International Journal of Pharmaceutics (v.406, #1-2)

Influence of particle size on regional lung deposition – What evidence is there? by Thiago C. Carvalho; Jay I. Peters; Robert O. Williams (1-10).
The understanding of deposition of particles in the respiratory tract is of great value to risk assessment of inhalation toxicology and to improve efficiency in drug delivery of inhalation therapies. There are three main basic mechanisms of particle deposition based primarily on particle size: inertial impaction, sedimentation and diffusion. The regional deposition in the lungs can be evaluated in regards to the aerodynamic particle size, in which particle density plays a significant role. In this review paper, we first introduce the available imaging techniques to confirm regional deposition of particles in the human respiratory tract, such as planar scintigraphy, single photon emission computed tomography (SPECT) and positron emission tomography (PET). These technologies have widely advanced and consequently benefited the understanding of deposition pattern, although there is a lack of lung dosimetry techniques to evaluate the deposition of nanoparticles. Subsequently, we present a comprehensive review summarizing the evidence available in the literature that confirms the deposition of smaller particles in the smaller airways as opposed to the larger airways.
Keywords: Inhalation therapy; Regional lung deposition; Particle size distribution; Aerodynamic diameter; Imaging techniques;

Zinc–pectin–chitosan composite microparticles were designed and developed as colon-specific carrier. Resveratrol was used as model drug due to its potential activity on colon diseases. Formulations were produced by varying different formulation parameters (cross-linking pH, chitosan concentration, cross-linking time, molecular weight of chitosan, and drug concentration). Single-step formulation technique was compared with multi-step technique. Effect of these parameters was investigated on shape, size, weight, weight loss (WL), moisture content (MC), encapsulation efficiency (EE), drug loading (L), and drug release pattern of the microparticles. The formulation conditions were optimized from the drug release study. In vivo pharmacokinetics of the zinc-pectinate particles was compared with the zinc–pectin–chitosan composite particles in rats. Formulations were spherical with 920.48–1107.56 μm size, 21.19–24.27 mg weight of 50 particles, 89.83–94.34% WL, 8.31–13.25% MC, 96.95–98.85% EE, and 17.82–48.31% L. Formulation parameters showed significant influence on drug release pattern from the formulations. Formulation prepared at pH 1.5, 1% chitosan, 120 min cross-linking time, and pectin:drug at 3:1 ratio demonstrated colon-specific drug release. Microparticles were stable at 4 °C and room temperature. Pharmacokinetic study indicated in vivo colon-specific drug release from the zinc–pectin–chitosan composite particles only.
Keywords: Pectin; Chitosan; Zinc; Drug release; Colon;

NP-647, a novel TRH analogue: Investigating physicochemical parameters critical for its oral and parenteral delivery by Kailas Khomane; Lokesh Kumar; Chhuttan Lal Meena; Rahul Jain; Arvind Bansal (21-30).
NP-647 (l-pGlu-(2-propyl)-l-His-l-ProNH2) is a novel thyrotropin releasing hormone (TRH) analogue, with potential antiepileptic activity. In the present study, the physicochemical parameters of NP-647, including its solid state properties, dissociation constant, partition coefficient, solubility (intrinsic solubility and pH-solubility profile) and stability (gastrointestinal enzymatic stability, pH-stability profile and temperature stability) were investigated for their criticality for oral and parenteral delivery. NP-647 was characterized as amorphous material having glass transition temperature of 66.73 °C at 50% RH. It was found very hygroscopic with deliquescent in nature. pK a of the compound, as determined using potentiometric titration, was found to be 7.2 ± 0.02 (basic). Intrinsic solubility and pH-solubility behavior were determined using dissolution titration template method. NP-647 has intrinsic solubility of 2.4 ± 0.01 mg mL−1. Partition/distribution studies indicate that NP-647 has a low log  P (−1.07 ± 0.06) and log  D 7.4 (−1.20 ± 0.02), characteristic of hydrophilic molecule. It was found most stable in tartrate buffer of pH of 5.0. Arrhenius plot of NP-647 suggest its half life of ∼3.2 years and shelf life of ∼6 months. These studies conclude that amorphous nature of NP-647 with deliquescent property will be critical in its solid oral dosage formulation and need to be investigated further.
Keywords: Preformulation; Consensus approach; Physicochemical properties; TRH analogues; Dissolution titration template; Peptide delivery;

A phenomenon was observed for the behavior of hypromellose matrices during dissolution. The tablet laminated radially, with both edges curled outwards, forming a “butterfly” shape. The butterfly effect is thus coined to describe this behavior. Due to the flamboyant shape assumed by the hydrated matrix, the apparent surface area for drug release was significantly increased. This study attempted to elucidate mechanistically the cause of this butterfly effect. Two formative mechanisms were proposed based on the behavior of moving solvent fronts and the anisotropic expansion of materials in solution. It was hypothesized that the particle size of hypromellose, applied compaction force used and proportions of both insoluble and soluble excipients contributed to the butterfly effect. The influence of the expanded shape on the mechanism and rate of drug release was also investigated. Matrix formulation was an important factor. Greater drug release was observed when the butterfly-shaped hydrated matrix was formed. The drug release profiles generally fitted the Higuchi and Korsmeyer–Peppas equations, indicating a combination of both diffusion and erosional drug release mechanisms. A combination of both fine and coarse hypromellose size fractions and adequate compaction force (more than 3 kN) were necessary for the manifestation of the butterfly effect.
Keywords: Hypromellose tablet matrix; Butterfly effect; Tablet behavior during dissolution; Compaction force; Hypromellose particle size;

Carvedilol dissolution improvement by preparation of solid dispersions with porous silica by Odon Planinšek; Borut Kovačič; Franc Vrečer (41-48).
Impregnation of porous SiO2 (Sylysia) with carvedilol from acetone solution was used to improve dissolution of this poorly water-soluble drug. Solvent evaporation in a vacuum evaporator and adsorption from acetone solution were the methods used to load various amounts of carvedilol into the Sylysia pores. The impregnated carriers were characterized using nitrogen-adsorption experiments, X-ray diffraction, wettability measurements, attenuated total reflectance FTIR spectroscopy and thermal analysis. The impregnation procedures resulted in a significant improvement of drug release compared to dissolution of pure carvedilol or its physical mixtures with Sylysia. The results showed that when the drug precipitated in a thin layer within the carrier the dispersion retained a high specific surface area, micropore volume, and drug-release rate from the solid dispersion. Increasing the amount of drug in the solid dispersion caused particle precipitation within the pores that decreased the carrier's specific surface area and pore volume and decreased the release rate of the drug. The results also suggest that the amorphous form of carvedilol, the improved wettability and weak interactions between the drug and carrier in the solid dispersion also contribute to improved dissolution of the drug from the dispersion.
Keywords: Carvedilol; Solid dispersion; Dissolution rate improvement; Porous SiO2;

Differential expression of organic cation transporters in normal and polyps human nasal epithelium: Implications for in vitro drug delivery studies by Remigius Agu; Chris MacDonald; Elizabeth Cowley; Di Shao; Ken Renton; David B. Clarke; Emad Massoud (49-54).
The aim of this study was to compare the expression of organic cation transporters (OCTs) in normal and polyps nasal epithelium. Primary cell cultures of human nasal epithelium (polyps and normal tissues) were compared by investigating the uptake of a fluorescent organic cation, [4-dimethylaminostyryl-N-methylpyridinium (4-Di-1-ASP)]. The effect of concentration, temperature, pH and competing inhibitors were investigated. Quantitative polymerase chain reaction (qPCR) was used to compare the OCTs gene expression levels in the cells. The K m (μM) and V max (μM/mg protein/15 min) for 4-Di-1-ASP uptake were higher in normal (K m  = 3031 ± 559.6, V max  = 70.8 ± 8.8) cells compared to polyps (K m  = 952.4 ± 207.8, V max  = 30.9 ± 2.1). qPCR results showed that OCT1-3 and organic cation/carnitine transporter 1-2 gene transcripts (OCTN1-2) were expressed in both normal and polyps cells at comparable levels, with OCT-3 having the highest expression level in both cultures. Kruskal–Wallis ANOVA showed that pH and specific inhibitors had similar effects on both normal and polyps cells (p  > 0.5). Similarly, OCTs and OCTNs gene expression levels were similar. This study showed that polyps biopsies can be used for isolating cells to study organic cation transporters in human nasal epithelium as no major functional or molecular differences relative to normal cells could be found.
Keywords: Organic cation transporters; Nasal epithelium; Polyps; Drug transport; Drug uptake; Tissue culture; Drug delivery;

Investigating the effect of dehydration conditions on the compactability of glucose by Niraj S. Trasi; Stephan X.M. Boerrigter; Stephen R. Byrn; Teresa M. Carvajal (55-61).
Hydrates are commonly found in pharmaceutical ingredients either in excipients or in the active pharmaceutical ingredient form. There is always the possibility that the processing involved in manufacturing can result in the dehydration of the hydrate components. It has been seen that different dehydration conditions can have an effect on the behavior of the final product; however this area has not been fully investigated. In this work, glucose monohydrate powder was dehydrated at four different conditions and then compressed to see the effect on the hardness of the compacts.Various analytical tools such as inverse gas chromatography, differential scanning calorimetry, X-ray powder diffractometry and scanning electron microscopy were used to determine any differences in the properties of the dehydrates and correlated with the obtained compact hardness.Annealing studies were performed to determine the effect of storage on the dehydrated materials both before and after compression. It was observed that while annealing of the powders did have an impact, annealing of the compacts did not influence the hardness. The results of the characterization and annealing studies showed that the difference in the behavior of glucose dehydrates were due to the presence of amorphous regions within the particulates.
Keywords: Glucose; Dehydration; Compaction; Amorphous; Hardness;

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa by Bitten Plesner; Peter Westh; Anders D. Nielsen (62-68).
The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated® recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature T m measured by differential scanning calorimetry (DSC) and the temperature of aggregation, T agg, measured by light scattering (LS) both increased with GlycoPEGylation. Both T m and T agg were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure), slightly stabilised thermally (unfolding temperature) and stabilised kinetically (temperature of aggregation).
Keywords: GlycoPEGylation; rFVIIa; Circular dichroism; Differential scanning calorimetry; Light scattering; Stability;

Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with phospholipid bilayers and monolayers by Maria Grazia Sarpietro; Sara Ottimo; Maria Chiara Giuffrida; Flavio Rocco; Maurizio Ceruti; Francesco Castelli (69-77).
Cytarabine (1-β-d-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1′,2-tris-nor-Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the environment of biological membranes, as well as of lipophilic carriers.The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and monolayers has been studied by the differential scanning calorimetry and the Langmuir–Blodgett techniques. The interaction has been evaluated considering the effect of the compounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as carriers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers with respect to the free drug.
Keywords: Cytarabine; n-Squalenoyl cytarabine; Prodrug; DSC; Langmuir–Blodgett; Biomembrane model;

A nuclear magnetic resonance approach to the comparison of mucoadhesive properties of polysaccharides for ophthalmic uses by Gloria Uccello-Barretta; Samuele Nazzi; Federica Balzano; Marco Sansò (78-83).
Mucoadhesive properties of tamarind seed polysaccharide (TSP) and larch arabinogalactan (AG), which are developed for ophthalmic applications, were investigated by NMR spectroscopy. Polysaccharide to mucin affinities were compared by using ketotifen fumarate as low molecular weight interaction probe. Proton selective relaxation rate measurements revealed enhanced affinity of TSP to mucin with respect to AG.
Keywords: Nuclear magnetic resonance; Polymer interaction; Mucoadhesion; Eye-drop excipient;

Preparation and in vitro–in vivo evaluation of double layer coated and matrix sustained release pellet formulations of diclofenac potassium by Jijun Fu; Xiaoli Wang; Lishuang Xu; Jia Meng; Yan Weng; Guofei Li; Haibing He; Xing Tang (84-90).
The purpose of the present study was to prepare matrix extended release pellets of diclofenac potassium using low amount of release-modifying agents and, to compare its performance in vivo with coated pellets and matrix tablets. Coated pellets were prepared by extrusion–spheronization, followed by double layer coating using different polymers separately. Matrix pellets with different release rate in vitro were prepared by extrusion–spheronization with different kinds of retarding materials. Bioavailability study of different coated pellets revealed that the drug concentration in plasma of beagle dogs was too low to be detected and, implied that the drug was nearly not released from the preparations before reaching colon due to the appearance of lag time in the dissolution process. The phenomenon indicated that slow-release pellets of diclofenac potassium perhaps should not be developed as double membrane-controlled type. The AUC (0→24) of the immediate release pellets, the two matrix pellets and the reference were 304.4, 87.7, 204.1 and 179.1 μg h/ml, respectively. The C max of the formulations mentioned above were 46.3, 13.0, 33.6 and 32.1 μg/ml, respectively. All the matrix formulations, including the reference, exhibited incomplete absorption due to the short small intestine transit time and termination of the drug release in the colon because of its limited solubility. The matrix pellets were bioequivalent with the commercially available tablet (Voltaren®) although the drug release in vitro of the former was much faster, while the bioavailability of the matrix pellets with similar in vitro drug release to the reference (Voltaren®) was much lower than the latter. The results perhaps was caused by lacking of physical robustness in the waxy tablet formulation, resulted in low wet strength and easily destroyed by the mechanical destructive forces and finally introduced faster drug release rate in vivo. It is apparent that preparations with similar performance in vitro may differ a lot in vivo because of the differences in drug release rate in vivo owing to various wet strengths of excipients contained, especially for sustained release products.
Keywords: Diclofenac potassium; Sustained release; Pellets; Bioavailability; Matrix;

Swelling kinetics, ionic conductivity and electrically assisted solute permeation (theophylline, methylene blue and fluorescein sodium) of poly(ethylene glycol) (PEG) crosslinked poly(methyl vinyl ether-co-maleic acid) (PMVE/MA) hydrogels are presented. The effects of PMVE/MA concentration and PEG molecular weight (MW) on swelling behaviour and network parameters were investigated in phosphate buffered saline (pH 7.4). The percentage swelling of hydrogels increased, and the crosslink density decreased, with a decrease in PMVE/MA content and with an increase in PEG MW. The ionic conductivity of the formulation was found to increase with an increase in PEG MW. The application of an electrical current led to a significant enhancement in the rate and extent of solute permeation across the swollen hydrogels. Furthermore, it was found that the extent of solute permeation enhancement following current application was dependent upon the crosslink density and ionic conductivity of the formulation. In general, a decrease in crosslink density and an increase in ionic conductivity led to a greater enhancement in solute permeation following current application. The electro-responsive nature of these hydrogels suggests that have a potential application in electrically controlled drug delivery systems.
Keywords: Poly(methyl vinyl ether-co-maleic acid); Poly(ethylene glycol); Hydrogel; Network parameters; Electro-responsive drug delivery;

A bio-activity guided in vitro pharmacokinetic method to improve the quality control of Chinese medicines, application to Si Wu Tang by Ling Wang; Zhijun Wang; Siukwan Wo; Clara B.S. Lau; Xiao Chen; Min Huang; Vincent H.L. Lee; Moses S.S. Chow; Zhong Zuo (99-105).
The purpose of this study was to demonstrate the feasibility of using a bio-activity guided in vitro pharmacokinetic (BAPK) method in identifying relevant (absorbable and bioactive) markers for quality control (QC) of Chinese medicines (CM), using Si Wu Tang (SWT), a popular CM for women's health, as an example.A stepwise BAPK approach was utilized for relevant marker determination and evaluating of six SWT products: (1) data mining to identify active components of SWT, (2) quantification of the identified active components in each SWT product, (3) determination of in vitro dissolution and metabolism of the components under simulated gastrointestinal conditions, (4) identification of absorbable components or marker(s) via in vitro Caco-2 cell model, (5) stability testing of the permeable marker(s).Our results showed considerable variations in the amount of active components in different SWT products. Of the nine active components identified from data mining, three (ferulic acid, ligustilide, senkyunolide A) were found to be well permeated and stable over three months. Paeoniflorin, the marker designated by Chinese Pharmacopoeia, was poorly permeable and thus could not be considered a relevant marker for SWT. Our preliminary evaluation of the BAPK method appears to be feasible and may offer as a useful approach for identifying relevant markers of other TCM products in the future.
Keywords: Si Wu Tang; Chinese medicine; Quality control; Paeoniflorin; Ligustilide; Ferulic acid;

Effect of casting solvent on crystallinity of ondansetron in transdermal films by Satyanarayan Pattnaik; Kalpana Swain; Subrata Mallick; Zhiqun Lin (106-110).
The purpose of the present investigation is to assess the influence of casting solvent on crystallinity of ondansetron hydrochloride in transdermal polymeric matrix films fabricated using povidone and ethyl cellulose as matrix forming polymers. Various casting solvents like chloroform (CHL), dichloromethane (DCM), methanol (MET); and mixture of chloroform and ethanol (C-ETH) were used for fabrication of the transdermal films. Analytical tools like scanning electron microscopy (SEM), X-ray diffraction (XRD) studies, differential scanning calorimetry (DSC), etc. were utilized to characterize the crystalline state of ondansetron in the film. Recrystallisation was observed in all the transdermal films fabricated using the casting solvents other than chloroform. Long thin slab-looking, long wire-like or spherulite-looking crystals with beautiful impinged boundaries were observed in SEM. Moreover, XRD revealed no crystalline peaks of ondansetron hydrochloride in the transdermal films prepared using chloroform as casting solvent. The significantly decreased intensity and sharpness of the DSC endothermic peaks corresponding to the melting point of ondansetron in the formulation (specifically in CHL) indicated partial dissolution of ondansetron crystals in the polymeric films. The employed analytical tools suggested chloroform as a preferred casting solvent with minimum or practically absence of recrystallization indicating a relatively amorphous state of ondansetron in transdermal films.
Keywords: Ondansetron hydrochloride; Transdermal delivery; Polymeric system; Casting solvent;

Opto-Thermal Transient Emission Radiometry (OTTER) to image diffusion in nails in vivo by P. Xiao; X. Zheng; R.E. Imhof; K. Hirata; W.J. McAuley; R. Mateus; J. Hadgraft; M.E. Lane (111-113).
This work describes the first application of Opto-Thermal Transient Emission Radiometry (OTTER), an infrared remote sensing technique, to probe the extent to which solvents permeate the human nail in vivo. Decanol, glycerol and butyl acetate were selected as model solvents. After application of the solvents, individually, to human volunteers, OTTER was used to depth profile the solvents. The permeation rate of the solvents was ranked as glycerol > decanol > butyl acetate. It is possible that some of the butyl acetate may have evaporated during the experiment. The ability of decanol to extract lipids from biological tissue is also considered. These preliminary results demonstrate the potential of OTTER as a tool to identify optimal excipients with which to target drugs to the nail.
Keywords: Opto-Thermal Transient Emission Radiometry; Nail; Depth profile; Solvents;

Lipid membrane composition influences drug release from dioleoylphosphatidylethanolamine-based liposomes on exposure to ultrasound by Tove J. Evjen; Esben A. Nilssen; Robert A. Fowler; Sibylla Røgnvaldsson; Martin Brandl; Sigrid L. Fossheim (114-116).
The effect of membrane composition on calcein release from dioleoylphosphatidylethanolamine (DOPE)-based liposomes on exposure to low doses of 1.13 MHz focused ultrasound (US) was investigated by multivariate analysis, with the goal of designing liposomes for US-mediated drug delivery. Regression analysis revealed a strong correlation between sonosensitivity and the non-bilayer forming lipids DOPE and pegylated distearoylphosphatidylethanolamine (DSPE-PEG 2000), with DOPE having the strongest impact. Unlike most of the previously studied distearoylphosphatidylethanolamine (DSPE)-based liposomes, all the current DOPE-based liposome formulations were found stable in 20% serum in terms of drug retention.
Keywords: DOPE; Drug release; Ultrasound; Liposomes; Calcein;

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex by Akira Saheki; Yoshiharu Fukui; Yuji Hoshi; Hiroshi Fukui; Junzo Seki; Ikumi Tamai (117-121).
Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5–7.5 in the presence of NaCl at higher concentration than 50 mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.
Keywords: Lipoplex; Polynucleotide; Liposome; Conformation; Poly(I); Poly(C);

Characterization, blood profile and biodistribution properties of surface modified PLGA nanoparticles of SN-38 by Pedram Ebrahimnejad; Rassoul Dinarvand; Mahmoud Reza Jafari; Seyed Abolghasem Sajadi Tabasi; Fatemeh Atyabi (122-127).
SN-38, the active metabolite of irinotecan, poses a challenge in terms of drug delivery due to its low solubility and labile lactone ring. The aim of this study was to develop a SN-38 nanoparticulate delivery system to evaluate the in vivo blood profile and biodistribution properties of nanoparticles (NPs).Poly lactide-co-glycolide (PLGA) NPs that were covalently bound to polyethylene glycol-folate (PEG-FOL) were prepared, and their in vivo biodistribution in rats was investigated. Either the SN-38 solution or SN-38 NP suspension was administered intravenously into the tail vein at a dose of 2 mg SN-38 eq./kg. As expected, SN-38 NPs showed a higher plasma concentration in vivo when compared with free SN-38 during a 24 h period. Compared with the SN-38 solution, both folate targeted and non-targeted NPs exhibited superior drug concentration in body organs such as the liver, spleen, and lung at 1 and 8 h post-administration.
Keywords: SN-38; Nanoparticle; PLGA; Pegylation; Folate targeting; Biodistribution; Nanotechnology;

Interaction of folate-conjugated human serum albumin (HSA) nanoparticles with tumour cells by Karsten Ulbrich; Martin Michaelis; Florian Rothweiler; Thomas Knobloch; Patchima Sithisarn; Jindrich Cinatl; Jörg Kreuter (128-134).
Folic acid has been previously demonstrated to mediate intracellular nanoparticle uptake. Here, we investigated cellular uptake of folic acid-conjugated human serum albumin nanoparticles (HSA NPs). HSA NPs were prepared by desolvation and stabilised by chemical cross-linking with glutaraldehyde. Folic acid was covalently coupled to amino groups on the surface of HSA NPs by carbodiimide reaction. Preparation resulted in spherical HSA NPs with diameters of 239 ± 26 nm. As shown by size exclusion chromatography, 7.40 ± 0.90 μg folate was bound per mg HSA NPs. Cellular NP binding and uptake were studied in primary normal human foreskin fibroblasts (HFFs), the human neuroblastoma cell line UKF-NB-3, and the rat glioblastoma cell line 101/8 by fluorescence spectrophotometry, flow cytometry, and confocal laser scanning microscopy. Covalent conjugation of folic acid to HSA NPs increased NP uptake into cancer cells but not into HFFs. Free folic acid interfered with cancer cell uptake of folic acid-conjugated HSA NPs but not with uptake of folic acid-conjugated HSA NPs into HFFs. These data suggest that covalent linkage of folic acid can specifically increase cancer cell HSA NP uptake.
Keywords: Nanoparticles; Human serum albumin (HSA); Folic acid; Folate receptor; Cancer targeting;

Phospholipid–polyaspartamide micelles for pulmonary delivery of corticosteroids by Emanuela Fabiola Craparo; Girolamo Teresi; Maria Luisa Bondi’; Mariano Licciardi; Gennara Cavallaro (135-144).
A novel drug delivery system for beclomethasone dipropionate (BDP) has been constructed through self-assembly of a pegylated phospholipid–polyaminoacid conjugate. This copolymer was obtained by chemical reaction of α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)2000] (DSPE-PEG2000-NH2). Benefiting from the amphiphilic structure with the hydrophilic shell based on both PHEA and PEG and many hydrophobic stearoyl tails, PHEA-PEG2000-DSPE copolymer was able to self assemble into micelles in aqueous media above a concentration of 1.23 × 10−7  M, determined by fluorescence studies. During the self-assembling process in aqueous solution, these structures were able to incorporate BDP, with a drug loading (DL) equal to 3.0 wt%. Once the empty and BDP-loaded micelles were prepared, a deep physicochemical characterization was carried out, including the evaluation of mean size, PDI, ζ potential, morphology and storage stability. Moreover, the excellent biocompatibility of both empty and drug-loaded systems was evaluated either on human bronchial epithelium (16HBE) or on red blood cells. The cellular uptake of BDP, free or blended into PHEA-PEG2000-DSPE micelles, was also evaluated, evidencing a high drug internalization when entrapped into these nanocarriers and demonstrating their potential for delivering hydrophobic drugs in the treatment of pulmonary diseases.
Keywords: α,β-Poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)2000] (DSPE-PEG2000-NH2); Polymeric micelles; Drug delivery; Beclomethasone dipropionate (BDP); Pulmonary diseases;

Physical stability of nanosuspensions: Investigation of the role of stabilizers on Ostwald ripening by Sudhir Verma; Sumit Kumar; Rajeev Gokhale; Diane J. Burgess (145-152).
The effect of stabilizer type and the amount of micellar solubilized drug on Ostwald ripening of nanosuspensions was investigated. The most probable mechanism for Ostwald ripening was found to be surface nucleation controlled.The effect of stabilizer type (small molecule vs. polymeric) and the amount of micellar solubilized drug on Ostwald ripening of nanosuspensions was investigated. Indomethacin nanosuspensions were prepared with small molecule stabilizers (sodium lauryl sulfate (SLS) and Dowfax 2A1 (DF)) and a polymeric stabilizer (hydroxypropyl methyl cellulose (HPMC)). Two different drug:stabilizer ratios were used to evaluate the effect of micellar solubilized drug. The Ostwald ripening potential of nanosuspensions was evaluated by subjecting them to various stress conditions (temperature (15, 25, 35 and 45 °C), thermal cycling, and mechanical shaking) for three months. The mean particle size increased in all SLS and DF formulations stored under different stress conditions. No effect of micellar solubilized drug on the Ostwald ripening rate was observed. In the case of HPMC formulations only those stored at higher temperatures (35 or 45 °C) exhibited an increase in mean particle size. The increase in size in the HPMC formulation stored at 45 °C was attributed to dehydration of the HPMC chains and subsequent loss of protection of the nanoparticles. The cube of the mean particle diameter versus time plot was determined to be non-linear for all formulations exhibiting Ostwald ripening. Therefore, according to the Lifshitz, Slyozov and Wagner theory the process was not diffusion controlled. The most probable mechanism for Ostwald ripening was surface nucleation controlled.
Keywords: Ostwald ripening; Nanosuspensions; Stabilizers; Micelles; Physical stability; Particle size;

Novel microparticles (3–5 μm) were created by pre-emulsifying barley proteins with a homogenizer followed a microfluidizer system. These microparticles exhibited a high oil carrying capacity (encapsulation efficiency, 93–97%; loading efficiency, 46–49%). Microparticle degradation and bioactive compound release behaviours were studied in the simulated gastro-intestinal (GI) tract. The data revealed that nano-encapsulations (20–30 nm) were formed as a result of enzymatic degradation of barley protein microparticle bulk matrix in the simulated gastric tract. These nano-encapsulations delivered β-carotene to a simulated human intestinal tract intact, where they were degraded by pancreatic enzymes and steadily released the β-carotene. These uniquely structured microparticles may provide a new strategy for the nutraceutical and pharmaceutical industries to develop targeted delivery systems for lipophilic bioactive compounds.
Keywords: Barley protein; Microparticle; Nano-encapsulation; Lipophilic bioactive compounds; Controlled release;

The two polyhydroxy surfactants polyglycerol 6-distearate (Plurol®Stearique WL1009 – (PS)) and caprylyl/capryl glucoside (Plantacare® 810 – (PL)) are a class of PEG-free stabilizers, made from renewable resources. They were investigated for stabilization of aqueous solid lipid nanoparticle (SLN) and nanostructured lipid carrier (NLC) dispersions. Production was performed by high pressure homogenization, analysis by photon correlation spectroscopy (PCS), laser diffraction (LD), zeta potential measurements and differential scanning calorimetry (DSC). Particles were made from Cutina CP as solid lipid only (SLN) and its blends with Miglyol 812 (NLC, the blends containing increasing amounts of oil from 20% to 60%). The obtained particle sizes were identical for both surfactants, about 200 nm with polydispersity indices below 0.20 (PCS), and unimodal size distribution (LD). All dispersions with both surfactants were physically stable for 3 months at room temperature, but Plantacare (PL) showing a superior stability. The melting behaviour and crystallinity of bulk lipids/lipid blends were compared to the nanoparticles. Both were lower for the nanoparticles. The crystallinity of dispersions stabilized with PS was higher, the zeta potential decreased with storage time associated with this higher crystallinity, and leading to a few, but negligible larger particles. The lower crystallinity particles stabilized with PL remained unchanged in zeta potential (about −50 mV) and in size. These data show that surfactants have a distinct influence on the particle matrix struture (and related stability and drug loading), to which too little attention was given by now. Despite being from the same surfactant class, the differences on the structure are pronounced. They are attributed to the hydrophobic–lipophilic tail structure with one-point anchoring in the interface (PL), and the loop conformation of PS with two hydrophobic anchor points, i.e. their molecular structure and its interaction with the matrix surface and matrix bulk. Analysis of the effects of the surfactants on the particle matrix structure could potentially be used to further optimization of stability, drug loading and may be drug release.
Keywords: Polyhydroxy surfactants; Solid lipid nanoparticles; SLN; Nanostructured lipid carriers; NLC; Physical stability; Crystallinity;

Chemotherapy with hybrid liposomes for acute lymphatic leukemia leading to apoptosis in vivo by Hideaki Ichihara; Junichi Ueno; Masayo Umebayashi; Yoko Matsumoto; Ryuichi Ueoka (173-178).
Hybrid liposomes (HL) composed of 95 mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene (25) dodecyl ether (C12(EO)25) were prepared by sonication. A clear solutions of HL-25 having hydrodynamic diameter of about 50 nm could be maintained over 3 weeks. Remarkable reduction of tumor volume in model mice of acute lymphatic leukemia (ALL) intravenously treated with HL-25 without drugs after the subcutaneously inoculation of human ALL (MOLT-4) cells was verified in vivo. Induction of apoptosis in tumor of model mice of ALL treated with HL-25 was observed in micrographs on the basis of TUNEL method. Remarkable decrease of the ascites in ALL model mice treated with HL-25 was observed. It is noteworthy that prolonged survival (>400%) was obtained in model mice of ALL after the treatment with HL-25 without drugs.
Keywords: Hybrid liposomes; Chemotherapy; Acute lymphatic leukemia; Apoptosis; In vivo;