efficiency than conventional liposomes. All formulations produced an increase in pulmonary absorption and circulation time of LMWH upon first dosing. Three repeated dosings of conventional liposomes resulted in decreased half-life and bioavailability; no changes in these parameters were observed with pegylated liposomes. PEG-2000 liposomes were effective in reducing thrombus weight when administered every 48 h over 8 days. In terms of thrombolytic effects and dosing frequency, PEG-2000 liposomes administered via the pulmonary route at a dose of 100U/kg were as effective as 50 U/kg LMWH administered subcutaneously. This paper suggests that inhalable pegylated liposomes of LMWH could be a potential noninvasive approach for DVT and PE treatment. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci99:4554–4564,     

Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood–brain-barrier: Implications for therapy of Alzheimer disease

Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation features and to cross in vitro and in vivo the blood–brain barrier (BBB). Surface plasmon resonance showed that bi-functionalized liposomes strongly bind Aβ (k_D = 0.6 μM), while Thioflavin-T and SDS-PAGE/WB assays show that liposomes inhibit peptide aggregation (70% inhibition after 72 h) and trigger the disaggregation of preformed aggregates (60% decrease after 120 h incubation). Moreover, experiments with dually radiolabelled LIP suggest that bi-functionalization enhances the passage of radioactivity across the BBB either in vitro (permeability = 2.5 × 10^− 5cm/min, 5-fold higher with respect to mono-functionalized liposomes) or in vivo in healthy mice. Taken together, our results suggest that mApoE-PA-LIP are valuable nanodevices with a potential applicability in vivo for the treatment of AD.From the Clinical EditorBi-functionalized liposomes with phosphatidic acid and a modified ApoE-derived peptide were demonstrated to influence Aβ aggregation/disaggregation as a potential treatment in an Alzheimer’s model. The liposomes were able to cross the blood-brain barrier in vitro and in vivo. Similar liposomes may become clinically valuable nanodevices with a potential applicability for the treatment of Alzheimer’s disease.     

Synthetic polysulfane derivatives induce cell cycle arrest and apoptotic cell death in human hematopoietic cancer cells

Natural polysulfanes including diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic possess antimicrobial, chemopreventive and anticancer properties. However these compounds exhibit chemical instability and reduced solubility, which prevents their potential clinical applicability. We synthesized six DATS and DATTS derivatives, based on the polysulfane motif, expected to exhibit improved physical and chemical properties and verified their biological activity on human leukemia cells.We identified four novel cytotoxic compounds (IC₅₀ values: compound 1, 24.96 ± 12.37 μM; compound 2, 22.82 ± 4.20 μM; compound 3, 3.86 ± 1.64 μM and compound 5, 40.62 ± 10.07 μM, compared to DATTS: IC₅₀: 9.33 ± 3.86 μM). These polysulfanes possess excellent differential toxicity, as they did not affect proliferating mononuclear blood cells from healthy donors.We further demonstrated ability of active compounds to induce apoptosis in leukemia cells by analysis of nuclear fragmentation and of cleavage of effector and executioner caspases. Apoptosis was preceded by accumulation of cells in G2/M phase with a pro-metaphase-like nuclear pattern as well as microtubular alterations. Prolonged and persistent arrest of cancer cells in early mitosis by the benzyl derivative identifies this compound as the most stable and effective one for further mechanistic and in vivo studies.     

Half-life extension of the HIV-fusion inhibitor peptide TRI-1144 using a novel linker technology

We have previously developed a linker technology for half-life extension of peptides, proteins and small molecule drugs (1). The linkers undergo β-elimination reactions with predictable cleavage rates to release the native drug. Here we utilize this technology for half-life extension of the 38 amino acid HIV-1 fusion inhibitor TRI-1144. Conjugation of TRI-1144 to 40 kDa PEG by an appropriate β-eliminative linker and i.v. administration of the conjugate increased the in vivo half-life of the released peptide from 4 to 34 h in the rat, and the pharmacokinetic parameters were in excellent accord with a one-compartment model. From these data we simulated the pharmacokinetics of the PEG-TRI-1144 conjugate in humans, predicting a t_1/2,_β of 70 h for the released peptide, and that a serum concentration of 25 nM could be maintained by weekly doses of 8 μmol of the conjugate. Using a non-circulating carrier (2) similar simulations indicated a t_1/2,_β of 150 h for the peptide released from the conjugate and that dosing of only 1.8 μmol/week could maintain serum concentrations of TRI-1144 above 25 nM. Hence, releasable β-eliminative linkers provide significant half-life extension to TRI-1144 and would be expected to do likewise for related peptides.     

Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting

The estradiol(E₂)-loaded chitosan nanoparticles (CS-NPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The CS-NPs had a mean size of (269.3 ± 31.6) nm, a zeta potential of +25.4 mV, and loading capacity of E₂ CS-NPs suspension was 1.9 mg ml⁻¹, entrapment efficiency was 64.7% on average. Subsequently, this paper investigated the levels of E₂ in blood and the cerebrospinal fluid (CSF) in rats following intranasal administration of E₂ CS-NPs. E₂-loaded CS-NPs were administered to male Wister rats either intranasally or intravenously at the dose of 0.48 mg kg⁻¹. The plasma levels achieved following intranasal administration (32.7 ± 10.1 ng ml⁻¹; t_max 28 ± 4.5 min) were significantly lower than those after intravenous administration (151.4 ± 28.2 ng ml⁻¹), while CSF concentrations achieved after intranasal administration (76.4 ± 14.0 ng ml⁻¹; t_max 28 ± 17.9 min) were significantly higher than those after intravenous administration (29.5 ± 7.4 ng ml⁻¹ t_max 60 min). The drug targeting index (DTI) of nasal route was 3.2, percent of drug targeting (DTP%) was 68.4%. These results showed that the E₂ must be directly transported from the nasal cavity into the CSF in rats. Finally, compared with E₂ inclusion complex, CS-NPs improved significantly E₂ being transported into central nervous system (CNS).     

Human absorption of a supplement containing purified hydroxytyrosol,a natural antioxidant from olive oil,and evidence for its transient association with low-density lipoproteins

There is growing interest in the health effects of olive oil polyphenols, particularly hydroxytyrosol (HT), for their potential application in the treatment of inflammatory conditions such as cardiovascular disease (CVD). As oxidative modification of low-density lipoproteins (LDL) plays a central role in the development of CVD, natural antioxidants are a main target for the nutraceutical industry. In this study we firstly investigated the absorption of pure hydroxytyrosol (99.5%) administered as a supplement in an aqueous solution (2.5 mg/kg BW) in the plasma and urine of healthy volunteers (n = 10).Plasma C_max for HT and homovanillic alcohol (HvOH) were detected at 13.0 ± 1.5 and 16.7 ± 2.4 min, respectively. The HT and HvOH levels were undetectable 2-h after the administration. HT, HvOH, homovanillic acid and 3,4-dihydroxyphenylacetic acid were found as free forms (44%) or as glucuronide (34.4%) or sulphate (21.2%) conjugates in the 24-h urine samples of the subjects.In a second phase of the study, the same amounts of HT were administered to the subjects and the presence of HT in purified plasma lipoproteins was investigated in LDL fractions freshly isolated. 10 min after the ingestion of the HT supplement, more than 50% of the total amount detected was present in the LDL-purified fractions and its concentration declined in accordance with its presence in plasma but no changes were found in total antioxidant capacity, malondialdehyde or LDL lag time. These results indicate that pure HT transiently associates with LDL lipoproteins in vivo.     

Effect of liposome size on peritoneal retention and organ distribution after intraperitoneal injection in mice

Peritoneal carcinomatosis is a serious concern when treating digestive or ovarian tumors. Treatment with systemic chemotherapy suffers from poor penetration of cytotoxic agents into the peritoneal cavity and is not quite effective. Local delivery of drugs, especially as controlled-release delivery systems like liposomes, could provide sustained and higher drug levels and reduce systemic toxicity.In order to investigate the effect of liposome size on peritoneal retention, liposomes composed of distearoylphosphatidylcholine and cholesterol (DSPC/CHOL, molar ratio 2:1) were prepared at four sizes of 100, 400, 1000 and 3000 nm. Subsequently, these liposomes were labeled with ^99mTc complex of hexamethylpropyleneamineoxime (^99mTc-HMPAO) and injected into mouse peritoneum. Then, mice were sacrificed at eight different time points and the percentage of injected radiolabel in the peritoneal cavity and the organ distribution in terms of percentage injected dose/gram tissue (%ID/g) were obtained.Results showed that the free label (^99mTc-HMPAO) was cleared very rapidly from the cavity so that after 5 min and 7 h only 6.89 ± 2.51% and 0.91 ± 0.51% of the injected dose was recovered, respectively. However, for the liposomal formulations, this recovery value ranged from 8.47 ± 1.62% to 29.99 ± 12.06% at 7 h. Peritoneal retention of the vesicles was increased with their size, and the highest retention rate was obtained with 1000 nm liposomes with an AUC value 15.51 times that of ^99mTc-HMPAO. In blood, as expected, 100 nm liposomes showed much higher levels because of their greater stability. Their greater blood concentration also caused increased levels in the heart and kidneys, although their organ to blood AUC ratio was the lowest.Overall, among the different sized neutral liposomes investigated, the 1000 nm vesicles seemed to be the most optimal, achieving a greater peritoneal level and retention.     

Dry Powder Aerosols Generated by Standardized Entrainment Tubes From Drug Blends With Lactose Monohydrate: 1. Albuterol Sulfate and Disodium Cromoglycate

The major objective of this study was: discriminatory assessment of dry powder aerosol performance using standardized entrainment tubes (SETs) and lactose-based formulations with two model drugs. Drug/lactose interactive physical mixtures (2%w/w) were prepared. Their properties were measured: solid-state characterization of phase behavior and molecular interactions by differential scanning calorimetry and X-ray powder diffraction; particle morphology and size by scanning electron microscopy and laser diffraction; aerosol generation by SETs and characterization by twin-stage liquid impinger and Andersen cascade impactor operated at 60 L/min. The fine particle fraction (FPF) was correlated with SET shear stress (τ_s), using a novel powder aerosol deaggregation equation (PADE). Drug particles were < 5 μm in volume diameter with narrow unimodal distribution (Span < 1). The lowest shear SET (τ_s = 0.624 N/m²) gave a higher emitted dose (ED ~ 84–93%) and lower FPF (FPF_6.4 ~ 7–25%). In contrast, the highest shear SET (τ_s = 13.143 N/m²) gave a lower ED (ED ~ 75–89%) and higher FPF (FPF_6.4 ~ 15–46%). The performance of disodium cromoglycate was superior to albuterol sulfate at given τ_s, as was milled with respect to sieved lactose monohydrate. Excellent correlation was observed (R² ~ 0.9804–0.9998) when pulmonary drug particle release from the surface of lactose carriers was interpreted by PADE linear regression for dry powder formulation evaluation and performance prediction.