International Journal of Pharmaceutics (v.456, #2)

Introduction of a new scaling approach for particle size reduction in toothed rotor-stator wet mills by Joshua Engstrom; Chenchi Wang; Chiajen Lai; Jason Sweeney (261-268).
A new scaling approach has been demonstrated for particle size reduction of an active pharmaceutical ingredient (API) in toothed rotor-stator wet mills for a wide range of wet milling conditions and scales. Compound A was crystallized by a cooling crystallization protocol in ethanol and wet milled with 2 types of lab scale mills and 1 pilot plant mill. The particle size was analyzed by FBRM (focused beam reflectance measurement), static light scattering and optical microscopy. The correlation between particle size and the energy input calculations, tailored for the toothed rotor-stator wet mill, had R 2  = 0.90 on a logarithmic plot. The new scaling approach forms the basis to correlate API particle size to energy input in toothed rotor-stator wet mills by accounting for wet mill tooth configuration and geometry, rotor tip speed and the probabilistic effects of collision such as shear frequency and slurry residence time.
Keywords: Comminution; Wet milling; Tip speed; Shear frequency;

Dexamethasone-loaded poly(ɛ-caprolactone)/silica nanoparticles composites prepared by supercritical CO2 foaming/mixing and deposition by M.B.C. de Matos; A.P. Piedade; C. Alvarez-Lorenzo; A. Concheiro; M.E.M. Braga; H.C. de Sousa (269-281).
A supercritical carbon dioxide (scCO2)-assisted foaming/mixing method (SFM) was implemented for preparing dexamethasone (DXMT)-loaded poly(ɛ-caprolactone)/silica nanoparticles (PCL/SNPs) composite materials suitable for bone regeneration. The composites were prepared from PCL and mesoporous SNPs (MCM-41/SBA-15) by means of scCO2-assisted SFM at several operational pressures, processing times and depressurization conditions. DXMT was loaded into SNPs (applying a scCO2 solvent impregnation/deposition method – SSID) and into PCL/SNPs composites (using the SFM method). The effects of the employed operational and compositional variables on the physicochemical and morphological features as well as in the in vitro release profiles of DXMT were analyzed in detail. This work demonstrates that the above-referred scCO2-based methods can be very useful for the preparation of DXMT-loaded PCL/SNPs composites with tunable physicochemical, thermomechanical, morphological and drug release properties and suitable for hard-tissue regeneration applications.
Keywords: Poly(ɛ-caprolactone); Mezoporous silica nanoparticles; Composites; Supercritical CO2 foaming/mixing; Supercritical CO2 impregnation/deposition;

The flavonol quercetin is potentially clinically relevant for its antimicrobial, beneficial cardiovascular effects, cancer treatment amongst others. However, its successful therapeutic application is severely curtailed by its poor water solubility and poor absorption following oral administration. In this study, solid dispersions of quercetin in poly(ethylene glycol) (PEG) at various compositions demonstrated an increase in the solubility, however with time, dissolution profiles show a decrease in dissolved flavonol concentration. The mechanism by which this decrease in solubility occurs was studied experimentally as well as by computational mesocscale particle dynamics simulations. The results suggest that phase separation of the polymer and flavonol during release from the solid dispersion is responsible for the time-dependent decrease in dissolved quercetin. It is suggested that the increase in release of quercetin in a PEG solid dispersion would only be beneficial if it were administered at the site of absorption, e.g. rectal administration, to ensure absorption prior to phase separation. The solid dispersions presented here would greatly improve the pharmaceutical availability of the flavonol at the site of absorption. Computational mesoscopic modeling was successfully applied to study the solid dispersions and corroborate experimental findings.
Keywords: PEG; Quercetin; Solubility; Solid dispersion; Mesoscopic computational dynamics; Coarse-graining;

A novel application of indolicidin for gene delivery by Wei-Wen Hu; Ze-Wei Lin; Ruoh-Chyu Ruaan; Wen-Yih Chen; Shiow-Lian Catherine Jin; Yung Chang (293-300).
A bovine derived antimicrobial peptide, indolicidin (IL), was studied of its new application for gene transfer. Plasmid DNA was complexed with both IL and polyethylenimine (PEI) as ternary particles. Compared to DNA/IL complexes, the DNA/IL/PEI particles demonstrated high zeta potentials, small particle sizes, and superior loading efficiencies, suggesting the incorporation of polycations can support IL for gene delivery. For in vitro experiments, these ternary particles significantly improved gene transfection efficiencies over the sole administrations of IL or PEI. This synergistic effect revealed that IL and PEI may play different roles for gene transfer. Our results suggest that IL should be a potential carrier for gene delivery. As our knowledge, our study should be the first article indicating the carrier ability of IL for gene transfer.
Keywords: Indolicidin; Cell-penetrating peptide; Gene delivery; Nonviral vectors; Transfection;

Inhibition of surface crystallisation of amorphous indomethacin particles in physical drug–polymer mixtures by Petra A. Priemel; Riikka Laitinen; Sarah Barthold; Holger Grohganz; Vesa-Pekka Lehto; Thomas Rades; Clare J. Strachan (301-306).
Surface coverage may affect the crystallisation behaviour of amorphous materials. This study investigates crystallisation inhibition in powder mixtures of amorphous drug and pharmaceutical excipients. Pure amorphous indomethacin (IMC) powder and physical mixtures thereof with Eudragit® E or Soluplus® in 3:1, 1:1 and 1:3 (w/w) ratios were stored at 30 °C and 23 or 42% RH. Samples were analysed during storage by X-ray powder diffraction, thermogravimetric analysis, differential scanning calorimetry, and scanning electron microscopy (SEM). IMC Eudragit® mixtures showed higher physical stability than pure IMC whereas IMC Soluplus® mixtures did not.Water uptake was higher for mixtures containing Soluplus® than for amorphous IMC or IMC Eudragit® mixtures. However, the T g of amorphous IMC was unaffected by the presence (and nature) of polymer. SEM revealed that Eudragit® particles aggregated on the surface of IMC particles, whereas Soluplus® particles did not. The drug particles developed multiple crystallites at their surface with subsequent crystal growth.The intimate contact between the surface agglomerated Eudragit® particles and drug is believed to inhibit crystallisation through reduced IMC surface molecular mobility. Polymer particles may also mechanically hinder crystal growth outwards from the surface. This work highlights the importance of microparticulate surface coverage of amorphous drug particles on their stability.
Keywords: Amorphous; Surface crystallisation; Spatial separation; Indomethacin; Excipients;

Assessing the physical–chemical properties and stability of dapivirine-loaded polymeric nanoparticles by José das Neves; Mansoor Amiji; Maria Fernanda Bahia; Bruno Sarmento (307-314).
Nanocarriers may provide interesting delivery platforms for microbicide drugs and their characterization should be addressed early in development. Differently surface-engineered dapivirine-loaded, poly(epsilon-caprolactone) (PCL)-based nanoparticles (NPs) were obtained by nanoprecipitation using polyethylene oxide (PEO), sodium lauryl sulfate (SLS), or cetyltrimethylammonium bromide (CTAB) as surface modifiers. Physical–chemical properties of NP aqueous dispersions were evaluated upon storage at −20–40 °C for one year. NPs presented 170–200 nm in diameter, roundish-shape, low polydispersity index (≤0.18), and high drug association efficiency (≥97%) and loading (≥12.7%). NPs differed in zeta potential, depending on surface modifier (PEO: −27.9 mV; SLS: −54.7 mV; CTAB: +42.4 mV). No interactions among formulation components were detected by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR), except for SLS–PCL NPs. Colloidal properties of NPs were lost at −20 °C storage. Negatively charged NPs were stable up to one year at 5–40 °C; as for CTAB–PCL NPs, particle aggregation was observed from 30 to 90 days of storage depending on temperature. Colloidal instability affected the in vitro drug release of CTAB–PCL NPs after 360 days. In any case, no degradation of dapivirine was apparent. Overall, PEO–PCL and SLS–PCL NPs presented suitable properties as nanocarriers for dapivirine. Conversely, CTAB–PCL NPs require additional strategies in order to increase stability.
Keywords: HIV/AIDS; Microbicides; Poly(epsilon-caprolactone); Colloidal stability; Drug release;

The transmembrane transport of drug loaded micelles to intracellular compartment is quite crucial for efficient drug delivery. In the current study, we investigated the cellular internalization and anticancer activity of doxorubicin loaded micelles with folate modified stealthy PEOz corona. Folate-decorated micelles incorporating doxorubicin were characterized for particle size, degree of folate decoration, drug loading content and encapsulation efficiency, morphology, and surface charge. The targeting capability and cell viability were assessed using HeLa, KB, A549 and MCF-7/ADR cell lines. In vitro study clearly illustrated the folate receptor (FR) mediated targeting of FA modified micelles to FR-positive human HeLa, KB and MCF-7/ADR cells, while specific delivery to FR-negative A549 cells was not apparently increased at the same experimental conditions. Cytotoxicity assay showed 60% and 58% decrease in IC50 values for HeLa and KB cells, while only a slight decrease for A549 cells, following treatment with folate modified formulations. The enhanced intracellular delivery of FA modified micelles in MCF-7/ADR cells was also observed. In vivo antitumor tests revealed DOX entrapped FA-PEOz-PCL micelles effectively inhibited the tumor growth and reduced the toxicity to mice compared with free DOX. The current study showed that the targeted nano-vector improved cytotoxicity of DOX and suggested that this novel PEOz endowed stealthy micelle system held great promise in tumor targeted therapy.
Keywords: Antitumor; Doxorubicin; Folic acid; PEOz-PCL; Targeting;

Internal mouthpiece designs as a future perspective for enhanced aerosol deposition. Comparative results for aerosol chemotherapy and aerosol antibiotics by Paul Zarogoulidis; Dimitris Petridis; Christos Ritzoulis; Kaid Darwiche; Ioannis Kioumis; Konstantinos Porpodis; Dionysios Spyratos; Wolfgang Hohenforst-Schmidt; Lonny Yarmus; Haidong Huang; Qiang Li; Lutz Freitag; Konstantinos Zarogoulidis (325-331).
In an effort to identify factors producing a finest mist from Jet-Nebulizers we designed 2 mouthpieces with 4 different internal designs and 1–3 compartments.Ten different drugs previous used with their “ideal” combination of jet-nebulizer, residual-cup and loading were used. For each drug the mass median aerodynamic diameter size had been established along with their “ideal” combination.For both mouthpiece, drug was the most important factor due the high F-values (F large  = 251.7, p  < 0.001 and F small  = 60.1, p  < 0.001) produced. The design affected the droplet size but only for large mouthpiece (F large  = 5.99, p  = 0.001, F small  = 1.72, p  = 0.178). Cross designs create the smallest droplets (2.271) so differing from the other designs whose mean droplets were greater and equal ranging between 2.39 and 2.447. The number of compartments in the two devices regarding the 10 drugs was found not statistically significant (p-values 0.768 and 0.532 respectively). Interaction effects between drugs and design were statistically significant for both devices (F large  = 8.87, p  < 0.001, F small  = 5.33, p  < 0.001).Based on our experiment we conclude that further improvement of the drugs intended for aerosol production is needed. In addition, the mouthpiece design and size play an important role in further enhancing the fine mist production and therefore further experimentation is needed.
Keywords: Aerosol; Mouthpiece; Designs;

Is there any association between imidapril hydrochloride stability profile under dry air conditions and cancer initiation? by Katarzyna Regulska; Marek Murias; Beata Stanisz; Miłosz Regulski (332-339).
Stability study for imidapril hydrochloride (IMD) was performed under stress conditions of increased temperature (T  = 373 K) and decreased relative air humidity (RH = 0%) in order to obtain and identify its degradation product. The degradation sample stored for 15 days under the above environmental conditions was analyzed by LC–MS technique and it was found that the only degradation impurity formed in the course of the investigated drug degradation was IMD diketopiperazine derivative (DKP) which was produced by dehydration and intramolecular cyclization. The kinetics of its formation was analyzed by a revalidated RP-HPLC method and the kinetic model of this reaction was established. It was concluded that the DKP formation follows Prout–Tompkins kinetics with the rate constant k  ± Δk =  2.034 ± 0.157 × 10−6 [s−1]. The obtained degradation impurity was further assessed with respect to its mutagenic potential using commercial Ames MPF 98/100 microplate format mutagenicity assay kit equipped with Salmonella typhimurium strains TA 98 and TA 100. Both strains were exposed to six concentrations (in a range of 0.16–5.0 mg/mL) of DKP in the presence and absence of metabolic activation system. No mutagenic effect was observed confirming that the presence of DKP in IMD final dosage form has no impact on cancer initiation.
Keywords: Imidapril hydrochloride; Degradation; Mutagenicity; Bacterial reverse mutation assay;

On the ubiquitous presence of fractals and fractal concepts in pharmaceutical sciences: A review by Natassa Pippa; Aristides Dokoumetzidis; Costas Demetzos; Panos Macheras (340-352).
Fractals have been very successful in quantifying nature's geometrical complexity, and have captured the imagination of scientific community. The development of fractal dimension and its applications have produced significant results across a wide variety of biomedical applications. This review deals with the application of fractals in pharmaceutical sciences and attempts to account the most important developments in the fields of pharmaceutical technology, especially of advanced Drug Delivery nano Systems and of biopharmaceutics and pharmacokinetics. Additionally, fractal kinetics, which has been applied to enzyme kinetics, drug metabolism and absorption, pharmacokinetics and pharmacodynamics are presented. This review also considers the potential benefits of using fractal analysis along with considerations of nonlinearity, scaling, and chaos as calibration tools to obtain information and more realistic description on different parts of pharmaceutical sciences. As a conclusion, the purpose of the present work is to highlight the presence of fractal geometry in almost all fields of pharmaceutical research.
Keywords: Fractal analysis; Morphology; Advanced Drug Delivery nano Systems (aDDnSs); Nanotechnology; Biopharmaceutics; Pharmacokinetics;

Focused beam reflectance measurement to monitor nimodipine precipitation process by Xiaoming Xu; Akhtar Siddiqui; Mansoor A. Khan (353-356).
Crystallization of nimodipine in liquid-filled soft gelatin capsule during storage was reported for some commercial products, resulting in product recalls due to product quality and more importantly safety concerns. In this study, a real time particle monitoring tool, focused beam reflectance measurement, was used to evaluate the precipitation conditions of nimodipine in co-solvents. Upon water addition, two particle populations were discovered, appearing at different percentage of water content. Two transitions (i.e. sudden increase in particle counts) were observed, possibility related to nucleation and crystal growth of nimodipine. Furthermore, lowering storage temperature increased the tendency of nimodipine precipitation. Most critically, it was determined that with certain excipient, the drug precipitation occurred at approximately 7% (w/w) water content. Considering that all the orally administered liquid filled soft gelatin capsule shells contain residue water content as plasticizer, moisture transfer from the shell to the formulation may occur during long term storage, resulting in drug precipitation, particularly under cold temperature conditions.
Keywords: Nimodipine; Crystallization; Focused beam reflectance measurement; Moisture content; Liquid-filled capsule;

Transungual delivery of ketoconazole using novel lacquer formulation by Farhaan Hafeez; Xiaoying Hui; Audris Chiang; Sidney Hornby; Howard Maibach (357-361).
Onychomycosis, a common fungal infection of the nail, can have a substantial impact on quality of life. The success of topical therapy for onychomycosis depends on effective penetration, which can be enhanced using an appropriate delivery method. This study evaluated the effectiveness of a novel topical lacquer on enhancing [14C]-ketoconazole penetration by comparing nail absorption, nail distribution, and nail penetration of [14C]-ketoconazole dissolved in the novel lacquer versus a commercial ketoconazole cream. Using the in vitro finite dose model, the formulations were applied daily to human nail plates for 7 days. Drug absorption was measured by monitoring rate of appearance in each nail layer and the supporting bed. After the multiple day treatment, cumulative concentrations of ketoconazole formulated in novel lacquer in the deep nail layer and the nail bed were significantly greater than cumulative concentrations of commercial ketoconazole (p  < 0.05), as well as several orders of magnitude greater than the minimal inhibitory concentration (MIC) deemed necessary to inhibit the growth of causative dermatophytic and yeast species. These results suggest that this novel ketoconazole lacquer has the potential to be an effective topical treatment for onychomycosis.
Keywords: Onychomycosis; Ketoconazole; Topical formulation; Human nail; In vitro; MIC;

Effect of permeation enhancers on transdermal delivery of fluoxetine: In vitro and in vivo evaluation by Eunjae Jung; Yun Pyo Kang; In-Soo Yoon; Jung Sun Kim; Sung Won Kwon; Suk-Jae Chung; Chang-Koo Shim; Dae-Duk Kim (362-369).
The aim of this study was to investigate the feasibility of transdermal fluoxetine (FX) delivery. The effects of chemical forms (base or salt) and permeation enhancers on in vitro skin permeation of FX were assessed using hairless mouse, rat and human cadaver skin. The optimized formulations from the in vitro studies were then evaluated in an in vivo pharmacokinetic study in rats. The in vitro skin permeation studies suggested that the FX base (FXB) and isopropyl myristate (IPM)–limonene mixture could be suitable for transdermal delivery of FX. The permeation parameters of FX through human cadaver skin were well correlated with that through hairless mouse and rat skin, suggesting that these animal models can be used for predicting the permeability of FX through human skin. After transdermal administration of FX with IPM or the IPM–limonene mixture to rats, the mean steady-state plasma concentration (C ss) was 66.20 or 77.55 ng/mL, respectively, which was maintained over 36 h and had a good correlation with the predicted C ss from the in vitro data. These in vitro and in vivo data demonstrated that permeation enhancers could be a potential strategy for transdermal delivery of FX.
Keywords: Fluoxetine; Transdermal delivery; Permeation enhancers; Pharmacokinetics;

Effect of freeze-drying, cryoprotectants and storage conditions on the stability of secondary structure of insulin-loaded solid lipid nanoparticles by Sandra Soares; Pedro Fonte; Ana Costa; José Andrade; Vítor Seabra; Domingos Ferreira; Salette Reis; Bruno Sarmento (370-381).
This study aims to monitor the secondary structure behaviour of insulin when it is encapsulated into solid lipid nanoparticles (SLN), under the influence of several critical processing parameters. Insulin was used as a therapeutic protein model. Physicochemical properties of insulin-loaded SLN (Ins-SLN) were assessed, with special focus on the insulin secondary structure after its encapsulation into SLN and after freeze-drying using different cryoprotectants (glucose, fructose and sorbitol). Additionally, a 6-month stability study was performed to evaluate the maintenance of insulin secondary structure over time at different storage conditions (4 °C/60% RH, 25 °C/60% RH, 40 °C/75% RH).Ins-SLN were successfully produced with a mean and narrow particle size around 400 nm, zeta potential around −13 mV, an insulin association efficiency of 84%. Physical–chemical properties of SLN were maintained after freeze-drying. FTIR results showed that encapsulated insulin maintained a native-like structure in a degree of similarity around 92% after production, and 84% after freeze-drying. After 6 months, freeze-dried Ins-SLN without cryoprotectant stored at 40 °C/75% RH presented the same degree of structure preservation and morphology. Results revealed that insulin structure can be significantly protected by SLN matrix itself, without a cryoprotectant agent, even using a non-optimized freeze-drying process, and under the harsher storage conditions. Multivariable experimental settled the process parameters to fit with the desired product quality attributes regarding protein and nanoparticle stability.
Keywords: Insulin secondary structure; Solid lipid nanoparticles; Freeze-drying; Cryoprotectants; Stability study; FTIR;

Owing to a variety of structural and functional properties, milk proteins are steadily studied for food and pharmaceutical applications. In the present study, calcium caseinate in association with lecithin was firstly investigated in order to encapsulate the acetaminophen through spray-drying for taste-masking purpose for pediatric medicines. A 24-full factorial design revealed that the spray flow, the calcium caseinate amount and the lecithin amount had significant effects on the release of drug during the first 2 min. Indeed, increasing the spray flow and/or the calcium caseinate amount led to increase the released amount, whereas increasing the lecithin amount decreased the released amount. The “interaction” between the calcium caseinate amount and the lecithin amount was also shown to be statistically significant. The second objective was to compare the efficiency of two caseinate-based formulations, i.e. sodium caseinate and calcium caseinate, on the taste-masking effect. The characteristics of spray-dried powders determined by SEM and DSC were shown to depend on the caseinate/lecithin proportion rather than the type of caseinate. Interestingly, calcium caseinate-based formulations were found to lower the released amount of drug during the early time to a higher extent than sodium caseinate-based formulations, which indicates better taste-masking efficiency.
Keywords: Pediatric formulation; Taste-masking; Spray-drying; Calcium caseinate; Sodium caseinate;

Gastrointestinal gene delivery by cyclodextrins – In vitro quantification of extracellular barriers by Martin J. O’Neill; Aoife M. O’Mahony; Colin Byrne; Raphael Darcy; Caitriona M. O’Driscoll (390-399).
Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers.Stability and transfection efficiency of CD·DNA complexes were assessed post incubation in simulated gastric and intestinal fluids, bile salts and mucin, or with intestinal enzymes to represent extracellular barriers to intestinal gene delivery. Stability was determined by gel electrophoresis and transfection was measured by luciferase expression in intestinal epithelial cells (Caco-2).Transfection efficiency of CD·DNA complexes was enhanced after incubation in bile salts but was reduced after incubation in gastric and intestinal fluids and mucin. CD·DNA complexes were stable after incubation with pancreatic enzymes and with a model lower intestinal enzyme. Furthermore, the CD protected pDNA from degradation by DNase.In summary, physiologically relevant in vitro models were established and used to quantify the barriers posed by the intestinal extracellular environment to gene delivery. This systematic assessment identified the advantages and limitations of the CD vector and facilitated the proposal of formulation strategies to overcome these barriers.
Keywords: Cyclodextrins; Gene delivery; Intestinal barriers; Non-viral vectors; Stability;

A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles by M.Gulrez Zariwala; Naba Elsaid; Timothy L. Jackson; Francisco Corral López; Sebastien Farnaud; Satyanarayana Somavarapu; Derek Renshaw (400-407).
Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3 ± 31.75 nm to 495.1 ± 80.42 nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20 μM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98 ± 40.83 ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77 ± 29.37 ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66 ± 20.43 ng/mg cell protein) (p  ≤ 0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery.
Keywords: Solid lipid nanoparticles; Ferrous sulfate; Iron supplementation; Chitosan; Caco-2; Ferritin;

Novel biotinylated chitosan-graft-polyethyleneimine copolymer as a targeted non-viral vector for anti-EGF receptor siRNA delivery in cancer cells by Mohammad H. Darvishi; Alireza Nomani; Mohsen Amini; Mohammad A. Shokrgozar; Rassoul Dinarvand (408-416).
The major impediments to develop an efficient non-viral siRNA-mediated gene silencing method, as a therapeutic approach, are the low cellular uptake and intracellular delivery and release of non-viral vectors. To overcome these problems, designing a proper vector with high transfection efficiency is obviously under scrutiny of various studies. The present study, evaluate a novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer as an appropriate non-viral vector for targeted delivery of siRNA to cancer cells. The composition of the synthesized Bio-Chi-g-PEI copolymer was thoroughly characterized using 1H NMR and FTIR spectroscopy, besides the hydroxyazobenzene-2-carboxylic acid (HABA) assay. In vitro cytotoxicity assay of the Bio-Chi-g-PEI copolymers was performed by MTT assay. Cytotoxicity evaluations indicated that the new copolymer was markedly less toxic than PEI 25KD. Physicochemical properties of the Bio-Chi-g-PEI/siRNA complexes such as complex stability, size, zeta potential, and their morphology at various weight ratios, investigated by appropriate methods, revealed the suitability of the complexes for the transfection. The efficient cellular internalization of the complexes for HeLa and OVCAR-3 cells in culture media was confirmed by intracellular tracking of the prepared complexes using confocal laser scanning microscopy and Cy3-labeled anti-epidermal growth factor receptor siRNA. Finally, evaluation of the transfection efficiency and gene silencing by flow cytometry and real-time polymerase chain reaction highlighted the significantly higher efficiency of transfection and silencing for biotinylated copolymer compared with the PEI 25KD and non-biotinylated copolymer.
Keywords: Gene silencing; siRNA delivery; Biotinylation; Chitosan-graft-polyethyleneimine; Tumor-targeting delivery; Epidermal growth factor receptor;

Tailored beads made of dissolved cellulose—Investigation of their drug release properties by Emrah Yildir; Ruzica Kolakovic; Natalja Genina; Jani Trygg; Martin Gericke; Leena Hanski; Henrik Ehlers; Jukka Rantanen; Mikko Tenho; Pia Vuorela; Pedro Fardim; Niklas Sandler (417-423).
In the frame of this work, we have investigated drug entrapping and release abilities of new type of porous cellulose beads (CBs) as a spherical matrix system for drug delivery. For that purpose, CBs prepared with three different methods were used as drug carriers and three compounds, anhydrous theophylline (Thp), riboflavin 5′-phosphate sodium (RSP) and lidocaine hydrochloride monohydrate (LiHCl) were used as model drug substances. The loading procedure was carried out by immersing swollen empty beads into the solutions of different concentrations of model drugs. The morphology of empty and loaded beads was examined using a field emission scanning electron microscopy (FE-SEM). Near-infrared (NIR) imaging was performed to identify the drug distributions on and within the loaded CBs. The drug amount incorporated into CBs was examined spectrophotometrically and in vitro drug release studies were performed to determine the drug release rates. The results of FE-SEM and chemical NIR imaging analyses revealed that incorporated drug were distributed on the surface and but also within the internal structure of the CBs. Physical properties of CBs and solubility of model drugs had effect on loading efficacy. Also, the drug release rates were controlled by solubility of model drugs (diffusion controlled release). In conclusion, CBs from dissolved cellulose show promise in achieving controlled drug delivery.
Keywords: Porous cellulose beads; Drug delivery; Macrospheres; Drug loading; Chemical imaging;

The effect of PEG-5K grafting level and particle size on tumor accumulation and cellular uptake by Chun-Liang Lo; Meng-Han Chou; Pei-Lin Lu; I-Wen Lo; Yi-Ting Chiang; Shang-Yu Hung; Chieh-Yu Yang; Shuian-Yin Lin; Shiaw-Pyng Wey; Jem-Mau Lo; Ging-Ho Hsiue (424-431).
PEG-modified gold nanoparticles (PEG-modified GNs) with diameters of 40 nm and 70 nm were prepared to elucidate the effect of extent of PEG (M.W. 5000) grafting and particle size on tumor accumulation and cellular uptake. Flow cytometry reveals that cellular uptake is strongly related to the size of PEG-modified GNs, rather than the extent of PEG-5K grafting level. Cytotoxicity analysis based on the intracellular release of drugs showed that the 70 nm PEG-modified GNs have the higher cytotoxicity, beccause of their greater cellular uptake. Also, particle size, rather than PEG-5K grafting level affects tumor accumulation. However, PEG-5K grafting level significantly affects the accumulation of particles in the liver and spleen. This finding is important in determining the proper PEG-5K grafting level and particle size for designing nano-medicines.
Keywords: Gold nanoparticles; Surface modification; Polyethylene glycol; Cellular uptake; Biodistribution;

Formulation and in vitro/in vivo evaluation of levodopa transdermal delivery systems by Kyung Eun Lee; Yun Jung Choi; Byu Ree Oh; In Koo Chun; Hye Sun Gwak (432-436).
This study aims to investigate the feasibility of Levodopa transdermal delivery systems (TDSs). Levodopa TDSs were formulated using various vehicles and permeation enhancers, and in vitro permeation and in vivo pharmacokinetic studies were carried out. In the in vitro study, ester-type vehicles showed relatively high enhancing effects; propylene glycol monocaprylate and propylene glycol monolaurate showed the highest permeation fluxes from both solution and pressure sensitive adhesive (PSA) TDS formulations. Lag time was dramatically shortened with PSA TDS formulations as compared with solution formulations. In the in vivo study, the addition of fatty acids increased blood drug concentrations regardless of the kind or concentration of fatty acid; the AUCinf increased up to 8.7 times as compared with propylene glycol (PG) alone. PSA TDS containing 10% linoleic acid exhibited prolonged T max as compared with oral form. Total clearance of L-dopa from PSA TDSs was significantly lower than from oral form (up to 86.8 times). Especially, PSA TDS containing 10% linoleic acid (LOA) revealed 76.2 fold higher AUCinf than oral administration. Based on our results, the L-dopa PSA TDS containing PG with 10% LOA could be used as a good adjuvant therapy for Parkinson's disease patients who experience symptom fluctuation by L-dopa oral administration.
Keywords: Levodopa; Transdermal delivery system; Permeation; Pharmacokinetics;

Micro- and nanobubbles: A versatile non-viral platform for gene delivery by Roberta Cavalli; Agnese Bisazza; David Lembo (437-445).
Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1–8 μm, characterised by their core–shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or ‘loaded’ in purposed-built bubble systems – indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described.
Keywords: Microbubbles; Nanobubbles; Nanodroplets; Gene delivery; Ultrasound; Non viral carriers;

Formulation, characterization and pharmacokinetics of Morin hydrate niosomes prepared from various non-ionic surfactants by Ayman Y. Waddad; Sarra Abbad; Fan Yu; Were L.L. Munyendo; Jing Wang; Huixia Lv; Jianping Zhou (446-458).
Morin hydrate (MH), a bioflavonoid with antioxidant and anticancer activity as well as the ability to improve the bioavailability of other drugs on their concurrent use. Three differently optimized niosomal formulations using three different non-ionic surfactants (Span 60, Span 80 and Tween 60) were achieved by (L9 (34)) Taguchi orthogonal array (TOA). The analysis of TOA revealed that Tween 60 Niosomes had the highest entrapment efficiency (93.4%) compared to other optimized Niosomal formulations (71–79%). In terms of MH remaining %, Tween 60 Niosomes were found to be the most stable (89%) at 4 °C over one month compared to Span 60 (56%) and Span 80 (57%) Niosomes. The release pattern in all Niosomal formulations was found to follow the Weibull model and Tween 60 Niosomes had the highest release rate. The molecular modeling simulation explained the binding of MH to the human serum albumin (HSA) by hydrogen bonds during the in vitro release process. As for the bioavailability, the AUC0–8 showed 1.3–2.7 fold increase compared to the MH solution. Ex vivo images of the excised organs showed that MH could accumulate in brain which indicates that MH-Tween 60 Niosomes might be a possible candidate to deliver hydrophobic drugs and overcome the blood–brain barrier (BBB) penetration.
Keywords: Morin hydrate; Niosomes; Orthogonal array; Drug delivery; Pharmacokinetics; Molecular modeling;

A novel pH-sensitive interferon-β (INF-β) oral delivery system for application in multiple sclerosis by Pierre P.D. Kondiah; Lomas K. Tomar; Charu Tyagi; Yahya E. Choonara; Girish Modi; Lisa C. du Toit; Pradeep Kumar; Viness Pillay (459-472).
pH-sensitive microparticles were prepared using trimethyl-chitosan (TMC), poly(ethylene glycol)dimethacrylate (PEGDMA) and methacrylic acid (MAA) by free radical suspension polymerization, for the oral delivery of interferon-β (INF-β). The microparticles were subsequently compressed into a suitable oral tablet formulation. A Box–Behnken experimental design was employed for generating a series of formulations with varying concentrations of TMC (0.05–0.5 g/100 mL) and percentage crosslinker (polyethylene glycol diacrylate) (3–8%, w/w of monomers), for establishment of an optimized TMC-PEGDMA-MAA copolymeric microparticles. For pragmatism, insulin was initially employed as the model peptide for undertaking the preliminary experimentation and the optimized formulation was subsequently evaluated using INF-β. The prepared copolymeric microparticulate system was characterized for its morphological, porositometric and mucoadhesive properties. The optimized microparticles with 0.5 g/100 mL TMC and 3% crosslinker had an INF-β loading efficiency of 53.25%. The in vitro release of INF-β was recorded at 74% and 3% in intestinal (pH 6.8) and gastric (pH 1.2) pH from the oral tablet formulation, respectively. The tablet was further evaluated for plasma concentration of INF-β in the New Zealand White rabbit, and compared to a known subcutaneous formulation. The system showed an astounding effective release profile over 24 h with higher INF-β plasma concentrations compared with the subcutaneous injection formulation.
Keywords: Oral delivery system; Interferon-β (INF-β); Insulin; Box–Behnken experimental design; Copolymeric microparticulate system; pH-responsive;

Preactivated thiomers: Evaluation of gastroretentive minitablets by Sabine Hauptstein; Christiane Müller; Sarah Dünnhaupt; Flavia Laffleur; Andreas Bernkop-Schnürch (473-479).
The object of this study was to evaluate the potential of a recently developed preactivated thiolated pectin derivative as mucoadhesive excipient in drug delivery to the gastric cavity. Pectin (Pec) was chemically modified with l-cysteine (Cys). The free thiol groups of resulting thiomer were activated with 2-mercaptonicotinic acid (MNA) in order to improve stability and reactivity of attached thiol groups over a broad pH range. Multiunit dosage form properties of the resulting conjugate (Pec–Cys–MNA) were compared to unmodified pectin and the intermediate thiolated using rosuvastatin calcium as a model drug in loaded minitablets. Obtained results were compared with unmodified pectin and the intermediate thiolated pectin. Approximately half of attached thiol groups (507 μmol/g polymer) have been preactivated. Minitablets were evaluated regarding mucoadhesive properties, hardness, disintegration behavior, swelling characteristics and release of rosuvastatin calcium. Mediated by covalent bonds between the polymer and cysteine-rich subdomains in mucus, total work of adhesion increased more than 5-fold. The modification had no impact on hardness of compressed tablets but implementation of the aromatic ligand went along with reduction in hydrophilic properties. Disintegration time was prolonged more than 2-fold while water uptake capacity increased. Weight gain for Pec–Cys–MNA was at least 16-fold. Further, a sustained release of rosuvastatin calcium over 36 h was determined. Neither biodegradability nor CaCo-2 cell viability was affected. The study shows that Pec–Cys–MNA is a promising excipient for the development of mucoadhesive gastric dosage form.
Keywords: Mucoadhesive drug delivery system; Preactivated thiomers; Swellable polymer; Rosuvastatin; Gastric;

Benzocaine polymorphism: Pressure–temperature phase diagram involving forms II and III by Inès Gana; Maria Barrio; Bernard Do; Josep-Lluís Tamarit; René Céolin; Ivo B. Rietveld (480-488).
Understanding the phase behavior of an active pharmaceutical ingredient in a drug formulation is required to avoid the occurrence of sudden phase changes resulting in decrease of bioavailability in a marketed product. Benzocaine is known to possess three crystalline polymorphs, but their stability hierarchy has so far not been determined. A topological method and direct calorimetric measurements under pressure have been used to construct the topological pressure–temperature diagram of the phase relationships between the solid phases II and III, the liquid, and the vapor phase. In the process, the transition temperature between solid phases III and II and its enthalpy change have been determined. Solid phase II, which has the highest melting point, is the more stable phase under ambient conditions in this phase diagram. Surprisingly, solid phase I has not been observed during the study, even though the scarce literature data on its thermal behavior appear to indicate that it might be the most stable one of the three solid phases.
Keywords: Thermodynamics; Physical stability; Phase diagram; Pressure; X-ray powder diffraction; Polymorphism;

The performance of two laboratory-scale extrusion apparatuses used to approximate the action of an industrial screen extruder, namely a multi-holed die ram extruder and a roller screen extruder, were compared. Both devices featured short dies (ram 2 mm, screen 1 mm) with die diameter 1 mm and hole area fraction approaching 0.25. A series of water/microcrystalline cellulose (MCC) pastes with water contents varying from 45 to 60 wt% were extruded and pellets obtained from subsequent spheronisation of the extrudates characterised in terms of size and shape. Each device exhibited a different range of processing windows for acceptable spheronised products, with the ram apparatus being able to extrude a wider range of paste water contents than the screen device. The pellets obtained from extrusion–spheronisation (E–S) of the pastes via the screen device were in general smaller, with a wider size distribution, than those from ram E–S. These results are attributed to the different mechanical histories experienced by the pastes in the two types of extruder, which lead to different extrudate densities being achieved. MCC/water/calcium carbonate pastes were also tested, where the latter component represented a ‘hard’ (non-deformable) active pharmaceutical ingredient. Addition of calcium carbonate increased the stiffness of the paste, which could be countered by adjusting the water content of the deformable MCC/water matrix within the extrudability limits of the latter material.
Keywords: Microcrystalline cellulose; Ram extrusion; Screen extrusion; Spheronisation;

Investigation on novel chitosan nanoparticle–aptamer complexes targeting TGF-β receptor II by Xia Chen; Xiaoyan Zhu; Lei Li; Guangjun Xian; Wei Wang; Dawei Ma; Lin Xie (499-507).
In our previous study, a dominant sequence called aptamer S58 antagonized TGF-β-induced myofibroblast transdifferentiation in human Tenon's capsule fibroblasts (HTFs) through sealing the targeting site of TGF-β receptor II (TβR II). However, rapid degradation by ubiquitous nucleases limited the aptamer's efficacy. Chitosan-nanoparticles (CS-NP) are good drug carriers. Herein we synthesises novel chitosan nanoparticle–aptamer S58 complexes called CS(S58)-NP in order to preserve and prolong S58's efficacy. We synthesised CS(S58)-NP at various molar ratios of CS-NP to S58 using an ionic gelation method. Then, the properties of the CS(S58)-NP including particle size, zeta potential, protection capacity, slow-release effect and cytotoxicity were studied. The targeting effect of the CS(S58)-NP was also studied. CS(S58)-NP at molar ratios of 20 and 30 showed high aptamer encapsulation efficiency, powerful aptamer protection, stable sustained-release ability and low cytotoxicity. FITC-labelled CS(S58)-NP could successfully bind to TβR II. As a result, TGF-β-induced cell proliferation and α-SMA expression were both inhibited. Furthermore, the CS(S58)-NP could inhibit TGF-β-induced α-SMA expression for a longer time than naked S58, even in serum. This research applied CS-NP as the aptamer carrier. The research results demonstrate that CS-NP are potentially able to preserve and prolong aptamer S58's efficacy. This study reveals that the use of CS-NP is promising for aptamer delivery and CS(S58)-NP can be a potential anti-scarring therapeutic approach after glaucoma filtration surgery.
Keywords: Chitosan; Nanoparticle; Aptamer; TGF-β; Anti-scarring; Human Tenon's capsule fibroblasts;

Reducing mechanical activation-induced amorphisation of salbutamol sulphate by co-processing with selected carboxylic acids by Vincent Curtin; Youness Amharar; Kieran H. Gallagher; Sarah Corcoran; Lidia Tajber; Owen I. Corrigan; Anne Marie Healy (508-516).
The unintentional generation of amorphous character in crystalline active pharmaceutical ingredients (APIs) is an adverse consequence of mechanical activation during dosage form manufacture. In this study, we assess and compare the ability of low glass transition temperature (T g) dicarboxylic acids to mitigate amorphisation of a model API, salbutamol sulphate (SS), on both co-milling and co-mixing.SS processed alone, as well as co-milled and co-mixed composites of the API with glutaric acid (GA), adipic acid (AA) and pimelic acid (PA) were characterised by powder X-ray diffraction (pXRD), differential scanning calorimetry (DSC) and dynamic vapour sorption (DVS).Milling and dry mixing of SS both resulted in pXRD amorphous materials. No amorphous content of SS was detected by DVS on co-milling with 50% (w/w) GA, while amorphisation was more than halved, relative to the API milled alone, on co-milling with 50% (w/w) AA and PA, respectively. Co-mixing with each excipient also resulted in a decrease in API amorphicity, although the extent of reduction was considerably less compared to the co-milling experiments.The solubility (Solexcipient) of each excipient in amorphous SS was determined by thermal methods. No further reduction in API amorphisation was achieved on co-mixing with 50% (w/w) excipient, compared to concentrations corresponding to the solubility of each excipient in the amorphous API (SolGA  = 36%, SolAA  = 21%, SolPA  = 22%). PXRD confirmed gradual dissolution over time of GA in amorphous SS on co-mixing. In contrast to co-mixing, co-milling SS at excipient weight fractions above their respective solubilities in the amorphous drug resulted in further reductions in API amorphisation. This is thought to be due to the generation of a molecular dispersion of amorphous API, supersaturated with excipient, thereby leading to a more pronounced composite T g lowering effect.The results indicate that co-processing with low T g excipients is an effective strategy at minimising amorphisation of an API on mechanical activation.
Keywords: Mechanical activation; Amorphous; Glass transition temperature; Salbutamol sulphate; Crystalline;

Size analysis of nanoparticles in commercial O/W sunscreens by C. Nagelreiter; C. Valenta (517-519).
Nanoparticles are employed in a variety of applications and especially in cosmetics the issue is discussed whether or not they can be regarded as safe. Analysis of nanosized structures and morphology studies prove to be difficult in many aspects. Nevertheless, there is the demand for new, cost-effective and simple yet reliable methods of analysis to assess the occurrence of nanoparticles in cosmetics in order to evaluate the possible risks conditioned by nanosized structures. In the present study, a simple method was developed to extract particles from commercial sunscreens that are O/W emulsions to measure the particle size of suspended material by laser diffraction. A following, simple calculation based on the specific surface area and particle size distribution allows distinguishing agglomerated nanoparticles from larger particles and thereby contributes well to the tools in analysis of cosmetic products. It was possible to create a simple, fast and cost-effective method to obtain an overview whether nanoparticles are included in a cosmetic product or not.
Keywords: Laser diffraction; Nanoparticles; Size estimation;

A study of the effects of sodium halides on the performance of air-jet and vibrating-mesh nebulizers by Mohammad Najlah; Asma Vali; Michael Taylor; Basel T. Arafat; Waqar Ahmed; David A. Phoenix; Kevin M.G. Taylor; Abdelbary Elhissi (520-527).
The influence of sodium halide electrolytes on aerosols generated from the Aeroneb Pro vibrating mesh nebulizer and the Sidestream air-jet nebulizer has been evaluated. Fluids with a range of concentrations of Na halides (i.e. NaF, NaCl, NaBr and NaI) were used as nebulizer solutions and their effect on aerosol properties such as total aerosol output, fine particle fraction (FPF), volume median diameter (VMD) and predicted regional airway deposition were investigated. For both nebulizers, the inclusion of electrolyte significantly enhanced the aerosol properties compared with HPLC grade (deionized) water. Aerosol output, FPF and aerosol fraction less than 2.15 μm were directly proportional to electrolyte concentration. Furthermore, the proportion of aerosols that are likely to deposit in the oropharyngeal region, and the VMD of the droplets were inversely related to the electrolyte concentration for both nebulizers. In general, the inclusion of electrolytes had a greater impact on the aerosol properties of the vibrating-mesh nebulizer. In the Aeroneb Pro, NaI 2.0% (w/v) was the optimum solution as it generated the highest aerosol output, FPF and output fraction below 2.15 μm with the lowest VMD and minimal predicted oropharyngeal deposition. This was attributed to the polarizing ability of iodide ions present in the largest quantity at the air–water interface. This study has shown that the Aeroneb Pro vibrating-mesh device demonstrated greatly enhanced aerosol properties when halides were included in the nebulizer solutions.
Keywords: Aerosol; Electrolyte; Halide; Polarizing ability; Surface tension; Nebulizer;

The tableting properties of melibiose monohydrate by Satu Lakio; Janne Sainio; Petteri Heljo; Tuomas Ervasti; Niina Kivikero; Anne Juppo (528-535).
In this research, the tableting properties of α-melibiose monohydrate were studied. Melibiose is a disaccharide which bears structural resemblance to lactose, because they both consist of galactose and glucose monosaccharide subunits. Compactibility and deformation behavior of two melibiose batches from different suppliers were studied and compared with α-lactose monohydrate and some other typical tableting excipients. Differences in the deformation behavior were determined comparing the shape of the Heckel plots, the yield pressure values and the strain rate sensitivity (SRS) indexes. In addition, the effect of moisture on the tabletability was studied. According to the yield pressures and SRS indexes melibiose was concluded to be fragmenting, even at higher degree than lactose monohydrate. However, the overall deformation behavior of melibiose was found to be similar to that of lactose monohydrate. Increase in moisture content resulted in higher tensile strengths of tablets for both melibiose batches, but it seemed to have more effect on compactibility of the other batch. In conclusion, melibiose has potential to be used as an excipient in tablet formulations.
Keywords: Melibiose; Lactose; Excipient; Tableting; Deformation; Compactibility;

Intra-articular fate of degradable poly(ethyleneglycol)-hydrogel microspheres as carriers for sustained drug delivery by Laurent Bédouet; Florentina Pascale; Laurence Moine; Michel Wassef; Saïda H. Ghegediban; Van-Nga Nguyen; Michel Bonneau; Denis Labarre; Alexandre Laurent (536-544).
A novel degradable microsphere (MS) for intra-articular drug delivery, composed of a polyethylene glycol (PEG) core containing degradable regions made of short poly-(lactic-co-glycolic acid) (PLGA) sequences – named PEG-hydrogel MS – was injected into the cavity of sheep shoulder joint, and compared to non-degradable MS devoid of hydrolysable crosslinker in terms of location, degradation and inflammation. One week after intra-articular injection both groups of MS were localized beneath the synovial lining of the synovial fringes located at bottom of the shoulder joint, while a fraction of particles remained in synovial fluid. Histological analyses made one and 4 weeks after intra-articular injection showed cell proliferation around the non-degradable MS entrapped within the synovium. By contrast, degradable PEG-hydrogel MS were surrounded by few cells. The degradation of degradable PEG-hydrogel MS within the synovium was slow and was not fully complete after four weeks. Our findings indicate that the tissue entrapment of MS below the synovial lining was independent of the material degradability, while degradable PEG-hydrogel MS are less inflammatory than the non-degradable one. Degradable PEG-hydrogel MS offer several advantages over the non-degradable MS as carriers for a sustained drug delivery in synovial tissue according to the low intensity of inflammatory reaction triggered in synovium.
Keywords: Drug delivery; Intra-articular; Hydrogel; Microspheres; Osteoarthritis;

The development of a sensitive methodology to characterise hard shell capsule puncture by dry powder inhaler pins by Barbara M. Torrisi; James C. Birchall; Brian E. Jones; Fernando Díez; Sion A. Coulman (545-552).
In order for hard-shell capsules to function effectively as drug reservoirs in dry powder inhalers, the capsule must be punctured with sharpened pins to release the powdered medicament upon inspiration. Capsule performance in this setting is poorly understood. This study aims to develop a methodology to characterise hard shell capsule penetration by needles from commercial dry powder inhalers, to determine whether changes to capsule materials impact on their performance.Two pin types from two commercial dry powder inhalers were mounted in a material-testing machine, equipped with a 500 N load cell. A stainless steel bush was used to secure a capsule directly below the steel pin. Hypromellose (n  = 10) and gelatin capsules (n  = 10) were conditioned in ‘normal’ or low humidity conditions and were subsequently punctured with both types of pin. Each puncture event was recorded on a load–displacement curve.The force required for puncture was 2.82 ± 0.26 N for hypromellose capsules and 4.54 ± 0.26 N for gelatin capsules, stored in normal humidity. Different capsule materials possessed distinguishable signature profiles but repeated force–displacement profiles were highly reproducible i.e. intra-individual variability was minimal. A rapid, robust yet sensitive methodology has therefore been developed that is able to characterise hard shell capsule materials based on the puncture performance.
Keywords: Capsule; Dry powder inhaler; Gelatin; Hypromellose; Puncture;

Evaluation of solubility and partition properties of ampicillin-based ionic liquids by Catarina Florindo; João M.M. Araújo; Filipa Alves; Carla Matos; Ricardo Ferraz; Cristina Prudêncio; João Paulo Noronha; Željko Petrovski; Luís Branco; Luís Paulo N. Rebelo; Isabel M. Marrucho (553-559).
In order to overcome the problems associated with low water solubility, and consequently low bioavailability of active pharmaceutical ingredients (APIs), herein we explore a modular ionic liquid synthetic strategy for improved APIs. Ionic liquids containing l-ampicillin as active pharmaceutical ingredient anion were prepared using the methodology developed in our previous work, using organic cations selected from substituted ammonium, phosphonium, pyridinium and methylimidazolium salts, with the intent of enhancing the solubility and bioavailability of l-ampicillin forms. In order to evaluate important properties of the synthesized API-ILs, the water solubility at 25 °C and 37 °C (body temperature) as well as octanol–water partition coefficients (K ow's) and HDPC micelles partition at 25 °C were measured. Critical micelle concentrations (CMC's) in water at 25 °C and 37 °C of the pharmaceutical ionic liquids bearing cations with surfactant properties were also determined from ionic conductivity measurements.
Keywords: Active pharmaceutical ionic liquids; Solubility; Bioavailability; Octanol–water partition coefficients; Critical micelle concentrations;

Lecithin-based emulsions for potential use as saliva substitutes in patients with xerostomia – viscoelastic properties by Sara M. Hanning; Tao Yu; David S. Jones; Gavin P. Andrews; Jules A. Kieser; Natalie J. Medlicott (560-568).
The purpose of the present study was to investigate lecithin-rice bran oil rheological properties with the view to consider these as potential saliva substitutes in patients with severe xerostomia and salivary hypofunction. Pseudo-ternary phase diagrams of rice bran oil, lecithin and water mixtures were constructed and characterised using polarising light microscopy. Viscoelastic properties, which we hypothesise are important determinants in product performance, were analysed using both flow and oscillatory rheology. Rheological properties were influenced by composition, frequency and shear stress. Frequency-dependent viscoelasticity was observed in some formulations where viscosity dominated (tan  δ  > 1) at frequencies under 5 Hz and elasticity dominated (tan  δ  < 1) at higher frequencies. Threshold frequencies were determined for each formulation, where a peak in loss tangent was observed, coinciding with a reduction in the storage modulus and increase in loss modulus. The frequency-dependent behaviour of emulsions are of interest because these combinations exhibit viscous behaviour at low frequencies, which may improve lubrication of the oral cavity at rest, whereas increased elasticity at higher frequencies may improve retention during higher-shear tasks such as swallowing and speaking.
Keywords: Xerostomia; Salivary hypofunction; Rheology; Emulsion; Phase diagram; Saliva substitute;

Novel method for screening of enteric film coatings properties with magnetic resonance imaging by Przemysław Dorożyński; Witold Jamróz; Krzysztof Niwiński; Mateusz Kurek; Władysław P. Węglarz; Renata Jachowicz; Piotr Kulinowski (569-571).
The aim of the study is to present the concept of novel method for fast screening of enteric coating compositions properties without the need of preparation of tablets batches for fluid bed coating. Proposed method involves evaluation of enteric coated model tablets in specially designed testing cell with application of MRI technique. The results obtained in the testing cell were compared with results of dissolution studies of mini-tablets coated in fluid bed apparatus. The method could be useful in early stage of formulation development for screening of film coating properties that will shorten and simplify the development works.
Keywords: Shellac; Enteric coating; Mini-tablets; Delayed release; Magnetic resonance imaging (MRI); Ultra-short echo time imaging (UTE);

The focus of present research was to understand and control the variability of solid dispersion (SD) formulation of non-ribosomal peptide antibiotic, vancomycin (VCM). Hunter screening design was constructed using seven independent variables namely melting temperature (X 1), congealing temperature (X 2), mixing time (X 3), type of capsule shell (X 4), filling method (X 5), molecular weight of polyethylene glycol (PEG, X 6) and surfactant type (X 7), and responses measured were cumulative percentage of VCM released in 45 min (Y 1) and potency (Y 2). The SD formulations were prepared by melt-fusion method, and tested for dissolution, potency, and characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and near infrared chemical imaging (NIR-CI). Statistically significant (p  < 0.05) effect of congealing temperature (X 2), type of capsule shell (X 4), filling method (X 5), molecular weight of PEG (X 6) was revealed on Y 1, and R 2 of 0.992 was obtained between experimental and predicted value. None of the factors have statistically significant (p  > 0.05) influence on Y 2. SEM, DSC and PXRD indicated crystalline nature of SD formulations. Homogeneity of SD formulations was shown by NIR-CI images. In summary, the quality of VCM SD formulations could be assured by controlling the critical factors during manufacturing.
Keywords: Vancomycin; Polyethylene glycol; Dissolution; Potency; Design of experiment;

Eighteen months post-implantation, hyaluronic acid gel is partially absorbed. Hyaluronic acid gel is pooled into metachromatic vacuoles surrounded by fibrous strands and only slight infiltration of inflammatory cells.Nowadays there is an increased demand for safe and effective volume enhancing fillers to achieve soft tissue augmentation in order to overcome tissue defects and aging-associated skin changes. In the present study we characterized the rheological and biological properties of Variofill®, a new highly viscoelastic hyaluronic acid gel, by investigating the local effects following subcutaneous implantation in the rat to detect the host-tissue reactions and biodegradation over 18 months. We also investigated, for the first time, the application of Variofill® in esthetic and restorative surgery in two medical case reports. In the first case report we successfully performed Variofill® treatment to improve facial scars in a patient previously involved in a car crash. In the second case report we carried out a novel procedure involving a high-dose (1000 ml) injection of Variofill® into the dermis and subcutis of the abdominal quadrants in order to allow a classic reconstructive procedure of the abdominal wall in a patient presenting a wide incisional hernia.
Keywords: Implantation; Histology; Hernia; Rheology; Hyaluronic acid; Variofill®; Soft tissue filler;