European Journal of Pharmaceutics and Biopharmaceutics (v.74, #3)

APV Diary (S1).

Chitosan-modified poly(d,l-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects by Kohei Tahara; Hiromitsu Yamamoto; Naohide Hirashima; Yoshiaki Kawashima (421-426).
Chitosan (CS) surface-modified poly(d,l-lactide-co-glycolide) (PLGA) nanospheres (NS) for a siRNA delivery system were evaluated in vitro. siRNA-loaded PLGA NS were prepared by an emulsion solvent diffusion (ESD) method, and the physicochemical properties of NS were investigated. The level of targeted protein expression and siRNA uptake were examined in A549 cells. CS-modified PLGA NS exhibited much higher encapsulation efficiency than unmodified PLGA NS (plain-PLGA NS). CS-modified PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. siRNA uptake studies by observation with confocal leaser scanning microscopy (CLSM) indicated that siRNA-loaded CS-modified PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The efficiencies of different siRNA preparations were compared at the level of targeted protein expression. The gene-silencing efficiency of CS-modified PLGA NS was higher and more prolonged than those of plain-PLGA NS and naked siRNA. This result correlated with the CLSM studies, which may have been due to higher cellular uptake of CS-modified PLGA NS due to electrostatic interactions. It was concluded that CS-modified PLGA NS containing siRNA could provide an effective siRNA delivery system.
Keywords: siRNA; PLGA; Nanosphere; Chitosan; Emulsion solvent diffusion method;

CpG-free plasmid DNA prevents deterioration of pulmonary function in mice by Eugenia Lesina; Petra Dames; Andreas Flemmer; Kerstin Hajek; Thomas Kirchner; Iris Bittmann; Carsten Rudolph (427-434).
Nonviral gene vectors have been shown to be therapeutically effective in various animal models of inherited and acquired lung diseases. Although an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response has been previously observed for first-generation plasmids, its effect on pulmonary function has not been investigated to date. Here, we present data on lung functional parameters together with histopathology, cellular and inflammatory events in response to pulmonary administration of DNA-containing particles. We show that aerosol delivery of polyethylenimine gene vectors containing a first-generation CpG-rich plasmid induced an inflammatory response which was associated with a decrease in lung compliance. In contrast to these observations, aerosol application of CpG-free plasmid DNA prevented immune response and impairment of pulmonary function. These results demonstrate that aerosol delivery of CpG-free plasmid DNA is critical to avoid alteration of pulmonary function. Therefore, we suggest to use CpG-free pDNA for gene delivery to the lungs in future.
Keywords: CpG motif; Aerosol; Gene therapy; Nonviral; PEI; Gene transfer; Pulmonary function;

Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer by Yan-Shan Huang; Xiao-Fang Wen; Yi-Liang Wu; Ye-Fei Wang; Min Fan; Zhi-Yu Yang; Wei Liu; Lin-Fu Zhou (435-441).
The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications.
Keywords: Fusion protein; Gelatin; Granulocyte-colony-stimulating factor; Half-life; Pichia pastoris;

Efficacy of surface charge in targeting pegylated nanoparticles of sulpiride to the brain by Tapan Parikh; Murali Mohan Bommana; Emilio Squillante (442-450).
The objective of the study was to formulate sulpiride-loaded nanoparticles (NPs) that can improve bioretention and achieve dose reduction by passively targeting the drug near the site of action. Methoxy PEG–PLA and maleimide PEG–PLA were synthesized via ring opening polymerization of l-lactide and used to prepare pegylated nanoparticles (NPs) loaded with sulpiride by emulsification and solvent evaporation method. Thiolated cationized bovine serum albumin (CBSA) was conjugated through the maleimide function to the NPs. Rhodamine B and Alexa Fluor® 488 were used as fluorescent markers for nanoparticle uptake studies. The nanoparticles were characterized for particle size, zeta potential and drug loading. Sprague Dawley rats were administered with each of CBSA-NPs, BSA-NPs and uncoated NPs (10 mg/kg) via tail vein; plasma and urine concentrations were measured and tissue sections were observed under fluorescence microscope. Characterized particles (mean particle size 329 ± 44 nm) indicated the conjugation of cationic albumin to NPs (zeta potential shift from −39 mV to −19 mV). Fluorescence showed a high accumulation of CBSA-NPs in brain compared to that of BSA-NPs and uncoated NPs supported by plasma and urine profile. The significant results proved that CBSA-NPs could be a promising brain drug delivery for sulpiride.
Keywords: Adsorption; Albumin; Blood–brain barrier; Cationized bovine serum albumin; CNS; Drug targeting; Pegylation; Microscopy; Nanoparticles; Sulpiride; Surface charge;

Detection of early colorectal cancer imaged with peanut agglutinin-immobilized fluorescent nanospheres having surface poly(N-vinylacetamide) chains by Shinji Sakuma; Takanori Yano; Yoshie Masaoka; Makoto Kataoka; Ken-ichiro Hiwatari; Hiroyuki Tachikawa; Yoshikazu Shoji; Ryoji Kimura; Huaiyu Ma; Zhijian Yang; Li Tang; Robert M. Hoffman; Shinji Yamashita (451-460).
Peanut agglutinin (PNA)-immobilized fluorescent nanospheres were designed as a novel imaging agent for colonoscopy. PNA is a targeting moiety that binds to β-d-galactosyl-(1–3)-N-acetyl-d-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The in vivo performance of the imaging agent was evaluated using a human colorectal cancer orthotopic animal model. Human colorectal adenocarcinoma cell lines, HT-29, HCT-116, and LS174T, were implanted on the cecal serosa of immune-deficient mice. A loop of the tumor-bearing cecum was made, and the luminal side was treated with the imaging agent. Strong fluorescence was observed at several sites of the cecal mucosa, irrespective of cancer cell type. Microscopic histological evaluation of the cecal mucosa revealed that bright areas with fluorescence derived from the imaging agent and dark areas without the fluorescence well denoted the presence and absence, respectively, of the invasion of implanted cancer cells on the mucosal side. This good correlation showed that PNA-immobilized fluorescent nanospheres recognized millimeter-sized tumors on the cecal mucosa with high affinity and specificity.
Keywords: Imaging; Endoscopic imaging agent; Colorectal cancer; Peanut agglutinin; Poly(N-vinylacetamide);

Physical properties of griseofulvin-lipid nanoparticles in suspension and their novel interaction mechanism with saccharide during freeze-drying by Seitaro Kamiya; Takurou Kurita; Atsuo Miyagishima; Shigeru Itai; Masayuki Arakawa (461-466).
Size reduction of drug particles to the nanoscale is important in improving the dissolution rate of poorly water-soluble drugs. The aim of this study was to investigate the physicochemical properties of griseofulvin (GF)-lipid nanoparticles and the interactions between GF-lipid nanoparticles and various saccharides during freeze-drying. The phase transition temperature of the GF-lipid nanoparticle suspension was 56.8 °C, whereas that of the lipid nanoparticle suspension alone was 57.9 °C, indicating that the GF crystals were incorporated into the lipid phase. The mean particle size of a rehydrated suspension of xylose-containing freeze-dried GF-lipid nanoparticles was about 220 nm. However, the mean particle size on the rehydration of nanoparticles containing mannose (monosaccharide), fructose (disaccharide), lactose (disaccharide), or raffinose (trisaccharide) was about 60 nm, suggesting that these saccharides prevented aggregation during the freeze-drying process. Powder X-ray diffraction revealed that xylose existed in the crystalline state in the freeze-dried nanoparticles, whereas the other saccharides existed in amorphous states. Thus, the crystallization of the saccharide was found to be strongly correlated with the aggregation property of the nanoparticles. In the case of freeze-dried xylose, the nanoparticles were squeezed out as the saccharine crystal lattice arranged itself regularly. Then, the ejected nanoparticles were aggregated. In contrast, in the case of the other freeze-dried saccharide, the saccharide remained incorporated with the GF-lipid nanoparticles because its crystal lattice was arranged irregularly. Thus, the particle size was maintained.
Keywords: Nanoparticles; Saccharide; Powder X-ray diffraction; Freeze-drying; Crystal lattice; Amorphous;

Arg–Gly–Asp (RGD) modified doxorubicin-loaded liposomes could improve anticancer effect, and vascular disrupting agents (VDAs) could induce a rapid and selective shutdown of the blood vessels of tumors. We propose that RGD-modified liposomes for co-encapsulation and sequential release of vascular disrupting agent combretastatin A-4 (CA-4) and cytotoxic agent doxorubicin (Dox) could enhance tumor inhibition responses. In this study, we encapsulated Dox and CA-4 in RGD-modified liposomes. The release rate of Dox was proved to be much slower than that of CA-4 in vitro. Flow cytometry and laser confocal scanning microscopy clearly showed that RGD-modification promoted intracellular uptake of liposomal drugs by B16/B16F10 melanoma tumor cells and human umbilical vein endothelial cells (HUVECs). Cytotoxicity assay showed that the IC50 of RGD-modified liposomes was lower than that of the corresponding unmodified liposomes. Therapeutic benefits were examined on B16F10 melanoma tumors subcutaneously growing in C57BL/6 mice. In vivo study demonstrated that RGD-modified liposomes exhibited the most pronounced tumor regression effect when both CA-4 and Dox were co-encapsulated. These results suggest that the targeted drug delivery system for co-encapsulation of vascular disrupting agents and anticancer agents may be a promising strategy for cancer treatment.
Keywords: Combination therapy; Liposomes; Targeting; Combretastatin A-4; Doxorubicin;

Nanostructured lipid carriers (NLCs) made from mixtures of Precirol and squalene were prepared to investigate whether the bioavailability of lovastatin can be improved by oral delivery. The size, zeta potential, drug-loading capacity, and release properties of the NLCs were compared with those of lipid nanoparticles containing pure Precirol (solid lipid nanoparticles, SLNs) and squalene (lipid emulsions, LEs). Stable nanoparticles with a mean size range of 180–290 nm and zeta potential range of −3 to −35 mV were developed. More than 70% lovastatin was entrapped in the NLCs and LEs, which was significantly higher compared to the SLNs. The in vitro release kinetics demonstrated that lovastatin release could be reduced by up to 60% with lipid nanoparticles containing Myverol as the lipophilic emulsifier, which showed a decreasing order of NLCs > LEs > SLNs. Drug release was further decreased by soybean phosphatidylcholine (SPC) incorporation, with NLCs and SLNs showing the slowest delivery. The oral lovastatin bioavailability was enhanced from 4% to 24% and 13% when the drug was administered from NLCs containing Myverol and SPC, respectively. The in vivo real-time bioluminescence imaging indicated superior stability of the Myverol system over the SPC system in the gastric environment.
Keywords: Lovastatin; Lipid nanoparticles; Nanostructured lipid carriers; Oral administration; Emulsifier; Pharmacokinetics;

Cytotoxicity study of ordered mesoporous silica MCM-41 and SBA-15 microparticles on Caco-2 cells by Teemu Heikkilä; Hélder A. Santos; Narendra Kumar; Dmitry Yu. Murzin; Jarno Salonen; Timo Laaksonen; Leena Peltonen; Jouni Hirvonen; Vesa-Pekka Lehto (483-494).
Cytotoxicity of ordered mesoporous silica MCM-41 and SBA-15 microparticles (fractions between 1 and 160 μm) was determined in vitro on undifferentiated human colon carcinoma (Caco-2) cell line, considering the feasibility of using these silica-based materials in oral drug formulations. The cellular endpoints employed for assessing the effects of the MCM-41 and SBA-15 microparticles on Caco-2 were: (1) cell membrane integrity by monitoring live-cell protease activity (AFC) and by employing the flow cytometry method; (2) metabolic activity by monitoring total ATP content via luminescence assay; (3) activity of apoptotic effectors by caspase-3/7 activity assay. The generation of reactive oxygen species (ROS) was also followed, specifically the hydrogen peroxide (H2O2) and the superoxide radical ( O 2 • - ). MCM-41 and SBA-15 microparticles caused cytotoxic effects on the Caco-2 cells, at most tested concentrations (0.2–14 mg/ml) and incubation times (3 and 24 h). The effects on the cells included weakened cell membrane integrity, diminished cell metabolism and increased apoptotic signalling. The root cause for the cytotoxicity was heightened production of reactive oxygen species (ROS), especially the formation of the superoxide radical O 2 • - already after 3 h incubation with threshold dose 1 mg/ml, apparently overwhelming the antioxidant defences and causing mitochondrial dysfunction, hence increasing the apoptotic signalling.
Keywords: Mesoporous silica; Microparticle; Cytotoxicity; Cell viability; Biocompatibility; Caco-2; Oral drug delivery;

Evaluation of various PAMPA models to identify the most discriminating method for the prediction of BBB permeability by Jurgen Mensch; Anouche Melis; Claire Mackie; Geert Verreck; Marcus E. Brewster; Patrick Augustijns (495-502).
The Parallel Artificial Membrane Permeability Assay (PAMPA) has been successfully introduced into the pharmaceutical industry to allow useful predictions of passive oral absorption. Over the last 5 years, researchers have modified the PAMPA such that it can also evaluate passive blood–brain barrier (BBB) permeability. This paper compares the permeability of 19 structurally diverse, commercially available drugs assessed in four different PAMPA models: (1) a PAMPA-BLM (black lipid membrane) model, (2) a PAMPA-DS (Double Sink) model, (3) a PAMPA-BBB model and (4) a PAMPA-BBB-UWL (unstirred water layer) model in order to find the most discriminating method for the prediction of BBB permeability. Both the PAMPA-BBB model and the PAMPA-BLM model accurately identified compounds which pass the BBB (BBB+) and those which poorly penetrate the BBB (BBB−). For these models, BBB+ and BBB− classification ranges, in terms of permeability values, could be defined, offering the opportunity to validate the paradigm with in vivo data.The PAMPA models were subsequently applied to a set of 14 structurally diverse internal J&J candidates with known log (brain/blood concentration) (Log BB) values. Based on these Log BB values, BBB classifications were established (BBB+: Log BB ⩾ 0; BBB−: Log BB < 0). PAMPA-BLM resulted in three false positive identifications, while PAMPA-BBB misclassified only one compound. Additionally, a Caco-2 assay was performed to determine the efflux ratio of all compounds in the test set. The false positive that occurred in both models was shown to be related to an increased efflux ratio. Both the PAMPA-BLM and the PAMPA-BBB models can be used to predict BBB permeability of compounds in combination with an assay that provides p-gp efflux data, such as the Caco-2 assay.
Keywords: Permeability; Solubility; PAMPA; Caco-2; Efflux ratio; Log BB; Blood–brain barrier;

The one-step preparation of sustained release matrix pellets, using a melting procedure in a fluidized bed apparatus, was tested in a 23 full factorial design of experiments, using microcrystalline wax as lipophilic binder, theophylline as model drug and talc as additional matrix forming agent. The three influence parameters were (A) size of binder particles, (B) fraction of theophylline in solid particles and (C) fraction of microcrystalline wax in formulation. The response variables were agglomerate size and size distribution, dissolution time, agglomerate crush resistance, sphericity, yield and porosity.Nearly spherical pellets comprising a smooth, closed surface could be obtained with the used method, exhibiting the hollow core typical for the immersion and layering mechanism.The reproducibility was very good concerning all responses.The size of agglomerates is proportional to the size of the binder particles, which serve as cores for pellet formation in the molten state in the fluidized bed. Additionally, the agglomerate size is influenced by the volume of the solid particles in relation to the binder particles, with more solid particles leading to larger agglomerates and vice versa. Dissolution times vary in a very wide range, resulting from the interplay between amount of drug in relation to the meltable matrix substance microcrystalline wax and the non-meltable matrix substance talc.The change of binder particle size does not lead to a structural change of the matrix; both dissolution times and porosity are not significantly altered.Agglomerate crush resistance is low due to the hollow core of the pellets. However, it is significantly increased if the volume fraction of microcrystalline wax in the matrix is high, which means that the matrix is mechanically better stabilized.A theoretical model has been established to quantitatively explain agglomerate growth and very good accordance of the full particle size distributions between predicted and actual values could be shown. A low volumetric binder to solids ratio is compensated by a more porous layer. On the basis of this model, in-depth understanding on the mechanism and influence of product properties could be gained; and an a priori estimation of particle size distributions for new formulas can be performed, with densities, formula, and binder particle size distribution as input parameters.
Keywords: Fluidized bed; Melt agglomeration; Matrix; Pellets; Sustained release; Growth mechanism; Microcrystalline wax;

There is mounting evidence that the permeability coefficients (kp ) that describe any given transdermal drug permeation process generally follow some form of positively skewed, non-symmetrical distribution rather than a simple normal distribution. Yet a suitable theoretical treatment of this area has not been undertaken to date. In this paper, we describe a two-layer model that can explain five drugs’kp variabilities as measured in two previously published papers. The model shows why rapidly permeating drugs would tend to exhibit more symmetrical kp distributions while progressively more slowly permeating drugs would tend to exhibit progressively more positively skewed kp distributions. Future research should take this effect into account when comparing the flux variabilities of hydrophilic and lipophilic drugs.
Keywords: Transdermal; Flux; Permeation; Variability; Skewness; Two-layer model;