International Journal of Pharmaceutics (v.391, #1-2)

Evaluation of the transdermal permeation of different paraben combinations through a pig ear skin model by Thiago Caon; Ana Carolina Oliveira Costa; Marcone Augusto Leal de Oliveira; Gustavo Amadeu Micke; Cláudia Maria Oliveira Simões (1-6).
Although parabens have several features of ideal preservatives, different studies have shown that they may affect human health due to their estrogenic activity. Therefore, various strategies have been applied to reduce their skin penetration. However, the effect of paraben combinations on transdermal permeation has not yet been investigated. Thus, the objective of this study was to evaluate paraben permeation in pig ear skin using a Franz diffusion cell system with capillary electrophoresis detection, in order to identify which paraben combinations (defined by a factorial design) have the lowest skin permeation. The permeation of isolated parabens was also evaluated and the permeation characteristics, obtained by the Moser model, confirmed that lipophilicity and molecular weight may influence the systemic absorption of these compounds. In previous tests using isolated parabens, methyl and ethyl parabens presented greater retention in the epidermis compared to the dermis, while propyl and butyl parabens had similar retention profiles in these layers. An increase in ethanol concentration and experimental time promoted greater parabens retention in the dermis compared to the epidermis. The binary combinations of methyl and ethyl parabens as well as of methyl and propyl parabens (added to several cosmetic products in order to increase the antimicrobial spectrum) reduced significantly their permeation rates through pig ear skin (with the exception of EP), probably due to the high retention of these parabens in the epidermis and dermis.
Keywords: Parabens; Transdermal permeation; Pig ear skin; Capillary electrophoresis; Factorial design;

Evaluation needle length and density of microneedle arrays in the pretreatment of skin for transdermal drug delivery by Guang Yan; Kevin S. Warner; Jie Zhang; Sanjay Sharma; Bruce K. Gale (7-12).
Solid silicon microneedle arrays with different needle lengths (ranging from 100 to 1100 μm) and needle densities (ranging from 400 to 11,900 needles/cm2) were used to penetrate epidermal membrane of human cadaver skin. After this pretreatment, the electrical resistance of the skin and the flux of acyclovir across the skin were monitored. A linear correlation between the acyclovir flux and the inverse of the skin electric resistance was observed. Microneedle arrays with longer needles (>600 μm) were more effective in creating pathways across skin and enhancing drug flux, and microneedle arrays with lower needle densities (<2000 needles/cm2) were more effective in enhancing drug flux if the microneedles with long enough needle length (>600 μm). In addition, the microneedle arrays were used to penetrate hairless rat skin in vivo, and the trans-epidermal water loss (TEWL) of the rat skin was measured before and after the pretreatment. Treating rat skin with microneedle arrays of lower needle density and longer needle length was more effective in increasing TEWL. Integrity of the stratum corneum barrier of the penetrated rat skin as measured by TEWL recovered back to its base line level within 24 h after the microneedle pretreatment.
Keywords: Microneedles; Transdermal; Drug delivery; Needle length; Needle density;

Light induced fluorescence for predicting API content in tablets: Sampling and error by Reuben Domike; Samuel Ngai; Charles L. Cooney (13-20).
The use of a light induced fluorescence (LIF) instrument to estimate the total content of fluorescent active pharmaceutical ingredient in a tablet from surface sampling was demonstrated. Different LIF sampling strategies were compared to a total tablet ultraviolet (UV) absorbance test for each tablet. Testing was completed on tablets with triamterene as the active ingredient and on tablets with caffeine as the active ingredient, each with a range of concentrations. The LIF instrument accurately estimated the active ingredient within 10% of total tablet test greater than 95% of the time. The largest error amongst all of the tablets tested was 13%. The RMSEP between the techniques was in the range of 4.4–7.9%. Theory of the error associated with the surface sampling was developed and found to accurately predict the experimental error. This theory uses one empirically determined parameter: the deviation of estimations at different locations on the tablet surface. As this empirical parameter can be found rapidly, correct use of this prediction of error may reduce the effort required for calibration and validation studies of non-destructive surface measurement techniques, and thereby rapidly determine appropriate analytical techniques for estimating content uniformity in tablets.
Keywords: Tablets; Surface analysis; Fluorescence; PAT; Content uniformity;

Nitric oxide photorelease from hydrogels and from skin containing a nitro-ruthenium complex by Danielle Cristine Almeida Silva de Santana; Thais Tallarico Pupo; Marília Gama Sauaia; Roberto Santana da Silva; Renata Fonseca Vianna Lopez (21-28).
Nitric oxide (NO) is a gaseous molecule that has specific functions dictated by its localization and its kinetics of release. As NO-donors have a range of potential uses in the skin, much attention has been paid to the development of topical NO delivery systems. The aim of this work was to study the release rate and the skin penetration of the NO-donor cis-[Ru(NO2)(bpy)2(4-pic)]+ from different gel formulations and their potential as topical NO delivery systems under light stimuli. Among the formulations developed, the anionic gel retarded the nitro-ruthenium complex diffusion and also obstructed NO release after light irradiation. On the other hand, NO release before light irradiation was observed when the complex was dispersed in the cationic chitosan gel, possibly due to oxi-redox reactions between the amino groups of the polymer and the drug molecule. Finally, the non-ionic gel released the NO after light irradiation to the same extent as a drug aqueous solution at the same pH. The drug dispersed in this gel also penetrated into the stratum corneum skin layer, and the nitro-ruthenium complex present in the skin was able to release the NO after light stimuli, suggesting the potential use of this formulation as a topical NO delivery system.
Keywords: NO-donor; Nitro-ruthenium complex; Nitric oxide photorelease; Skin penetration; Light stimuli;

Surface modification of lactose inhalation blends by moisture by C.P. Watling; J.A. Elliott; C. Scruton; R.E. Cameron (29-37).
We present an investigation of the effects of relative humidity (RH) on lactose powders during storage, with the aims of determining the humidity conditions under which lactose inhalation blends are stable, and characterising the surface changes that occur as a result of water condensation. Lactose inhalation powders manufactured by milling and sieving were stored in environments of RH from 32% to 100% (at room temperature) and changes in surface properties were observed using BET nitrogen adsorption, environmental scanning electron microscopy and laser diffraction particle size analysis. We found that the specific surface area of all lactose powders decreased during storage, with the rate of decrease and final drop being larger at higher RH (ranging from a 62% decrease at 100% RH to a 34% decrease at 32% RH, at room temperature). The specific surface area decrease corresponded to a reduction in the volume of fine particles (<5 μm) in the blend. Two effects were found to contribute to the decrease in specific surface area: the smoothing of coarse particles, attributed to the surface fine particles undergoing deliquescence due to their enhanced solubility by the Kelvin effect (i.e. due to their greater curvature and consequently greater surface energy), and solid bridging between fine particles in agglomerates, such that loose fine particles disappeared from the powder blend, having bonded with coarser particles. These changes in particle properties resulting from moisture exposure are expected to influence the fine particle fraction of drug released from the powder blends, and the observation that lactose inhalation blends were unstable even at 32% RH could potentially be a concern for the pharmaceutical industry.
Keywords: Lactose; Dry powder inhalation; Relative humidity; Deliquescence; Ostwald ripening; Solid bridge;

The effect of particle size on the dehydration/rehydration behaviour of lactose by J.L. Crisp; S.E. Dann; M. Edgar; C.G. Blatchford (38-47).
Ethanolic suspensions of spray dried and micronized alpha lactose monohydrate (Lα·H2O) with average particle size between 3 and 200 μm, have been prepared and their dehydration behaviour was investigated by 13C CP-MASNMR spectroscopy. Sub-micron lactose suspension prepared by a novel high pressure homogenisation method has been compared with the standard ethanolic suspensions of Lα·H2O prepared by reflux or static room temperature methods. In all cases, suspensions were shown to contain the stable anhydrous form of lactose (LαS). Several approaches were employed to remove ethanol from these suspensions and the resulting dry lactose powders were then analysed by FT-IR, PXRD and SEM to evaluate the effect of drying procedure on type and distribution of lactose polymorphs and particle size. For samples with mean particle size greater than 1 μm, the stable anhydrous polymorphic form of lactose was retained on removal of the ethanol, although differences in the morphology and particle size of the crystals were apparent depending on method of suspension formation. Sub-micron LαS, while stable in dry conditions, has been shown to be less stable to atmospheric water vapour than LαS with particle size between 3 and 200 μm.
Keywords: Lactose; Sub-micron; Polymorph; CP-MASNMR; PXRD; SEM;

Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2) by Ludmila Luca; Martinus A.H. Capelle; Gia Machaidze; Tudor Arvinte; Olivier Jordan; Robert Gurny (48-54).
The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90° light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90° light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1–2 μm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.
Keywords: BMP-2; Aggregation; Physical stability; Fluorescence spectroscopy; Nile Red; 1,8-ANS; Light scattering; Electron microscopy;

Release modulation and cytotoxicity of hydroxycamptothecin-loaded electrospun fibers with 2-hydroxypropyl-β-cyclodextrin inoculations by Chengying Xie; Xiaohong Li; Xiaoming Luo; Ye Yang; Wenguo Cui; Jie Zou; Shaobing Zhou (55-64).
Hydroxycamptothecin (HCPT) is valid to various malignant tumors, but its insoluble and unstable lactone ring in physiological environment have restricted the clinic application. This work was aimed to formulate HCPT-loaded poly(dl-lactic acid)–poly(ethylene glycol) (PELA) fibrous mats through blend electrospinning with 2-hydroxypropyl-β-cyclodextrin (HPCD) to modulate the drug release and matrix degradation, and to enhance the structural integrity and cytotoxicity of the released HCPT. The entire drug fraction retained its active lactone form within electrospun fibers, and that was maintained over 85% during incubation for over 1 month. A biphasic release pattern was determined for HCPT-loaded electrospun fibers, which can be modulated by the addition of HPCD. HPCD served as solubilizer to maintain a large concentration gradient for HCPT between saturation and diffusion, and liberated HPCD created microstructure of ultrafine fibers, leading a faster release profile in the second phase. In vitro cytotoxicity test showed over 7 times higher inhibitory activity against cancer cells for HCPT-loaded electrospun fibers than free drug during 72 h incubation. Higher apoptosis rates and the arrest of the cell cycle during the S and G2/M phases were detected through flow cytometry analysis. It indicated therapeutic potentials of HCPT-loaded electrospun fibers as implantable anti-cancer agents for local chemotherapy.
Keywords: Blend electrospinning; Hydroxycamptothecin; Release modulation; Structural integrity; Local chemotherapy;

Possibility and effectiveness of drug delivery to skin by needle-free injector by Naoko Inoue; Hiroaki Todo; Dai Iidaka; Yoshihiro Tokudome; Fumie Hashimoto; Tohru Kishino; Kenji Sugibayashi (65-72).
We evaluated a needle-free injector (NFI), which has been studied as an administration device to the subcutaneous tissue, as a device to deliver drugs into skin tissues. ShimaJet® used for self-injection of insulin was selected as a spring-powered NFI in this study. Weak (NFI-w) and strong (NFI-s) injectors were evaluated. Rhodamine 6G, as a model compound, was injected onto the skin surface of hairless rats and the skin distribution and amount released from the skin of the compound were followed. A modified nozzle (able to inject at an angle of 45°) was prepared in addition to the conventional dedicated nozzle. The spring constants, nozzle shapes and penetration enhancer, 1-[2-(decylthio)ethyl] azacyclopentane-2-one (HPE-101), affected not only the skin distribution, but also the release profiles of rhodamine 6G. In addition, the release profiles of rhodamine 6G after injection using NFI-w or NFI-s obeyed diffusion-controlled or membrane-controlled kinetics, respectively. This difference was probably due to the skin site (depth) of rhodamine 6G delivered by the NFI. Furthermore, HPE-101 increased the retention time of rhodamine 6G in the epidermis. The present results suggested that an NFI can be a useful tool for enhanced drug delivery into skin.
Keywords: Needle-free injection; Transdermal drug delivery; ShimaJet®; Systemic drug delivery; Local drug delivery; High molecular weight compounds;

Transdermal patches for site-specific delivery of anastrozole: In vitro and local tissue disposition evaluation by Honglei Xi; Yonggang Yang; Dongmei Zhao; Liang Fang; Lin Sun; Liwei Mu; Jie Liu; Nanxi Zhao; Yanyan Zhao; Ni Zheng; Zhonggui He (73-78).
Anastrozole is a potent aromatase inhibitor and there is a need for an alternative to the oral method of administration to target cancer tissues. The purpose of the current study was to prepare a drug-in-adhesive transdermal patch for anastrozole and evaluate this for the site-specific delivery of anastrozole. Different adhesive matrixes, permeation enhancers and amounts of anastrozole were investigated for promoting the passage of anastrozole through the skin of rats in vitro. The best in vitro skin permeation profile was obtained with the formulation containing DURO-TAK® 87-4098, IPM 8% and anastrozole 8%. For local tissue disposition studies, the anastrozole patch was applied to mouse abdominal skin, and blood, skin, and muscle samples were taken at different times after removing the residual adhesive from the skin. High accumulation of the drug in the skin and muscle tissue beneath the patch application site was observed in mice compared with that after oral administration. These findings show that anastrozole transdermal patches are an appropriate delivery system for application to the breast tumor region for site-specific drug delivery to obtain a high local drug concentration.
Keywords: Anastrozole; Transdermal patch; Site-specific drug delivery; Local tissue disposition;

This research focused on constructing and characterizing an optimized porosity-enabled amalgamated matrix (P-EAM) for sustained transbuccal drug delivery. An interphase, co-particulate, co-solvent, homogenization technique and lyophilization guided through a Box–Behnken experimental design was employed in the fabrication, characterization and optimization of 15 P-EAMs. The effects of varying factor levels on the characteristic in vitro physicochemical performances of the P-EAMs were explored. Formulations had an average weight of 128.44 ± 3.48 mg with a dimensional size of 8 mm by 5 mm. Surface morphology showed varieties of pore structures, widespread distributions and uneven interconnectors. Satisfactory drug-loading was achieved (53.14 ± 2.19–99.02 ± 0.74%). Overall amount of drug released in 8 h was measured by the MDT50% value which ranged between 22.50 and 225.00 min. Formulation demonstrated significant levels of ex vivo bioadhesive strength measured as detachment force (F det  = 0.964 ± 0.015 to 1.042 ± 0.025 N) and work of adhesion (ω adh  = 0.0014 ± 0.00005 to 0.0028 ± 0.00008 J). The potential of the P-EAMs to initiate and sustain ex vivo transbuccal permeation of drug was shown and measured as a cumulative value of between 25.02 ± 0.85 and 82.21 ± 0.57% in 8 h. Formulations were mesoporous in nature with pore sizes ranging from 40 to 100 Å characterized by the presence of interconnectors. Statistical constraints were simultaneously set to obtain levels of independent variables that optimized the P-EAM formulation.
Keywords: Mechanistic characterization; Porosity-enabled amalgamated matrix; Transbuccal drug delivery; Experimental design; Simultaneous optimization;

Roller compaction of moist pharmaceutical powders by C.-Y. Wu; W.-L. Hung; A.M. Miguélez-Morán; B. Gururajan; J.P.K. Seville (90-97).
The compression behaviour of powders during roller compaction is dominated by a number of factors, such as process conditions (roll speed, roll gap, feeding mechanisms and feeding speed) and powder properties (particle size, shape, moisture content). The moisture content affects the powder properties, such as the flowability and cohesion, but it is not clear how the moisture content will influence the powder compression behaviour during roller compaction. In this study, the effect of moisture contents on roller compaction behaviour of microcrystalline cellulose (MCC, Avicel PH102) was investigated experimentally. MCC samples of different moisture contents were prepared by mixing as-received MCC powder with different amount of water that was sprayed onto the powder bed being agitated in a rotary mixer. The flowability of these samples were evaluated in terms of the poured angle of repose and flow functions. The moist powders were then compacted using the instrumented roller compactor developed at the University of Birmingham. The flow and compression behaviour during roller compaction and the properties of produced ribbons were examined. It has been found that, as the moisture content increases, the flowability of moist MCC powders decreases and the powder becomes more cohesive. As a consequence of non-uniform flow of powder into the compaction zone induced by the friction between powder and side cheek plates, all produced ribbons have a higher density in the middle and lower densities at the edges. For the ribbons made of powders with high moisture contents, different hydration states across the ribbon width were also identified from SEM images. Moreover, it was interesting to find that these ribbons were split into two halves. This is attributed to the reduction in the mechanical strength of moist powder compacts with high moisture contents produced at high compression pressures.
Keywords: Roller compaction; Moist powder; Flowability; Wet granular system; Granulation;

An assessment of beclomethasone dipropionate clathrate formation in a model suspension metered dose inhaler by Abdennour Bouhroum; Jonathan C. Burley; Neil R. Champness; Richard C. Toon; Philip A. Jinks; Philip M. Williams; Clive J. Roberts (98-106).
The aims of this study were to investigate and characterize the physico-chemical properties of beclomethasone dipropionate (BDP) crystallized from tricholoromonofluoromethane (CFC-11). Physical interactions in a model pressurised metered dose inhaler (pMDI) system and changes in surface energy after size reduction (micronization) were determined. Although CFC-11 has largely been phased out of use in pMDIs due to its ozone depletion potential, the BDP CFC-11 clathrate is a stable entity and thus suitable as a model for our initial investigations. In addition, although propellant clathrates have been known for sometime, as far as the authors are aware, their surface energies and adhesive interactions have not been reported. The structure of the clathrate was investigated using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray powder diffraction (X-RPD). In addition, atomic force microscopy (AFM) was employed to determine the dispersive surface free energy (SE) and force of adhesion (F adh) of the BDP CFC-11 clathrate with different pMDI components in a model propellant (decafluoropentane).The dispersive surface free energies for anhydrous BDP (micronized), the CFC-11 clathrate and ball-milled BDP CFC-11 clathrate are (47.5 ± 4.9) mJ m−2, (11.3 ± 4.1) mJ m−2 and (15.2 ± 1.3) mJ m−2 respectively. Force of adhesion results shows that BDP CFC-11 clathrates, even after being ball-milled for 2.5 h, have a lower F adh compared to micronized anhydrous BDP with different pMDI components. This shows that the formation of the crystalline CFC-11 clathrate is advantageous when compared to the micronized anhydrous form, in terms of its surface energy and potential interactions within a suspension MDI formulation. In the wider context, this work has implications for the future development of HFA formulations with APIs which are prone to the formation of propellant clathrates.
Keywords: Clathrate; Pressurised metered dose inhalers (pMDIs); Atomic force microscopy (AFM); Surface energy (SE); Force of adhesion (F adh);

Different modes of dynamic image analysis in monitoring of pharmaceutical dry milling process by Venkateshwar Rao Nalluri; Peter Schirg; Xin Gao; Antoine Virdis; Georgios Imanidis; Martin Kuentz (107-114).
This article focuses on the process analytical technology (PAT) of pharmaceutical dry milling. The first objective is to compare different modes of dynamic image analysis namely, on-line, in-line and at-line for monitoring powder milling. The second objective is to introduce time evolving size and shape analysis (TESSA). Thus, a conical mill was equipped with a dynamic image analysis system which consisted of a xenon flash light and charge-coupled device (CCD) camera. Different pharmaceutical excipients and granulates were chosen as models. The results from the on-line, in-line and the at-line measurement modes showed similar size distributions for the various materials studied, however differences were observed that were mainly attributed to sampling and dispersion. A high correlation of 0.975 (p  < 0.001) was observed between on-line d50 and at-line d50 when compared to 0.917 (p  < 0.001) between in-line d50 and at-line d50. The concept of TESSA was found to be useful in detecting changes in milling conditions including the successful detection of a damaged screen when intentionally introduced in the milling process. This monitoring approach of particle size and shape has potential to reduce product variability, facilitates process development, and ultimately helps in establishing quality by design concept for the manufacture of solid dosage forms.
Keywords: Process analytical technology (PAT); Conical mill; Process analyzers; Dynamic image analysis; Shape factor; Quality by design;

Potential of an injectable chitosan/starch/β-glycerol phosphate hydrogel for sustaining normal chondrocyte function by Jatuporn Ngoenkam; Atchariya Faikrua; Sukkid Yasothornsrikul; Jarupa Viyoch (115-124).
An injectable hydrogel for chondrocyte delivery was developed by blending chitosan and starch derived from various sources with β-glycerol phosphate (β-GP) in the expectation that it would retain a liquid state at room temperature and gel at raised temperatures. Rheological investigation indicated that the system consisting of chitosan derived from crab shell and corn starch at 4:1 by weight ratio (1.53%, w/v of total polymers), and 6.0% (w/v) β-GP (C/S/GP system) exhibited the sharpest sol–gel transition at 37 ± 2 °C. The C/S/GP hydrogel was gradually degraded by 67% within 56 days in PBS containing 0.02 mg/ml lysozyme. The presence of starch in the system increased the water absorption of the hydrogel when compared to the system without starch. SEM observation revealed to the interior structure of the C/S/GP hydrogel having interconnected pore structure (average pore size 26.4 μm) whereas the pore size of the hydrogel without starch was 19.8 μm. The hydrogel also showed an ability to maintain chondrocyte phenotype as shown by cell morphology and expression of type II collagen mRNA and protein. In vivo study revealed that the gel was formed rapidly and localized at the injection site.
Keywords: Chitosan/starch/β-glycerol phosphate; Injectable hydrogels; Chondrocytes;

Influence of polymer content on stabilizing milled amorphous salbutamol sulphate by P.N. Balani; S.Y. Wong; W.K. Ng; E. Widjaja; R.B.H. Tan; S.Y. Chan (125-136).
The study investigates the influence of polyvinyl pyrrolidone (PVP) concentration on stabilizing the amorphous form of salbutamol sulphate (SS) before and after storage under ambient and elevated humidity conditions. Different mass ratios of SS and PVP (0–90 wt%) were co-milled using a planetary ball mill. X-ray powder diffraction (XRPD), high sensitivity differential scanning calorimetry (HSDSC), dynamic vapor sorption (DVS), infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and Raman microscopy (RM) were used to analyze the stability of the co-milled mixtures against heat and humidity treatments as well as storage at different humidity conditions. Prior storage, DSC and DVS analyses revealed that re-crystallization of amorphous SS was suppressed above PVP content of 33 wt%. Probable hydrogen bond interaction between SS and PVP was found in FT-IR analysis. XRPD diffractograms and SEM analysis showed stability against re-crystallization was achieved in the co-milled mixtures with a minimum PVP content of 80 wt% after storage. Homogeneous distribution of SS and PVP from RM analysis showed fine clustering of SS and PVP, suggesting the formation of an amorphous dispersion at molecular level. The results provide insights on the application of thermal and humidity treatments, accelerated stability testing and investigations on drug–excipient interactions to predict the minimum ratio of an excipient for stabilizing the amorphous state of a milled API.
Keywords: Salbutamol sulphate; Polyvinyl pyrrolidone K30; Co-milling; X-ray powder diffraction; Raman microscopy; Stability;

The relationship between drug concentration, mixing time, blending order and ternary dry powder inhalation performance by Matthew D. Jones; João G.F. Santo; Bilal Yakub; Mansa Dennison; Husein Master; Graham Buckton (137-147).
Some studies have shown that the mixing order of drug, fines and coarse carrier in a ternary dry powder inhaler (DPI) formulation affects fine particle delivery; others have seen no difference. This was investigated by examining the influence of salbutamol sulphate concentration (0.5–4.5%w/w), mixing time and blending order (drug and lactose carrier first, then lactose fines; versus fines and carrier first, then drug) on formulation in vitro fine particle delivery. With 15 min of mixing, there was no effect of drug concentration or blending order on fine particle fraction (FPF). With 30 min of mixing, lower drug concentrations produced larger FPFs with the fines and carrier first blending order. Higher drug concentrations resulted in equal performance between the blending orders. With 60 min of mixing, the drug and carrier first blending order resulted in larger a FPF at 0.5%w/w salbutamol sulphate. The previous conflicting studies used a mixing time of 30 min; these results suggest that their conflicting results may have been due to the use of different drug concentrations. The complexity in the whole dataset suggests that blending order studies are of limited use for the investigation of the mechanism behind the effects of fines.
Keywords: Blending order; Drug concentration; Fines; Mixing time; Ternary dry powder inhaler formulations; Ternary interactive mixtures;

In situ droplet size and speed determination in a fluid-bed granulator by Henrik Ehlers; Jussi Larjo; Osmo Antikainen; Heikki Räikkönen; Jyrki Heinämäki; Jouko Yliruusi (148-154).
The droplet size affects the final product in fluid-bed granulation and coating. In the present study, spray characteristics of aqueous granulation liquid (purified water) were determined in situ in a fluid-bed granulator. Droplets were produced by a pneumatic nozzle. Diode laser stroboscopy (DLS) was used for droplet detection and particle tracking velocimetry (PTV) was used for determination of droplet size and speed. Increased atomization pressure decreased the droplet size and the effect was most strongly visible in the 90% size fractile. The droplets seemed to undergo coalescence after which only slight evaporation occurred. Furthermore, the droplets were subjected to a strong turbulence at the event of atomization, after which the turbulence reached a minimum value in the lower halve of the chamber. The turbulence increased as speed and droplet size decreased due to the effects of the fluidizing air. The DLS and PTV system used was found to be a useful and rapid tool in determining spray characteristics and in monitoring and predicting nozzle performance.
Keywords: Fluid bed; Droplet size; Droplet speed; High speed imaging; Diode laser stroboscopy; Particle tracking velocimetry;

Selective targeting of adenovirus to αvβ3 integrins, VEGFR2 and Tie2 endothelial receptors by angio-adenobodies by Hidde J. Haisma; Gera K. Kamps; Arend Bouma; Tessa M. Geel; Marianne G. Rots; Anu Kariath; Anna Rita Bellu (155-161).
Tumor angiogenesis is a prominent mechanism, driving the development and progression of solid tumors and the formation of cancer cell metastasis. Newly formed tumor vessels represent an elective target for the activity and the delivery of cancer therapeutics. We targeted adenovirus (Ad5) to endothelial receptors which are up-regulated during the formation of new blood vessels, to enhance the efficiency of anticancer gene therapy applications.Bifunctional angio-adenobodies were constructed by the fusion of a single chain antibody directed against the adenoviral fiber knob, to different peptides recognizing the αvβ3 integrins, VEGFR2 and Tie2 receptors on endothelial cells. The angio-adenobodies were coupled to the adenoviral vector, containing luciferase and GFP as reporter genes. In vitro data showed selective targeting of the Ad5 to the endothelial receptors both in mouse (H5V) and human cell lines (HUVEC). H5V cells, refractory to Ad5 infection, showed high level of luciferase expression when cells were infected with targeted virus. Viral transgene expression increased in HUVEC cells when cells were infected with Ad5 conjugated with angio-adenobody thereby demonstrating the affinity of the peptides for human endothelial cells also. In vivo data obtained from mice bearing a C26 colon carcinoma subcutaneously show viral transgene expression only in tumors infected with angio-adenobodies retargeted adenovirus.The results of the present study demonstrate that endothelial targeted angio-adenobodies represent a versatile tool to direct adenovirus from its native receptors to the integrins αvβ3, VEGFR2 and Tie2 receptors that are fundamental in many angiogenesis related diseases such as cancer.
Keywords: Adenovirus; Angiogenesis; αvβ3 Integrins; VEGFR2; Tie2;

This study probes the molecular interactions between model drugs and poloxamers that facilitate dissolution rate improvements using solid dispersions. Ibuprofen and ketoprofen solid dispersions were prepared at different mole ratios using poloxamers 407 and 188. The carbonyl stretching vibration of the ibuprofen dimer shifted to higher wavenumber in the infrared spectra of 2:1 drug:carrier mole ratio solid dispersions, indicating disruption of the ibuprofen dimer concomitant with hydrogen bond formation between the drug and carrier. Solid dispersions with mole ratios >2:1 drug:carrier (up to 29:1) showed both ibuprofen hydrogen-bonded to the poloxamer, and excess drug present as dimers. X-ray diffraction studies confirmed these findings with no evidence of crystalline drug in 2:1 mole ratio systems whereas higher drug loadings retained crystalline ibuprofen. Similar results were found with ketoprofen-poloxamer solid dispersions. Thermal analysis of ibuprofen-poloxamer 407 solid dispersions and their resultant phase diagram suggested solid solutions and a eutectic system were formed, depending on drug loading. Dissolution studies showed fastest release from the solid solutions; dissolution rates from solid solutions were 12-fold greater than the dissolution of ibuprofen powder whereas the eutectic system gave a 6-fold improvement over the powder. When designing solid dispersions to improve the delivery of poorly-water soluble drugs, the nature of drug:carrier interactions, which are governed by the stochiometry of the composition, can affect the dissolution rate improvement.
Keywords: Ibuprofen; Ketoprofen; Solid dispersion; Poloxamer 407; Poloxamer 188; Solid solution; Eutectic mixture; Hydrogen bonding;

Adsorption of carbamazepine onto crospovidone to prevent drug recrystallization by Martin Schulz; Bernhard Fussnegger; Roland Bodmeier (169-176).
The adsorption mechanism of the poorly water-soluble drug carbamazepine onto crosslinked polyvinylpyrrolidone (crospovidone) was investigated by adsorption isotherms in different solvents and Fourier-transformation infrared spectroscopy (FTIR). The drug adsorption was a result of hydrogen bonds between carbamazepine's amine group and crospovidone's carbonyl group. Solvents with a hydrogen donor site competed with the drug for binding sites on crospovidone and thereby decreased the extent of drug adsorption. To optimize the drug–carrier ratio, adsorbates with different drug loadings were prepared by the solvent deposition method and analyzed for drug crystals using differential scanning calorimetry, X-ray diffraction, scanning electron microscopy and polarized light microscopy. Adsorbates with a drug loading of 9.1% or less did not show drug crystals. The drug release increased in the order of micronized drug < physical mixture < adsorbate. This was attributed to wetting and deagglomeration effects in both the physical mixture and the adsorbate and the molecularly dispersed state of the drug in the adsorbate. The findings allow for a more rationale design of immediate release formulations and of transdermal patches containing drug adsorbates onto crospovidone.
Keywords: Crospovidone; Drug adsorbates; Recrystallization inhibition; Poorly water-soluble drug; Solvent deposition method;

Melt granulation of pharmaceutical powders: A comparison of high-shear mixer and fluidised bed processes by Nadia Passerini; Giacomo Calogerà; Beatrice Albertini; Lorenzo Rodriguez (177-186).
The main aim of this research was to compare in situ melt granulation process in high-shear mixers and fluidised bed equipments with particular attention to the final properties of granules. In addition, the study evaluated the suitability of melt granulation in fluidised bed for improving the dissolution rate of drugs. Agglomerates having identical composition (10%, w/w, of ibuprofen or ketoprofen, 20%, w/w, of PEG 6000 and 70%, w/w, of lactose monohydrate) were produced using both equipments and their morphology, particle size, flowability, friability, drug loading, dissolution behaviors at pH 1.2 and 7.4 and physicochemical properties (DSC and XRD analysis) have been evaluated and compared. The results showed that melt granulation can be successfully performed in both granulators. The utilization of a different equipment had strong impact on the particle size distribution of the granules and on their morphology, while the effect on others physical properties was little, as all the granules possess low friability and excellent flowability. Moreover both the solid state characteristics of the products and the dissolution behaviors of ibuprofen and ketoprofen granules were found to be practically independent of the equipment and all granules showed a significant increase of the drug dissolution rate in acidic conditions. In conclusion in situ melt granulation in fluidised beds could be considered a suitable alternative to the melt granulation in high-shear mixers.
Keywords: Melt agglomeration; Fluid bed; High-shear mixer; Poorly water soluble drugs; Technological properties; Drug dissolution;

The development of a dense gas solvent exchange process for the impregnation of pharmaceuticals into porous chitosan by Chengdong Ji; Angela Barrett; Laura A. Poole-Warren; Neil R. Foster; Fariba Dehghani (187-196).
The aim of this study was to prepare stable formulations of poorly water-soluble drugs in amorphous forms to enhance their dissolution rates, promote the bioavailability, minimize the dosage, thereby theoretically decreasing their side effects. A dense gas solvent exchange process was developed for the impregnation of poorly water-soluble drugs such as camptothecin and griseofulvin into a chitosan matrix. The amount of drug impregnated was measured by UV-spectrophotometery and gravimetric techniques. Pore characteristics and the crystallinity of the drugs in the impregnated chitosan were measured. Homogenous nano-sized pores with thin walls were formed in chitosan using the dense gas solvent exchange process. The method was efficient for the impregnation of a drug into chitosan. Results of XRD, Fourier transform infrared spectroscopy and differential scanning calorimetry demonstrated that as a result of interaction between chitosan and the drug, both camptothecin and griseofulvin were in amorphous forms after processing. The dissolution rate of processed griseofulvin was increased threefold due to the hydrophilic properties of chitosan and its interaction with the drug. A new approach was developed for promoting drug bioavailability that has the potential to decrease the required dose and side effects, particularly for chemotherapeutic drugs with narrow therapeutic index.
Keywords: Chitosan; Camptothecin; Amorphous; Porosity; Impregnation by dense gas CO2;

Crystallographic, thermal and spectroscopic characterization of a ciprofloxacin saccharinate polymorph by C.B. Romañuk; Y. Garro Linck; A.K. Chattah; G.A. Monti; S.L. Cuffini; M.T. Garland; R. Baggio; R.H. Manzo; M.E. Olivera (197-202).
A new polymorphic form of ciprofloxacin saccharinate (CIP-SAC II) is presented, and compared with CIP-SAC I, a different polymorph which we had previously reported. The characterization techniques used were single crystal and powder X-ray diffraction, differential scanning calorimetry, thermogravimetry analysis and infrared and 13C solid-state nuclear magnetic resonance spectroscopy. The results obtained from these techniques are consistent. Differential scanning calorimetry and thermogravimetric analysis showed that the reaction between the precursors is completed and the crystalline forms of both salts obtained (I and II) are highly pure. Infrared spectroscopy gave clear evidence of a salt formation. Solid-state nuclear magnetic resonance spectroscopy would indicate some degree of qualitative similarity in the intermolecular interaction scheme in both polymorphs, while thermal analysis data might indicate a difference in quantitative terms. A thorough single crystal structure determination of the new form CIP-SAC II allowed disclosing the most important inter- and intramolecular interactions.
Keywords: Fluoroquinolone; Salts; Polymorphism; Solid-state characterization;

The aim of this study was to develop and optimize SNEDDS formulations containing surfactants reported to be bioenhancers for improvement of dissolution and oral absorption of lacidipine (LCDP). Preliminary screening was carried out to select proper components combination. D-optimal mixture experimental design was applied to optimize a SNEDDS that contains a minimum amount of surfactant, a maximum amount of lipid, and possesses enhanced emulsification and dissolution rates. Three formulation variables; the oil phase X 1 (a mixture of Labrafil®/Capmul®), the surfactant X 2 (a mixture of Cremophor®/Tween® 80) and the co-surfactant X 3, were included in the design. The systems were assessed for droplet size, light absorbance, optical clarity, drug release and emulsification efficiency. Following optimization, the values of formulation components (X 1, X 2, and X 3) were 34.20%, 40.41% and 25.39%, respectively. There is a good correlation between light absorbance and droplet size analysis of diluted SNEDDS (R 2  = 0.883). Transmission electron microscopy demonstrated spherical droplet morphology. The stability of the optimized formulation was retained after storage at 40 °C/75% RH for three months. The optimized formulation of LCDP showed a significant increase in dissolution rate compared to the drug suspension under the same conditions. Our results proposed that the optimized SNEDDS formulation, containing bioenhancing surfactants, could be promising to improve oral absorption of LCDP.
Keywords: Lacidipine; Ternary phase diagram; Bioenhancers; SNEDDS; Dissolution; D-optimal mixture design;

Improved plasma stability and sustained release profile of gemcitabine via polypeptide conjugation by Lik-Voon Kiew; Soon-Keng Cheong; Khalifah Sidik; Lip-Yong Chung (212-220).
To enhance the stability of the anticancer drug gemcitabine (2′-deoxy-2′,2′-difluorocytidine), it was conjugated to poly-l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly-l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48 h, and reached equilibrium at 72 h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug.
Keywords: Gemcitabine; Poly-l-glutamic acid; Polymeric drug conjugates; Sustained release;

In order to provide an improved understanding of the flow in pressurized-metered dose inhalers (pMDIs), especially monitoring the output temperature and mass flow rate to obtain maximum atomization efficiency from the available energy, a numerical model for a two phases, multi-component compressible flow in a pressurized-metered dose inhaler is presented and validated. It is suitable for testing with various formulations and different geometries for a range of pMDI devices. We validated the model against available data in the literature for a single component HFA 134a propellant, and then investigated the response of the model to other formulations containing non-volatile components. Further validation is obtained by an experiment using the dual beam method which acquired the actuation flow properties such as spray velocity and duration. The deviation of the numerical predictions for the peak exit velocity against the experimental results is 5.3% and that for effective spray duration 5.0%. From the numerical and experimental results, it is found that for the formulations with the mass fraction of HFA 134a > 80%, the effective spray duration of the pMDI is around 0.1 s. Furthermore the droplet peak exit velocity at the axial station x  = 25 mm from the actuation nozzle decreases from 20 to 15 m/s with the reduction of the propellant (HFA 134a) from 95%. Formulations with the mass fraction of HFA 134a below 80% produce poor quality spray which is indicated from the unsteady peak exit velocity, changeable spray number density in each experimental test, and numerical simulations also confirmed the non-viability of this condition.
Keywords: Flash evaporation; Multiphase; Atomization; Model;

Development of pegylated liposomal vinorelbine formulation using “post-insertion” technology by Chun Lei Li; Jing Xia Cui; Cai Xia Wang; Lan Zhang; Yan Hui Li; Li Zhang; Xian Xiu; Yong Feng Li; Na Wei (230-236).
Prolonged vinorelbine exposure is correlated with improved antineoplastic effects, as evidenced by increased response rate in patients receiving continuous infusion. The administration of slow release pegylated liposomal vinorelbine formulation might mimic the pharmacokinetics of a continuous infusion, thus improving antitumor efficacy. But it is hard to prepare pegylated liposome vinorelbine using DSPE-PEG (an extensively used peglipid) because it could induce accelerated drug release. To resolve this problem, “post-insertion” technology was employed to prepare pegylated liposome vinorelbine formulations, which involved the incubation of vinorelbine-containing vesicles with DSPE-PEG micellar solutions. HPLC analysis revealed that after incubation at 60 °C for 60 min, ∼100% DSPE-PEG could be inserted into the outer monolayer of the vesicles. Moreover, the grafting of peglipid did not induce the release of entrapped vinorelbine irrespective of intraliposomal anions. Drug release experiments indicated that “post-insertion” formulations were more able to retain entrapped drugs than “co-dissolving” formulations. The same phenomenon was observed when both series of formulations were injected in normal mice to compare pharmacokinetic profiles. In L1210 ascitic model, a “post-insertion” formulation with a PEG grafting density of ∼0.5% exhibited the strongest antineoplastic effects, thus it was chosen to be further evaluated in S-180 and RM-1 models, in which the formulation was still more therapeutically active than conventional formulations. In conclusion, using “post-insertion” technology, the potential interaction between DSPE-PEG and vinorelbine could be prevented, thus making it possible to develop pegylated vinorelbine formulations.
Keywords: Liposomes; Vinorelbine; DSPE-PEG; Post-insertion; Active loading; Pharmacokinetics; Toxicity; Efficacy;

Preparation and properties of branched oligoglycerol modifiers for stabilization of liposomes by Atsushi Ishihara; Masahiro Yamauchi; Hiroko Kusano; Yukiteru Mimura; Masashi Nakakura; Masaaki Kamiya; Ayato Katagiri; Masaki Kawano; Hisao Nemoto; Toshiyuki Suzawa; Motoo Yamasaki (237-243).
We examined the effect on drug delivery of liposomes with surfaces that were modified with branched oligoglycerols (BGLs) and explored possible formulation advantages to increase drug exposure. BGL012 is a branched oligoglycerol derivative with a cascade-like structure of 12 glycerol units, characterized as a widely spread structure in aqueous solution. We prepared BGL-phospholipid derivatives (BGL-PEs), including BGL012, by coupling 1,2-distearoylphosphatidylethanolamine to BGLs. BGL012-PE modification of the liposomes (BGL012L) achieved a long circulation time after intravenous injection in rats. The circulation lifetime of BGL012L was almost the same as that of polyethylene glycol (PEG)-modified liposomes. The surface of BGL012L induced the formation of a fixed aqueous layer and reduced protein adsorption on the liposome surface, without strong interference with the binding reaction on the liposome. Thus, the newly synthesized branched oligoglycerol derivatives are considered to have useful hydrophilic and physical properties for modifying the liposome surface to increase drug exposure.
Keywords: Oligoglycerol; Liposome; Carrier; Long circulation;

Optimal conditions to prepare fine globular granules with a multi-functional rotor processor by Shin-ichiro Kimura; Yasunori Iwao; Masayuki Ishida; Takeaki Uchimoto; Atsuo Miyagishima; Takashi Sonobe; Shigeru Itai (244-247).
The optimal manufacturing conditions to obtain fine globular granules with a narrow size of particle distribution were investigated for a multi-functional rotor processor. A fractional factorial design analysis was undertaken to find out the significant operational conditions influencing the following physical characteristics of the obtained granules: size distribution, roundness and water content. Operational conditions tested were binder flow rate, atomization pressure, slit air flow rate, rotating speed and temperature of inlet air. It was observed that: the proportion of fine particles (106–212 μm) was positively affected by the atomization pressure, while negatively affected by the slit air flow rate; and roundness and water content were positively affected by the binder flow rate. Furthermore, the multiple regression analysis enabled the identification of an optimal operating window for production of fine globular granules. Therefore, the present study demonstrated that the combination of experimental design and multiple regression analysis allows a better understanding of complicated granulating process of multi-functional rotor processor to obtain fine globular granules.
Keywords: Multi-functional rotor processor; Fine globular granules; Experimental design; Multiple regression analysis;

Biophysical characterization of a liposomal formulation of cytarabine and daunorubicin by Awa Dicko; Sungjong Kwak; April A. Frazier; Lawrence D. Mayer; Barry D. Liboiron (248-259).
The biophysical characterization of CPX-351, a liposomal formulation of cytarabine and daunorubicin encapsulated in a synergistic 5:1 molar ratio (respectively), is presented. CPX-351 is a promising drug candidate currently in two concurrent Phase 2 trials for treatment of acute myeloid leukemia. Its therapeutic activity is dependent on maintenance of the synergistic 5:1 drug:drug ratio in vivo. CPX-351 liposomes have a mean diameter of 107 nm, a single phase transition temperature of 55.3 °C, entrapped volume of 1.5 μL/μmol lipid and a zeta potential of −33 mV. Characterization of these physicochemical properties led to identification of an internal structure within the liposomes, later shown to be produced during the cytarabine loading procedure. Fluorescence labeling studies are presented that definitively show that the structure is composed of lipid and represents a second lamella. Extensive spectroscopic studies of the drug–excipient interactions within the liposome and in solution reveal that interactions of both cytarabine and daunorubicin with the copper(II) gluconate/triethanolamine-based buffer system play a role in maintenance of the 5:1 cytarabine:daunorubicin ratio within the formulation. These studies demonstrate the importance of extensive biophysical study of liposomal drug products to elucidate the key physicochemical properties that may impact their in vivo performance.
Keywords: Cytarabine; Daunorubicin; Liposomes; Copper gluconate; EPR; CPX-351;

Stearic acid (SA) grafted chitosan oligosaccharide (CSO-SA) with different molecular weight of chitosan oligosaccharide (CSO) and graft ratio of stearic acid were synthesized by coupling reaction of SA and CSO. The cationic polymeric micelles of CSO-SA via self-assemble formed and used for gene delivery of fish sperm DNA. Factors affecting complexation and stability of the complexes of CSO-SA micelles and DNA were investigated. The results indicated that pKa of CSO-SA with 3 kDa of CSO decreased from 8.16 to 6.02 as the substitution degree of amino groups of CSO in CSO-SA increased from 9.79% to 63.41%, whereas the molecular weight (M W) of CSO less affected the pKa. As for the stability of complexes, ethidium bromide assay data demonstrated that the complexes consisting of CSO-SA with lower amino substitution degree or smaller molecular weight of CSO were more stable than that with the higher amino substitution degree or molecular weight of CSO. The results also presented that the low pH and ionic strength environment were in favor for the stability of complexes.
Keywords: Chitosan oligosaccharide; Stearic acid; Cationic micelles; Gene delivery vector; Complexation; Stability;

High loading fragrance encapsulation based on a polymer-blend: Preparation and release behavior by Aurapan Sansukcharearnpon; Supason Wanichwecharungruang; Natchanun Leepipatpaiboon; Teerakiat Kerdcharoen; Sunatda Arayachukeat (267-273).
The six fragrances, camphor, citronellal, eucalyptol, limonene, menthol and 4-tert-butylcyclohexyl acetate, which represent different chemical functionalities, were encapsulated with a polymer-blend of ethylcellulose (EC), hydroxypropyl methylcellulose (HPMC) and poly(vinyl alcohol) (PV(OH)) using solvent displacement (ethanol displaced by water). The process gave ≥40% fragrance loading capacity with ≥80% encapsulation efficiency at the fragrance to polymer weight ratio of 1:1 and at initial polymer concentrations of 2000–16,000 ppm and the obtained fragrance-encapsulated spheres showed hydrodynamic diameters of less than 450 nm. The release profile of the encapsulated fragrances, evaluated by both thermal gravimetric and electronic nose techniques, indicated different release characteristics amongst the six encapsulated fragrances. Limonene showed the fastest release with essentially no retention by the nanoparticles, while eucalyptol and menthol showed the slowest release.
Keywords: Nanoparticles; Fragrance; Encapsulation; Controlled release; TGA; Essential oil;

Novel and simple loading procedure of cisplatin into liposomes and targeting tumor endothelial cells by M. Hirai; H. Minematsu; Y. Hiramatsu; H. Kitagawa; T. Otani; S. Iwashita; T. Kudoh; L. Chen; Y. Li; M. Okada; D.S. Salomon; K. Igarashi; M. Chikuma; M. Seno (274-283).
Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects.High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl LewisX (CDDP-SLX-Lip) because we previously demonstrated Sialyl LewisX enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues.
Keywords: Cisplatin; Cis-diamminedinitratoplatinum (II); Liposome; E-selectin; Sialyl LewisX;

Interactions of lipid-based liquid crystalline nanoparticles with model and cell membranes by Justas Barauskas; Camilla Cervin; Marija Jankunec; Marija Špandyreva; Kristina Ribokaitė; Fredrik Tiberg; Markus Johnsson (284-291).
Lipid-based liquid crystalline nanoparticles (LCNPs) are interesting candidates for drug delivery applications, for instance as solubilizing or encapsulating carriers for intravenous (i.v.) drugs. Here it is important that the carriers are safe and tolerable and do not have, e.g. hemolytic activity. In the present study we have studied LCNP particles of different compositions with respect to their mixing behavior and membrane destabilizing effects in model and cell membrane systems. Different types of non-lamellar LCNPs were studied including cubic phase nanoparticles (Cubosome®) based on glycerol monooleate (GMO), hexagonal phase nanoparticles (Hexosome®) based on diglycerol monooleate (DGMO) and glycerol dioleate (GDO), sponge phase nanoparticles based on DGMO/GDO/polysorbate 80 (P80) and non-lamellar nanoparticles based on soy phosphatidylcholine (SPC)/GDO. Importantly, the LCNPs based on the long-chain monoacyl lipid, GMO, were shown to display a very fast and complete lipid mixing with model membranes composed of multilamellar SPC liposomes as assessed by a fluorescence energy transfer (FRET) assay. The result correlated well with pronounced hemolytic properties observed when the GMO-based LCNPs were mixed with rat whole blood. In sharp contrast, LCNPs based on mixtures of the long-chain diacyl lipids, SPC and GDO, were found to be practically inert towards both hemolysis in rat whole blood as well as lipid mixing with SPC model membranes. The LCNP dispersions based on a mixture of long-chain monoacyl and diacyl lipids, DGMO/GDO, displayed an intermediate behavior compared to the GMO and SPC/GDO-based systems with respect to both hemolysis and lipid mixing. It is concluded that GMO-based LCNPs are unsuitable for parenteral drug delivery applications (e.g. i.v. administration) while the SPC/GDO-based LCNPs exhibit good properties with limited lipid mixing and hemolytic activity. The correlation between results from lipid mixing or FRET experiments and the in vitro hemolysis data indicates that FRET assays can be one useful screening tool for parenteral drug delivery systems. It is argued that the hemolytic potential is correlated with chemical activity of the monomers in the mixtures.
Keywords: Lipid nanoparticles; Liquid crystals; Liposomes; Hemolysis; Interaction kinetics; FRET fluorescence;

BBSKE (1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)] ethane, PCT: CN02/00412) is a novel organoselenium anticancer drug that plays a role in anticancer through inhibiting TrxR (thioredoxin reductase). In this study, we prepared a tri-block copolymer micelles loading BBSKE utilizing the amphiphilic tri-block copolymers (PEG6000-PLA6000) which we synthesized. And then the characters of the copolymer micelles were investigated. When packaged in polymeric micelles, the water solubility of BBSKE was improved to 0.21 mg/ml. The IC50 were 7.14 μM, 5.05 μM and 4.23 μM when MCF-7 breast cancer cells were treated with BBSKE after 24 h, 48 h and 72 h. The inhibition effect of polymeric micelles to MCF-7 tumor cells was bettered when folate, whose receptor was highly expressed in various tumors, was coated on the surface of these nanoparticles. Finally, by adopting a new way of imaging in vivo, we studied the distribution of micelles in nude mice with and without MCF-7 tumor. The results demonstrated that this copolymer micelles loading BBSKE can accumulate into tumor efficiently.
Keywords: Polymeric micelles; Imaging in vivo; Anticancer; MCF-7; BBSKE;

Dry powdered aerosols of diatrizoic acid nanoparticle agglomerates as a lung contrast agent by Nashwa El-Gendy; Kristin L. Aillon; Cory Berkland (305-312).
Aerosolized contrast agents may improve the resolution of biomedical imaging modalities and enable more accurate diagnosis of lung diseases. Many iodinated compounds, such as diatrizoic acid, have been shown to be safe and useful for radiographic examination of the airways. Formulations of such compounds must be improved in order to allow imaging of the smallest airways. Here, diatrizoic acid nanoparticle agglomerates were created by assembling nanoparticles into inhalable microparticles that may augment deposition in the lung periphery. Nanoparticle agglomerates were fully characterized and safety was determined in vivo. After dry powder insufflation to rats, no acute alveolar tissue damage was observed 2 h post-dose. Diatrizoic acid nanoparticle agglomerates possess the characteristics of an efficient and safe inhalable lung contrast agent.
Keywords: Diatrizoic acid; Contrast medium; Pulmonary delivery; Dry powder; Aerosols; CT imaging;

Nanostructured lipid carriers constituted from high-density lipoprotein components for delivery of a lipophilic cardiovascular drug by Wen-Li Zhang; Xiao Gu; Hui Bai; Ru-Hui Yang; Chen-Dong Dong; Jian-Ping Liu (313-321).
To investigate the possibility of reconstituted protein-free high-density lipoprotein (HDL) being a carrier for delivering a lipophilic cardiovascular drug, Tanshinone IIA-loaded HDL-like nanostructured lipid carriers (TA-NLC) were prepared by a nanoprecipitation/solvent diffusion method. The physicochemical parameters of TA-NLC were characterized in terms of particle size, zeta potential, morphology, entrapment efficiency, differential scanning calorimetry (DSC) and stability. A novel two-step method has been employed to determine entrapment efficiency of TA-NLC. The binding properties of TA-NLC to apolipoproteins were investigated by in vitro incubation competition assay in the presence of native HDL and electrophoresis test. Phagocytosis and cytotoxicity was evaluated using mouse macrophage cell line RAW 264.7 with TA-NLC and incubated TA-NLC with native HDL (TA-NLC-apo). The results showed that TA-NLC had an average diameter of 8.0 ± 1.2 nm, zeta potential of −29.0 ± 0.0 mV, drug loading of 6.17 ± 0.3% and entrapment efficiency of 97.84 ± 1.2%. TA-NLC were demonstrated spheres with drug incorporated in lipid core forming a shell-core structure. DSC analysis showed that TA was dispersed in NLC in an amorphous state. The incorporation of glycerol trioleate to NLC led to crystal order disturbance. Agarose gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-SPAGE) patterns indicated that TA-NLC could bind to apolipoprotein A-I (apoA-I) specifically in vitro. Phagocytosis studies showed significant differences in uptake between TA-NLC and TA-NLC-apo and demonstrated that TA-NLC incubated with native HDL could turn endogenous by association to apolipoproteins, which cannot trigger immunological responses and could escape from recognition by macrophages.
Keywords: HDL; Nanostructured lipid carrier; Electrophoresis; Phagocytosis; Apolipoprotein A-I;

PX-18 is a new highly potent phospholipase A2 inhibitor with a poor aqueous solubility. Therefore, it was formulated as nanosuspensions with an active content of 1% (w/w), 5% (w/w) and 10% (w/w) using high-pressure homogenization. By contact angle measurements on compressed discs of PX-18, Tween 80 was identified as best wetting agent of PX-18 and therefore used to stabilize the nanosuspensions. The achieved particle size of all nanosuspensions was well in the nanometer range. For the 1% (w/w) PX-18 nanosuspension an average particle size below 50 nm was measured by photon correlation spectroscopy (PCS). A good reproducibility of the mean particle size was found in between different batches. As postulated by the zeta potential of the nanosuspensions, a good physical stability over an observation period of 180 days was obtained when stored at 5 ± 3 °C. PX-18 formulated as nanosuspensions was chemically stabile. An increase in saturation solubility was found after formulating PX-18 as nanosuspension compared to bulk material, favourable for increasing bioavailability.
Keywords: High-pressure homogenization; Nanosuspension; Drug nanocrystals; Saturation solubility;