European Journal of Pharmaceutics and Biopharmaceutics (v.80, #2)

APV Diary (I).

Expert opinion: Responsive polymer nanoparticles in cancer therapy by William B. Liechty; Nicholas A. Peppas (241-246).
Polymeric nanoparticles are emerging as an attractive treatment options for cancer due to their favorable size distribution, drug carrying capacity, and tunable properties. In particular, intelligent nanoparticles that respond to biological cues are of interest because of their ability to provide controlled release at a specific site. Tumor sites display abnormal pH profiles and pathophysiology that can be exploited to provide localized release. In this expert opinion, we discuss passive and active targeting of nanoparticles and several classes of pH-responsive nanoparticles.
Keywords: Drug delivery; pH-responsive polymers; Cancer therapy; Nanoparticles; Nanogels; Polymer micelles;

Intracellular delivery of siRNA complexed by DEAPA-PVA-g-PLGA was characterized to be energy-dependent and predominantly clathrin-mediated. Nanoparticles entrapped in endo-/lyso-somes can escape into the cytosol to deliver bioactive siRNA at its site of action.Efficient downregulation of gene expression depends on the uptake, intracellular distribution and efficient release of siRNA from their carrier. Therefore, the cellular uptake behavior and mechanism and intracellular localization of siRNA-loaded biodegradable nanoparticles were investigated.A biodegradable polymer, composed of poly(vinyl alcohol) (PVA) modified with diamine moieties and grafted with PLGA, abbreviated as DEAPA-PVA-g-PLGA, was used for the preparation of siRNA-loaded nanoparticles by solvent displacement. Particle sizes and morphology were determined by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The dependence of particle uptake into H1299-EGFP cells (lung cancer cells expressing green fluorescent protein) on both incubation time and temperature was studied by flow cytometry. Inhibition experiments focusing on clathrin- or caveolae-mediated uptake or uptake by macropinocytosis were performed. The intracellular localization was investigated by confocal laser scanning microscopy. The GFP knockdown efficiency was determined in vitro to establish the potential of the nanoparticles for the downregulation of gene expression.Nanoparticles with diameters of 120–180 nm were successfully generated. In contrast to the uptake of standard PEI-polyplexes, which increased continuously over a period of 4 h, nanoparticle uptake was complete within 2 h. A decrease in particle uptake at 4 °C (in comparison with 37 °C) suggests an active uptake process. Inhibition experiments revealed the predominance of clathrin-mediated uptake for siRNA-loaded nanoparticles. The siRNA-loaded nanoparticles could be clearly located within cells, mainly in intracellular vesicles. Particle uptake could be increased by the addition of lung surfactant to the formulation. Bioactivity in terms of successful GFP knockdown in vitro was demonstrated and could be further optimized by the use of surfactant-modified particles.In conclusion, a high and rapid cellular uptake was shown for siRNA-loaded nanoparticles. Cell internalization is based on an energy-dependent and predominantly clathrin-mediated process. Particle localization in endosomes and lysosomes was demonstrated. Evidence for the efficient delivery of bioactive siRNA and specific GFP knockdown provides a solid basis for the application of DEAPA-PVA-g-PLGA-based particles for gene silencing in vivo.
Keywords: Nanoparticles; Biodegradable polymer; siRNA delivery; Cellular uptake mechanism; Lung surfactant; Knockdown;

Development and optimization of nanosomal formulations for siRNA delivery to the liver by Anup K. Kundu; Partha K. Chandra; Sidhartha Hazari; Yashoda V. Pramar; Srikanta Dash; Tarun K. Mandal (257-267).
Compared with non-sonicated nanosomes (B1), sonicated nanosomes (B6) enhance siRNA (red) release from endosomes (green), siRNA deposition (red) and knock-down of gene (i.e., GAPDH) in the liver.The objective of this study is to develop an effective siRNA delivery system for successful delivery to the liver for the treatment of HCV. Nanosize liposomes (nanosomes) have been prepared using a mixture of cholesterol and DOTAP. A functional siRNA was encapsulated into nanosomes following condensation with protamine sulfate. The delivery of siRNA was optimized in an in vitro cell culture system. The efficacy of the formulations was evaluated by measuring functional gene silencing and cytotoxicity. Encapsulation of siRNA ⩾7.4 nM resulted in successful delivery of siRNA to nearly 100% of cells. The formulations containing lipid-to-siRNA ratio ⩾10.56:1 instantly cleared approximately 85% of HCV while maintaining cell viability at about 90%. The formulations were sonicated to further reduce the particle size. The size of these formulations was decreased up to 100 nm. However, there were no significant changes observed in zeta potential, or in siRNA encapsulation and integrity following sonication. The sonicated formulations also showed higher liver hepatocytes deposition and gene silencing properties. This study therefore provides a novel approach of siRNA delivery to liver hepatocytes, which can also be applied to treat HCV in chronic liver diseases.
Keywords: High-pressure homogenization; Hepatitis C virus; siRNA delivery; Nanosomes; Sonication;

Cationic solid lipid nanoparticles for co-delivery of paclitaxel and siRNA by Yong Hee Yu; Eunjoong Kim; Dai Eui Park; Gayong Shim; Sangbin Lee; Young Bong Kim; Chan-Wha Kim; Yu-Kyoung Oh (268-273).
Cationic solid lipid nanoparticles (cSLN) were formulated for co-delivery of paclitaxel and siRNA. Enhanced antitumor effect was observed in human epithelial carcinoma cells following treatment with siMCL1-complexed PcSLN (paclitaxel-loaded cSLN).In this study, we formulated cationic solid lipid nanoparticles (cSLN) for co-delivery of paclitaxel (PTX) and siRNA. 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine-based cSLN were prepared by emulsification solidification methods. PTX-loaded cSLN (PcSLN) were characterized by zeta potential and gel retardation of complexes with small interfering RNA (siRNA). The sizes of PcSLN did not significantly differ from those of empty cSLN without PTX (EcSLN). The use of cSLN increased the cellular uptake of fluorescent dsRNA in human epithelial carcinoma KB cells, with PcSLN complexed to fluorescence-labeled dsRNA promoting the greatest uptake. For co-delivery of therapeutic siRNA, human MCL1-specific siRNA (siMCL1) was complexed with PcSLN; luciferase-specific siRNA (siGL2) complexed to EcSLN or PcSLN was used as a control. MCL1 mRNA levels were significantly reduced in KB cells treated with siMCL1 complexed to PcSLN, but not in groups treated with siMCL alone or siGL2 complexed to PcSLN. siMCL1 complexed to PcSLN exerted the greatest in vitro anticancer effects in KB cells, followed by siMCL1 complexed to EcSLN, siGL2 complexed to PcSLN, PTX alone, and siMCL1 alone. In KB cell-xenografted mice, intratumoral injection of PcSLN complexed to siMCL1 significantly reduced the growth of tumors. Taken together, our results demonstrate the potential of cSLN for the development of co-delivery systems of various lipophilic anticancer drugs and therapeutic siRNAs.
Keywords: Solid lipid nanoparticles; Co-delivery; Paclitaxel; SiRNA; Combined cancer therapy;

While sterilisation by gamma-irradiation altered the capacity of non-encapsulated antigen to stimulate immune responses in vitro and in vivo, the method did not impinge on the antigen when encapsulated in PLGA microparticles as assessed in vitro in antigen-presentation assays and in vivo in mice.During the last two decades, synthetic polymers such as poly(lactide-co-glycolide) (PLGA) have been investigated for the development of nano- or microparticles as adjuvants or antigen vehicles. To enable transfer of this technology to human settings, the issue of sterilisation is of central importance. Since most polymers are heat-sensitive, sterilisation of polymeric microspheres for parenteral administration is assured either by costly and laborious aseptical preparation or the more preferred γ-irradiation. Many studies have investigated the effect of γ-irradiation on various physiochemical properties of the microspheres, but investigations on immunological effects are rare. We prepared poly(lactide-co-glycolide) (PLGA) microspheres containing ovalbumin (OVA) and tested the effect of γ-irradiation on the various immunological properties in mice. For reference, OVA was γ-irradiated and tested equivalently. The ability of encapsulated or non-encapsulated OVA to trigger activation of dendritic cells (DCs) was not affected by irradiation. However, while γ-irradiation of free OVA strongly influenced the antigen presentation, encapsulated OVA was not affected by irradiation. γ-Irradiation of OVA also reduced the immunogenicity in mice with regard to OVA-specific IgG1 production. In contrast, the antibody and the T-cell responses in mice immunised with PLGA-encapsulated OVA were similar irrespective of the γ-irradiation status. Hence, encapsulation of antigen into PLGA microspheres protects antigen from the potential detrimental effect of γ-irradiation leading to inactivation or altered immunogenicity. Sterilisation by γ-irradiation therefore enables a cost-effective production of PLGA-based antigen-delivery systems as compared to the more laborious and expensive aseptical production of such vaccines.
Keywords: Gamma-irradiation; Microparticles; PLGA; Immunogenicity; Antigen presentation; Antibodies;

Residual transglutaminase in collagen – Effects, detection, quantification, and removal by W. Schloegl; A. Klein; R. Fürst; U. Leicht; E. Volkmer; M. Schieker; S. Jus; G.M. Guebitz; I. Stachel; M. Meyer; M. Wiggenhorn; W. Friess (282-288).
A new ELISA detects high amounts of residual transglutaminase (mTG) in mTG-treated collagen despite intensive washing. Dialysis enables the complete removal of mTG.In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p  < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.
Keywords: Collagen; ELISA; Enzyme; Protein; Transglutaminase; Western blot;

The carriers had significant effects on the crystalline properties and oral bioavailability of flurbiprofen in solid self-nanoemulsifying drug delivery system (solid SNEDDS).In order to investigate the effects of solid carriers on the crystalline properties, dissolution and bioavailability of flurbiprofen in a solid self-nanoemulsifying drug delivery system (solid SNEDDS), different solid SNEDDS formulations were prepared by spray-drying the solutions containing liquid SNEDDS and various carriers. The liquid SNEDDS, composed of Labrafil M 1944 CS/Labrasol/Trasncutol HP (12.5/80/7.5%) with 2% w/v flurbiprofen, gave a z-average diameter of about 100 nm. Silicon dioxide, a hydrophobic solid carrier, produced an excellent conventional solid SNEDDS with a nanoemulsion droplet size of less than 100 nm, similar to the liquid SNEDDS and smaller than the other solid SNEDDS formulations. The drug was in an amorphous state in this solid SNEDDS. Furthermore, it greatly improved the dissolution rate and oral bioavailability of flurbiprofen in rats because it allowed the spontaneous formation of an interface between the oil droplets and the water. Magnesium stearate, a hydrophobic carrier, produced a solid SNEDDS with the largest diameter. However, it greatly enhanced the dissolution rate and oral bioavailability due to the formation of a simple eutectic mixture. The hydrophilic carriers such as polyvinyl alcohol (PVA), sodium carboxymethyl cellulose (Na-CMC) and hydroxypropyl-β-cyclodextrantrin (HP-β-CD) did not form a solid SNEDDS but rather a solid dispersion (or microcapsule). HP-β-CD improved the dissolution rate but did not improve the oral bioavailability as much as the hydrophobic polymers. PVA and Na-CMC hardly improved the dissolution rate but maintained constantly high plasma levels in rats for a long period. Thus, the selection of carrier is an important factor in the development of solid SNEDDS, since the carriers had significant effects on the crystalline properties, dissolution and oral bioavailability of flurbiprofen and on the formation of solid SNEDDS.
Keywords: Solid self-nanoemulsifying drug delivery system; Flurbiprofen; Carrier; Crystalline property; Dissolution; Bioavailability;

β-casein nanoparticles entrapped and stabilized paclitaxel and its nanocrystals at high loading efficiency. Target-activated release performance was demonstrated as the cytotoxicity to human gastric carcinoma cell line was found only after simulated gastric digestion, suggesting potential for oral delivery of chemotherapy.We studied a potential drug delivery system comprising the hydrophobic anticancer drug paclitaxel entrapped within β-casein (β-CN) nanoparticles and its cytotoxicity to human gastric carcinoma cells. Paclitaxel was entrapped by stirring its dimethyl sulfoxide (DMSO) solution into PBS containing β-CN. Cryo-TEM analysis revealed drug nanocrystals, the growth of which was blocked by β-CN. Entrapment efficiency was nearly 100%, and the nanovehicles formed were colloidally stable. Following encapsulation and simulated digestion with pepsin (2 hours at pH = 2, 37 °C), paclitaxel retained its cytotoxic activity to human N-87 gastric cancer cells; the IC50 value (32.5 ± 6.2 nM) was similar to that of non-encapsulated paclitaxel (25.4 ± 2.6 nM). Without prior simulated gastric digestion, β-CN-paclitaxel nanoparticles were non-cytotoxic, suggesting the lack of untoward toxicity to bucal and esophageal epithelia. We conclude that β-CN shows promise to be useful for target-activated oral delivery of hydrophobic chemotherapeutics in the treatment of gastric carcinoma, one of the leading causes of cancer mortality worldwide.
Keywords: β-Casein; Paclitaxel; Oral delivery; Chemotherapy; Drug targeting; Nanoparticles;

Nanoparticulate lipid dispersions for bromocriptine delivery: Characterization and in vivo study by Elisabetta Esposito; Paolo Mariani; Laura Ravani; Catia Contado; Mattia Volta; Simone Bido; Markus Drechsler; Serena Mazzoni; Enea Menegatti; Michele Morari; Rita Cortesi (306-314).
A comparison between monoolein aqueous dispersions and nanostructured lipid carriers as bromocriptine delivery systems for the treatment of Parkinson’s disease.The physico-chemical properties and in vivo efficacies of two nanoparticulate systems delivering the antiparkinsonian drug bromocriptine (BC) were compared in the present study. Monoolein Aqueous Dispersions (MADs) and Nanostructured Lipid Carriers (NLCs) were produced and characterized. Cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction revealed the morphology of MAD and NLC. Dimensional distribution was determined by Photon Correlation Spectroscopy (PCS) and Sedimentation Field Flow Fractionation (SdFFF). In particular, BC was shown to be encapsulated with high entrapment efficiency both in MAD and in NLC, according to SdFFF combined with HPLC. Two behavioral tests specific for akinesia (bar test) or akinesia/bradykinesia (drag test) were used to compare the effects of the different BC formulations on motor disabilities in 6-hydroxydopamine hemilesioned rats in vivo, a model of Parkinson’s disease. Both free BC and BC–NLC reduced the immobility time in the bar test and enhanced the number of steps in the drag test, although the effects of encapsulated BC were longer lasting (5 h). Conversely, BC–MAD was ineffective in the bar test and improved stepping activity in the drag test to a much lower degree than those achieved with the other preparations. We conclude that MAD and NLC can encapsulate BC, although only NLC provide long-lasting therapeutic effects possibly extending BC half-life in vivo.
Keywords: Solid Lipid Nanoparticles (SLNs); Nanostructured Lipid Carrier (NLC); Bromocriptine (BC); X-ray; Drag test; Bar test;

Histidine-tagged HIV I Gag p41 bound to lipid based nanocapsules through interaction with surface accessible nickel generated a strong dose-dependent immune response when administered to BALB/c mice.The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high-affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs were 214.9 nm and −14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24 h (<5%). We further evaluated the antibody response of the Ni-NCs using His-Gag p41 as a model antigen. Formulations were administered subcutaneously to BALB/c mice at day 0 (prime) and 14 (boost) followed by serum collection on day 28. Serum His-Gag p41-specific antibody levels were found to be significantly higher at 1 and 0.5 μg doses of Gag p41-His–Ni-NCs (His-Gag p41 equivalent) compared with His-Gag p41 (1 μg) adjuvanted with aluminum hydroxide (AH). The serum IgG2a levels induced by Gag p41-His–Ni-NCs (1 μg) were significantly higher than AH adjuvanted His-Gag p41. The Ni-NCs alone did not result in the elevation of systemic IL-12/p40 and CCL5/RANTES inflammatory cytokine levels upon subcutaneous administration in BALB/c mice. In conclusion, the proposed Ni-NCs can bind His-tagged proteins and have the potential to be used as antigen delivery system capable of generating strong antigen-specific antibodies at doses much lower than with aluminum-based adjuvant and causing no significant elevation of systemic pro-inflammatory IL-12/p40 and CCL5/RANTES cytokines.
Keywords: Green fluorescent protein; Antigen; Dendritic cells; Adjuvant; Cytokine; Antibody;

A remote-loading method in the presence of phenylboronic acid (PBA) was developed for stably encapsulating 3-deazaneplanocin A (DZNep) inside pegylated liposomes through forming a transient PBA–DZNep complex.3-Deazaneplanocin A (DZNep) is an attractive epigenetic anticancer agent through the inhibition of the cellular enhancer of zeste homolog 2 (EZH2) protein. The purpose of this study was to improve the pharmacokinetic characteristics of DZNep in vivo through developing a unilamellar pegylated liposomal formulation encapsulating DZNep (L-DZNep). A remote-loading method in the presence of phenylboronic acid (R-w-PBA) was developed to stably encapsulating DZNep inside liposomes (encapsulation efficiency = 50.7% at molar ratio of 1:10 of drug to lipids) through forming a transient PBA–DZNep complex. The pharmacokinetics of L-DZNep was investigated in Sprague–Dawley rats. In comparison with free drug, encapsulation of the DZNep in pegylated liposomes resulted in 99.3% reduction of the plasma clearance, whereas it increased the elimination half-life from 1.1 h to 8.0 h and the area under the plasma concentration curve by 138-fold. These findings demonstrate a novel approach (R-w-PBA method) through the development of L-DZNep, which may be extensively applied for the encapsulation of hydrophilic nucleoside analogs containing vicinal hydroxyl groups and protonable amino in the pegylated liposomes. Additionally, the pegylated liposomes could effectively prolong the retention of DZNep in the systemic circulation and therefore is highly likely to increase the DZNep’s tumor localization.
Keywords: 3-Deazaneplanocin A; Phenylboronic acid; Pegylated liposomes; Pharmacokinetics; Nucleoside analogs; Epigenetic anticancer agent;

More rapid drug elimination of liposomal topotecan prepared using a transmembrane NH4EDTA gradient compared with that prepared by a (NH4)2SO4 gradient.Antitumor drugs not only cause cytocidal effect on cancer cells, but also damage on normal healthy tissues, resulting in side effects. Liposome encapsulation can result in reduced systematic distribution due to the enhanced permeability and retention (EPR) effect, accompanied by drug accumulation in liver, spleen, and other immune organs, which can cause damage to those organs. It has been demonstrated that EDTA, frequently used as a chelator, possesses a synergistic antitumor effect. Indeed, our previous study showed that EDTA could reduce the toxicity of anthracyclines to the heart and immune organs. In this study, we intended to encapsulate topotecan within liposome adopting transmembrane NH4EDTA gradient in order to increase the antitumor activity and decrease the toxicity against normal immune organs. Regarding the encapsulation efficiency of topotecan liposomes, both the pH value of the buffer and the cholesterol content showed significant effects on encapsulation and drug retention. Liposome encapsulation dramatically increased the antitumor activity of topotecan compared to free drug (p  < 0.05), while similar efficacy was obtained from liposomes prepared by a NH4EDTA gradient or a (NH4)2SO4 gradient (tumor inhibition ratios were 85.6% and 84.1%, respectively). However, a significant decrease in toxicity against the immune organs was found in liposomes prepared by a NH4EDTA gradient compared to those prepared by a (NH4)2SO4 gradient. These results suggest the superiority of the proposed gradient for topotecan encapsulation in decreasing its toxicity on immune systems.
Keywords: Liposomes; Topotecan; NH4EDTA; Antitumor efficacy; Toxicity;

Efficacy of surface-modified liposomes in enhancing in vivo pulmonary absorption of elcatonin in rats.The aim of this study was to investigate the feasibility of surface-modified liposomes for pulmonary delivery of a peptide. Chitosan oligosaccharide (oligoCS) and polyvinyl alcohol with a hydrophobic anchor (PVA-R) were used as surface modifiers. The effect of liposomal surface modification on the behavior of the liposomes on pulmonary administration and potential toxicity were evaluated in vitro and in vivo. In an association study with A549 cells, PVA-R modification reduced interaction with A549 cells, whereas oligoCS modification electrostatically enhanced cellular interaction. The therapeutic efficacy of elcatonin (eCT) after pulmonary administration to rats was significantly enhanced and prolonged for 48 h after separate administration with oligoCS- or PVA-R-modified liposomes. oligoCS-modified liposomes adhered to lung tissues and caused opening of tight junctions, which enhanced eCT absorption. On the other hand, PVA-R-modified liposomes induced long-term retention of eCT in the lung fluid, leading to sustained absorption. Consequently, surface modification of liposomes with oligoCS or PVA-R has potential for effective peptide drug delivery through pulmonary administration.
Keywords: Surface modification; Pulmonary administration; Elcatonin; Liposomes; PVA; Chitosan;

Bioavailability enhancement of zaleplon via proliposomes: Role of surface charge by Karthik Y. Janga; Raju Jukanti; Ashok Velpula; Sharath Sunkavalli; Suresh Bandari; Prabhakar Kandadi; Prabhakar Reddy Veerareddy (347-357).
The study was focused to investigate the combined advantage of proliposomes and surface charge for improved oral delivery of zaleplon. A two to five fold enhancement in bioavailability confers the potential of proliposomes as suitable carriers for oral delivery of zaleplon.The present systematic study focused to investigate the combined advantage of proliposomes and surface charge for improved oral delivery of zaleplon. The zaleplon loaded proliposomes were prepared using hydrogenated soyphosphatidylcholine (HSPC) and cholesterol (CHOL) in varying ratios, and the optimized formulation was tailored with dicetyl phosphate and stearylamine to obtain negative and positive charged vesicles, respectively. The formulations were characterized for micromeritics, size, zeta potential, and entrapment efficiency. Further, in vitro release and dissolution study carried out provide an insight on the stability and enhanced dissolution of zaleplon from proliposome formulations. The solid state characterization (SEM, DSC, and PXRD) studies unravel the transformation of zaleplon to amorphous or molecular state from the native crystalline form. To depict the conclusions, in situ single-pass perfusion and bioavailability studies were carried out in rats. The significant increase in effective permeability coefficient (Peff) and rate and extent of absorption from cationic vesicles indicate the importance of surface charge for effective uptake across the gastrointestinal tract. Overall a two- to fivefold enhancement in bioavailability in comparison with control confers the potential of proliposomes as suitable carriers for improved oral delivery of zaleplon.
Keywords: Proliposomes; Zaleplon; Surface charge; Perfusion; Bioavailability; Stearylamine;

Freeze-drying study of peptide loaded multilamllar DOTAP-cholesterol liposomes and the importance of the cryoprotector addition during liposome preparation.In the present study, positively charged 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes as a delivery system for a hydrophilic decapeptide were developed. The main objective was the preparation of a stable, highly loaded, lyophilised formulation to yield the basis for an acceptable shelf life. The influences of addition of cholesterol, pH value, amounts of lipid and peptide, type and amount of sugar-based cryoprotective agent (trehalose and sucrose), and time point for cryoprotector addition as well as the freeze-drying process parameters were investigated. The collapse temperatures of the liposome dispersions in the presence of the disaccharides trehalose and sucrose were determined using a freeze-drying microscope (Lyostat 2). The liposome morphology before freeze-drying was determined by transmission electron microscopy (TEM). The evidence of intact liposomes after freeze-drying was shown by scanning electron microscope (SEM) imaging. In summary, this study demonstrated the successful development of DOTAP liposomes including their lyophilisation as a drug delivery system for small hydrophilic peptides.
Keywords: DOTAP; Liposome; Lyophilisation; Hydrophilic peptide; Freeze-drying microscope; Collapse temperature;

A novel small Odorranalectin-bearing cubosomes: Preparation, brain delivery and pharmacodynamic study on amyloid-β25–35-treated rats following intranasal administration by Hongbing Wu; Jianxu Li; Qizhi Zhang; Xiluan Yan; Liangran Guo; Xiaoling Gao; Mingfeng Qiu; Xinguo Jiang; Ren Lai; Hongzhuan Chen (368-378).
Odorranalectin (OL) can facilitate the access of drugs carried by cubosomes to the brain following intranasal administration, OL functionalization enhanced the therapeutic effects of Gly14-humanin on Alzheimer’s disease rats.Because of the immunogenicity and toxicity in vivo of large molecules such as lectins, the application of these molecules is remarkably restricted in drug delivery systems. In this study, to improve the brain drug delivery and reduce the immunogenicity of traditional lectin modified delivery system, Odorranalectin (OL, 1700 Da), a novel non-immunogenic small peptide, was selected to establish an OL-modified cubosomes (Cubs) system. The streptavidin (SA)-conjugated Cubs were prepared by incorporating maleimide–PEG–oleate and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothiolane thiolated SA; mono-biotinylated OL was then coupled with the SA-modified Cubs. The OL-decorated Cubs (OL-Cubs) devised via a non-covalent SA-biotin “bridge” made it easy to conjugate OL and determine the number of ligands on the surface of the Cubs using sensitive chemiluminescent detection. Retention of the bio-recognitive activity of OL after covalent coupling was verified by hemagglutination testing. Nose-to-brain delivery characteristic of OL-Cubs was investigated by in vivo fluorescent biodistribution using coumarin-6 as a marker. The relative uptake of coumarin carried by OL-Cubs was 1.66- to 3.46-fold in brain tissues compared to that incorporated in the Cubs. Besides, Gly14-Humanin (S14G-HN) as a model peptide drug was loaded into cubosomes and evaluated for its pharmacodynamics on Alzheimer’s disease (AD) rats following intranasal administration by Morris water maze test and acetylcholinesterase activity determination. The results suggested that OL functionalization enhanced the therapeutic effects of S14G-HN-loaded cubosomes on AD. Thus, OL-Cubs might offer a novel effective and noninvasive system for brain drug delivery, especially for peptides and proteins.
Keywords: Odorranalectin; Cubosomes; Intranasal administration; Gly14-Humanin; Brain delivery; Alzheimer’s disease;

After incubated for 30 min, the fluorescence intensity of SCC9 cells treated with FITC-P-FU-peptide was much higher than that treated with FITC-P-FU. The control sample was treated with culture medium and dealt with the same manner.In several groups of malignant tumors including head and neck tumors, a protein named Hsp47/CBP2 leaked from the cell was expressed on the tumor cell surface. Several synthetic peptides have been identified as effective ligands for binding to Hsp47/CBP2. This study has focused on the synthesis and in vitro characterization of a targeting delivery system of 5-fluorouracil (5-FU) to human head and neck squamous cell carcinoma (HNSCC) in order to improve anti-cancer efficacy and reduce dose-limiting toxicity of 5-FU. An N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, with Hsp47/CBP2 binding peptide sequence (namely WHYPWFQNWAMA) as a targeting ligand, was synthesized by a novel and simplified synthetic route. Under the controlled synthetic conditions, 1,3-dimethylol-5-FU, derived from 5-FU, was attached to the HPMA copolymer backbone via the lysosomally degradable GFLG linker, while the WHYPWFQNWAMA was conjugated via a non-degradable Gly-Gly (GG) linker. A control polymer without targeting moiety was also synthesized (P-FU). The in vitro cytotoxicity, internalization and apoptosis assays of the polymeric conjugates were evaluated. The characteristic apoptotic morphological changes were also assessed. Compared to 5-FU and P-FU, the HPMA copolymer containing the Hsp47/CBP2 binding peptide (P-FU-peptide) exhibited the highest cytotoxic efficacy to cell line of human head and neck squamous cell carcinoma (p  < 0.05) and was internalized much faster than P-FU, especially after being incubated for 30 min. Both of the morphology and apoptosis analyses demonstrated that the treatment of P-FU-peptide resulted in more apoptotic and necrotic induction of tumor cells than P-FU. Meanwhile, the rate of apoptosis induced by P-FU-peptide was higher than that of necrosis. In summary, the HPMA copolymer–Hsp47/CBP2 binding peptide conjugates showed a promising future for the treatment of HNSCC with improved efficacy.
Keywords: HPMA copolymer; 5-Fluorouracil; SCC9 cells; Hsp47/CBP2 binding peptide; Targeted delivery; Cytotoxicity;

Effect of ultraviolet filters on skin superoxide dismutase activity in hairless mice after a single dose of ultraviolet radiation by Fernanda Maria Pinto Vilela; Yris Maria Fonseca; José Roberto Jabor; Fabiana T.M.C. Vicentini; Maria José Vieira Fonseca (387-392).
Effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm2).Organic sunscreens may decrease their protective capability and also behave as photo-oxidants upon ultraviolet radiation (UVR) exposure. The present study investigated the effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm2). The retention of these UV filters was first evaluated in vivo using hairless mice to guarantee the presence of the filters in the skin layers at the moment of irradiation. The in vivo effect of the UV filters on skin SOD was then assayed spectrophotometrically via the reduction of cytochrome c. The cream gel formulation promoted the penetration of the three UV filters into the epidermis and the dermis at one hour post-application. A significant decrease in SOD activity was observed in irradiated animals treated with sunscreen formulation. However, no effect on SOD activity in skin was observed by the isolated presence of the sunscreens, the formulation components, or the exposure to UVR. The sunscreens may have formed degradation products under UVR that may have either inhibited the enzyme or generated reactive species in the skin.
Keywords: Benzophenone-3; Octyl methoxycinnamate; Octyl salicylate; UV radiation; Superoxide dismutase; Photodegradation;

Lipophilic beta adrenoreceptor antagonists penetrate to the posterior of the eye, where they bind to choroid and reside in the retina. Concentrations of C Max (axis in μg/g) obtained for atenolol (red column), timolol (blue column) and propranolol (green column).The treatment of posterior eye diseases, such as diabetic retinopathy and age-related macular degeneration, is of growing interest as the number of people affected by these conditions continues to rise. This study utilises the methods of cassette dosing and the perfused ovine eye model – to reduce animal usage and therefore animal time – to show that for a series of beta adrenoreceptor antagonists, lipophilicity is a key physicochemical property that governs drug distribution within the eye. Following intravitreal injection, lipophilic beta adrenoreceptor antagonists penetrate to the posterior eye, where they bind to the choroid and reside in the retina at greater concentrations than more hydrophilic beta adrenoreceptor antagonists, which preferentially penetrate to the anterior eye.
Keywords: Ocular; Pharmacokinetics; Mass spectrometry; Isolated perfused; Beta adrenoreceptor antagonist;

In vitro vs. canine data for assessing early exposure of doxazosin base and its mesylate salt by Marijana Erceg; Maria Vertzoni; Helena Cerić; Miljenko Dumić; Biserka Cetina-Čižmek; Christos Reppas (402-409).
Simulated cumulative doxazosin profiles in plasma until 2 h after administration were constructed using in vitro data and previously described procedures. Actual cumulative doxazosin profiles in plasma 0-2 h post dosing of DM to 24 adults were highly variable but biorelevant data led to better evaluation of the average input profile during the 0.5-2 h post-dosing. Simulated profiles constructed using biorelevant or data collected in pure aqueous buffers suggest that differences between DB and DM in humans are not substantial to affect early exposure.In this study, we evaluated the usefulness of biorelevant in vitro data and of canine data in forecasting early exposure after the administration of two phases of a BCS Class II compound, i.e., doxazosin base (DB) and its mesylate salt (DM). DB, DM, and doxazosin hydrochloride (DH) were prepared and characterized. In vitro data were collected in various media, including human aspirates. Solubilities of DB and DM in human gastric fluid were forecasted by data in fasted state simulating gastric fluid containing physiological components (FaSSGF-V2) but not by data in HClpH 1.8. Unlike data in FaSSGF-V2, dissolution of DB and DM tablets in HClpH 1.6 is rapid. Dissolution of DB tablet in FaSSGF-V2 is incomplete and conversion to DH seems to occur. Differences between DB and DM in dissolution in the small intestine are overestimated in the absence of physiological solubilizers. Using the in vitro data and previously described modeling procedures, the cumulative doxazosin profile in plasma was simulated and the 0–2 h profile was used for evaluating early exposure. Individual cumulative doxazosin profiles in plasma, after single DM tablet administrations to 24 adults, were constructed from corresponding actual plasma profiles. Compared with in vitro DM data in pure aqueous buffers, DM data in biorelevant media led to better prediction of early exposure. Based on intersubject variability in early exposure after DM administration and simulated profiles, the administered phase, DB or DM, does not have a significant impact on early exposure. Partial AUCs were used for evaluating early exposure after DB and DM administration in 4 dogs. Early exposure was significantly higher after administration of DM to dogs. Dogs are not appropriate for evaluating differences in early exposure after DB and DM administrations.
Keywords: Doxazosin base; Doxazosin salts; Dissolution; Early exposure; Humans; Dogs;

Five different bioequivalence protocols using pharmacokinetic end-points obtained on single and/or multiple dose studies on simulated extended-release formulations were tested. When evaluating bioequivalence, the inclusion use of the concentration at the end of the intended dosing interval obtained in the single-dose study avoids the need for steady-state studies while keeping the ability to detect differences between formulations.Use of single and multiple-dose studies is required to establish the bioequivalence between two extended-release oral dosage forms under the current European Guidelines. However, FDA is less strict in this regard and only requires a single-dose study. The objective of this work is to use a computer simulation in order to test the two approaches. Three pharmacokinetic models, representing different release mechanisms, were considered, and Monte Carlo simulations with intra- and inter-individual variabilities were performed. Five different bioequivalence protocols were used and a new pharmacokinetic metric –Cτ , the concentration at the end of the intended dosing interval obtained in the single-dose study – is proposed in order to avoid the need for steady-state studies while keeping the ability to detect differences between formulations. Results have shown that the European requirements are more capable to discriminate between two potentially different formulations but at the cost of the multiple-dose study and with an increased number of subjects when compared to the FDA requirements. However, the use of C max and AUC0– t obtained on a single-dose study with the added Cτ metric equals the discriminatory ability of the current EMA requirements, without the need of a multiple-dose study. This proposed approach results in the reduction in the number of studies and volunteers enrolled in clinical bioequivalence trials, without compromising the quality assurance of a new extended-release oral formulation.
Keywords: Extended-release oral drug products; Bioequivalence; Monte Carlo simulations; Pharmacokinetic metrics; Single-dose studies; Multiple-dose studies;

Chitosan enhances transcellular permeability in human and rat intestine epithelium by M. Magdalena Canali; Luciano P. Pedrotti; Jesús Balsinde; Cristina Ibarra; Silvia G. Correa (418-425).
The effective uptake of Chitosan by intestinal epithelial cells (IECs) is associated to the transcellular internalization of proteins such as horseradish peroxidase (HRP).The intestinal epithelium regulates the transit of molecules from and into the organism. Several agents act as absorption enhancers inducing changes in both transcellular and paracellular routes. Chitosan is a non-toxic biocompatible polysaccharide widely used as dietary supplement and mucosal delivery. Chitosan triggers both the activation of intestinal epithelial cells and the release of regulatory factors relevant for its immunomodulatory activity. Yet, the interaction of chitosan with intestinal epithelial cells is poorly characterized. We studied the uptake of this polysaccharide, and we evaluated its effects in both the net water and ion movements across human and rat colon samples and the epithelial permeability. Herein, we demonstrate that chitosan increases the transcellular permeability to ions, water and protein markers in human and rat intestinal mucosa and decreases the water permeability across the paracellular pathway. These findings are relevant to understand the activity of the polysaccharide in the mucosal environment.
Keywords: Chitosan; Intestine; Electrophysiology; Water transport; Antigen;

Novel Tanshinone II A ternary solid dispersion pellets prepared by a single-step technique: In vitro and in vivo evaluation by Jin Li; Pan Liu; Jian-Ping Liu; Wen-Li Zhang; Ji-Kun Yang; Yong-Qing Fan (426-432).
Tanshinone II A (TA) ternary solid dispersion pellets presented a smooth surface and a tightly packed coating structure under SEM. The addition of poloxamer 188 to binary TA-PVP system could remarkably promote the dissolution and bioavailability of Tanshinone II A at the TA/PVP/poloxamer 188 ratio of 1:4:1.Novel Tanshinone II A (TA) ternary solid dispersion (tSD) pellets with the combination of polyvinylpyrrolidone and poloxamer 188 as dispersing carriers were prepared by a single-step technique. A formulation screening study showed that the addition of poloxamer 188 to binary TA-PVP system could remarkably promote the dissolution rate of TA from 60% to 100% after 60 min. Scanning electron microscopy study revealed a smooth surface and a tightly packed coating structure. Differential scanning calorimetry analysis confirmed the formation of solid dispersions. In vivo test showed that TA tSD pellets presented significantly larger AUC0– t , which was 0.76 times more than that of binary solid dispersion (bSD) pellets, 2.87 times more than that of physical mixtures (PMs) and 5.40 times more than that of TA. C max of TA tSD pellets also increased by 1.82–8.97-fold as that of bSD pellets, PMs and TA. TA tSD pellets generated obviously shortened T max of (3.80 ± 0.398) h, compared to bSD pellets with (4.15 ± 0.456) h, PMs with (4.65 ± 0.226) h and TA with (5.52 ± 0.738) h. In conclusion, the addition of poloxamer 188 to pellets containing PVP-based solid dispersions could achieve complete dissolution, accelerated absorption rate and superior oral bioavailability. The fluid-bed technique becomes an alternative approach to obtain solid dispersion-coated pellets.
Keywords: Ternary solid dispersion pellets; Combined carriers; Polyvinylpyrrolidone; Poloxamer 188; In vitro dissolution study; In vivo evaluation;

Taste masking of paracetamol by hot-melt extrusion: An in vitro and in vivo evaluation by Mohammed Maniruzzaman; Joshua S. Boateng; Marion Bonnefille; Attila Aranyos; John C. Mitchell; Dennis Douroumis (433-442).
Hot-melt extrusion of paracetamol (PMOL) in Eudragit EPO and kollidon VA64 induces taste masking of the active. The masking effect is assessed both in vivo and in vitro.The purpose of this study was the in vitro and in vivo evaluation of the masking efficiency of hot melt extruded paracetamol (PMOL) formulations. Extruded granules containing high PMOL loadings in Eudragit EPO® (EPO) or Kollidon® VA64 (VA64) were prepared by hot-melt extrusion (HME). The taste masking effect of the processed formulation was evaluated in vivo by a panel of six healthy human volunteers. In addition, in vitro evaluation was carried out by an Astree e-tongue equipped with seven sensors. Taste sensing technology demonstrated taste improvement for both polymers by correlating the data obtained for the placebo polymers and the pure APIs alone. The best masking effect was observed for VA64 at 30% PMOL loading. The e-tongue results were in good agreement with the in vivo evaluation. In vitro dissolution of the extruded granules showed rapid PMOL releases.
Keywords: Hot-melt extrusion; Taste masking; Electronic tongue; Solubility parameter;

Dissolution of a poorly water-soluble drug dry coated with magnesium and sodium stearate by Tracy Tay; David A.V. Morton; Thomas R. Gengenbach; Peter J. Stewart (443-452).
Influence of dry coating micronized powders of a poorly water-soluble drug, indomethacin (IMC) with magnesium stearate (MgSt) and sodium stearate (NaSt) on the dissolution rate.The purpose of this research was to investigate the influence of dry coating micronized cohesive powders of a poorly water-soluble drug, indomethacin with force control agents, on its dissolution performance. A dry mechanical fusion method (mechanofusion) was used to coat indomethacin powders with magnesium stearate (0.25%, 1%, 5%) and sodium stearate (5%). After mechanofusion, significantly increased bulk and tapped densities and decreased intrinsic cohesion were observed for all samples. X-ray photoelectron spectroscopy analysis confirmed that a thicker magnesium stearate surface coating was achieved with increasing concentrations of the material. Dissolution was studied using the USP paddle method in buffer pH 5.0; several modelling approaches were used to explore the dissolution mechanisms. Whilst the bi-exponential equation represented dissolution of mechanofused indomethacin powders occurring from dispersed and agglomerated particles, it provided unrealistic parameter estimates for the two coating materials of contrasting properties. Initial increases in indomethacin dissolution were dependent on the concentration of magnesium stearate mechanofused onto the drug powders. The dissolution enhancing effect of indomethacin powders mechanofused with 5% sodium stearate was attributed to its surfactant properties that increased dispersion of indomethacin agglomerates. Initial drug release from the coated powders was described by a matrix-diffusion system according to the Higuchi model.
Keywords: Mechanical dry particle coating; Mechanofusion; Dissolution; Poorly water-soluble drugs; Magnesium stearate; Sodium stearate;

Effect of surfactant on 5-aminolevulinic acid uptake and PpIX generation in human cholangiocarcinoma cell by Chung-Wook Chung; Cy Hyun Kim; Kyung Ha Choi; Jin-Ju Yoo; Do Hyung Kim; Kyu-Don Chung; Young-IL Jeong; Dae Hwan Kang (453-458).
PDT toxicity of ALA with surfactants. Cells were incubated for 4h with 0.25mM ALA in the presence of different amounts of surfactants and then irradiated under 0.3-1.0 J/cm2 of light. Phototoxicity against HuCC-T1 cells increased with an increase in the surfactants and light doses.Photodynamic therapy (PDT) is a palliative therapy and has been used to cure cholangiocarcinoma (CC), which has a poor prognosis and limited available curative therapy. PDT was shown to improve the median survival time of advanced-stage patients. Recently, 5-aminolevulinic acid (ALA) has been used as a pro-photosensitizer, which can be transferred to intercellular protoporphyrin IX (PpIX), which is a strong photosensitizer, via the heme pathway. The main limitation of using ALA in PDT is the hydrophilic properties of ALA, which results in low cellular uptake. In this study, non-ionic surfactants, pluronic F68 (PF68) and Tween 80 (TW80), were used to address this limitation. The human CC cell line, HuCC-T1, was cotreated with ALA and different concentrations of surfactants for 4 h. The effect of surfactants was evaluated by monitoring the uptake of ALA, the fluorescence intensity of PpIX, and the cell survival rate after suitable light irradiation. Cotreatment with the surfactant resulted in an increased intracellular ALA level, PpIX formation, and phototoxicity.
Keywords: Cholangiocarcinoma; 5-Aminolevulinic acid; Photodynamic therapy; Penetration enhancer; Tween 80; Pluronic F68;

Effect of different preparation methods on the dissolution behaviour of amorphous indomethacin by Pranav Karmwar; Kirsten Graeser; Keith C. Gordon; Clare J. Strachan; Thomas Rades (459-464).
Dissolution profiles of (a) cryo-milled and (b) differently cooled amorphous samples of indomethacin at 37 ± 0.5 °C.The aim of this study was to investigate whether amorphous indomethacin samples prepared using different preparative techniques and processing parameters exhibit different structural and thermodynamic characteristics and whether these differences can be correlated to their dissolution behaviour. Samples were prepared either by cooling the drug melt at different cooling rates or by cryo-milling the drug for different milling times. The resulting amorphous materials were characterised using X-ray diffraction, Raman spectroscopy and polarising light microscopy. All samples were entirely X-ray amorphous, except for the sample cryo-milled for 15 min, which exhibited residual crystallinity. The shape of the halos in the diffractograms, however, varied depending on the preparation method and processing parameters, suggesting structural variations in the near order of the molecules between the prepared amorphous forms. This finding was supported by principal component analysis of the Raman spectra, as the samples clustered in the scores plot according to processing parameters for both of the preparative methods used. When investigating the dissolution behaviour, the samples cooled at different cooling rates showed no significant differences in their dissolution profiles and dissolution rates (≈0.55 μg/ml/cm2). In contrast, for cryo-milled samples, dissolution rate depended on the milling time, with samples milled for 120, 180 and 240 min, showing significantly increased dissolution rates of 0.28, 0.48 and 0.59 μg/ml/cm2, respectively, when compared to crystalline indomethacin (≈0.06 and 0.05 μg/ml/cm2 for α and γ-indomethacin, respectively). The milling processes appear to continue to affect the degree of disorder in the solid material, enhancing its dissolution rate, although all samples milled for >30 min were X-ray amorphous. Thus, choosing the right preparation technique and parameters for preparing amorphous solids is critical for producing materials with enhanced dissolution profiles.
Keywords: Amorphous; Indomethacin; Dissolution behaviour; Cryo-milling; Solution-mediated solid state transformation; Cooling rate;

Ex vivo study of bevacizumab transport through porcine nasal mucosa by Géraldine Samson; Alicia García de la Calera; Sophie Dupuis-Girod; Frédéric Faure; Evelyne Decullier; Gilles Paintaud; Céline Vignault; Jean-Yves Scoazec; Christine Pivot; Henri Plauchu; Fabrice Pirot (465-469).
Bevacizumab recovery throughout 2.5 h experiment in nasal cavity porcine mucosa mounted onto static vertical diffusion cells. Transmucosal absorption of bevacizumab as a function of time.Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder associated with abnormal angiogenesis and disabling epistaxis, for which bevacizumab is reported to be a new therapeutic option. In the present study, bevacizumab transport in porcine nasal mucosa was investigated to determine antibody bioavailability.Transmucosal absorption of bevacizumab was examined by using nasal mucosa specimens mounted onto static vertical diffusion cells then treated with bevacizumab solution (25 mg mL−1, 500 μg) for 2.5 h. Bevacizumab concentrations were measured by enzyme-linked immunosorbent assays. Mucosal integrity was examined by histological examination of treated mucosa.Transmucosal transport of bevacizumab followed a Fickian diffusion process (permeability coefficient: [0.63 ± 22] × 10−6  cm s−1; and steady-state flux: 56.4 ± 19.6 μg cm−2  h−1). Total recovery of bevacizumab throughout the 2.5 h experiment was 83% of the initial dose distributed (i) at the mucosal surface (263 ± 73 μg; ∼53%) and (ii) into (95 ± 14 μg; ∼19%) and through (56 ± 26 μg; ∼11%) the mucosa. There was no evidence of any noticeable histological effects, confirming the harmlessness of nasal bevacizumab delivery.In the present study, absorption of bevacizumab into nasal mucosa was demonstrated, providing new fundamentals that are mandatory for further clinical trials in HHT patients.
Keywords: Bevacizumab; Transmucosal transport; Pharmacokinetics; Monoclonal antibody; Hereditary hemorrhagic telangectasia;