International Journal of Pharmaceutics (v.441, #1-2)

Folate-functionalized nanoparticles for controlled ergosta-4,6,8(14),22-tetraen-3-one delivery by Xuhua Liang; Yang Sun; Lusha Liu; Xuan Ma; Xiaoyun Hu; Jun Fan; Yingyong Zhao (1-8).
The FA–ergone–BSANPs were proved to possess the slow and sustain drug release during circulating in the blood stream and the increase of drug release at the target sites.To improve the therapeutic effect of ergosta-4,6,8(14),22-tetraen-3-one (ergone), a folate–decorated ergone–bovine serum albumin nanoparticles (abbreviated FA–ergone–BSANPs) was prepared. The properties were extensively studied by Zetasizer Nano Particle Size Analyzer and TEM, which indicated the prepared nanoparticles were spherical in shape and uniform in size with a zeta potential of −23.8 mV. The drug-loading capacity also has been determined with drug loading content of 2.73% and encapsulation efficiency of 61.8%. In vitro release studies proved the much slow drug release from the nanoparticles during circulating in the blood stream and the increase of drug release at the target sites. The FA–ergone–BSANPs showed enhanced cellular uptake, increased targeting capacity, and increased cytotoxicity against KB cells over-expressing folate receptor (FR), which indicated that its potent cell-killing activity is specific for cells that express the FR. In vivo experiment also confirmed that FA–ergone–BSANPs represent a FR-targeted chemotherapeutic that can produce potent activity against FR-positive tumors. In conclusion, this report has a great significance in pharmacology and clinical medicine as well as methodology. Further detailed dose-optimization studies will be required for better understanding in vivo pharmacokinetic and bio-distribution behaviors.
Keywords: Ergosta-4,6,8(14),22-tetraen-3-one (ergone); Nanoparticles; Folate receptor; Targeted therapeutics; Slow release capacity; Anticancer;

Levonorgestrel (LNG) is the most commonly used progestin in contraception. In this study, we report the use of an alternative progestin, desogestrel, for transdermal contraception. The drug was found to be significantly more permeable as compared to LNG (p  < 0.05). Crystallization studies were used to select the best adhesive among acrylate (Duro-Tak 87-4098 and Duro-Tak 87-202A) and polyisobutylene (PIB, Duro-Tak 87-608A) pressure sensitive adhesives by determining the drug's saturation solubility in them. The use of copovidone and mineral oil as formulation excipients was investigated to increase drug loading in the PIB adhesive. Physical characterization of the patches was performed using in vitro drug release, content analysis, patch weight and thickness variations and rolling ball tack and peel adhesion studies. Optimized patches were evaluated for in vitro transdermal delivery across hairless rat skin. The saturation solubility of desogestrel was found to be approximately 49.3% (w/w) and 55.6% (w/w) in Duro-Tak 87-4098 and Duro-Tak 87-202A acrylate adhesives, respectively. The saturation solubility of desogestrel was significantly lower (3–4%, w/w) in the PIB adhesive. Mineral oil (10%, w/w) and copovidone (30%, w/w) were found to be optimum for increasing drug loading and patch cosmetics. Results from the physical characterization studies suggest that a uniform and reproducible 7 day drug-in-adhesive patch could be developed.
Keywords: Desogestrel; Contraceptive; Transdermal patch; Acrylate adhesive; Polyisobutylene adhesive; Crystallization;

In this study, a suspension of mycophenolate mofetil (MMF) suitable for inhalation was developed using the emulsion template process and characterized for particle size and aerosolization performance. To evaluate the benefits of this suspension over a solution, the IV Cellcept® solution was also characterized in vitro. Both formulations exhibited excellent aerosolization performance. The aerodynamic diameters for the solution and the suspension were within the respirable range (below 5 μm) and their fine particle doses were nearly equivalent, suggesting the same drug exposure during in vivo experiments. Single dose 24-h pharmacokinetic studies following inhalation of the formulations and oral administration of oral Cellcept® were performed in rats. Following oral administration, MMF was completely and rapidly metabolized into its active metabolite, mycophenolic acid (MPA) and partial metabolism was observed following pulmonary administration. Inhaled MMF suspension displayed more favorable pharmacokinetics than inhaled IV Cellcept® solution, but the MPA drug levels in each compartment were much lower than those obtained with oral Cellcept®. The dose normalized MPA levels in the lung, thymus gland and plasma following inhalation of the MMF suspension with the oral control suggested that pulmonary delivery of a MMF suspension could be beneficial in preventing lung allograft rejection.
Keywords: Mycophenolate mofetil; Immunosuppressant; Pulmonary delivery; Preclinical pharmacokinetics; Lung; Lymphatic system;

Temperature- and pH-responsive nanoparticles of biocompatible polyurethanes for doxorubicin delivery by Anning Wang; Hui Gao; Yanfang Sun; Yu-long Sun; Ying-Wei Yang; Guolin Wu; Yinong Wang; Yunge Fan; Jianbiao Ma (30-39).
A series of temperature- and pH-responsive polyurethanes based on hexamethylene diisocyanate (HDI) and 4,4′-diphenylmethane diisocyanate (MDI) were synthesized by a coupling reaction with bis-1,4-(hydroxyethyl) piperazine (HEP), N-methyldiethanolamine (MDEA) and N-butyldiethanolamine (BDEA), respectively. The chemical structure, molecular weight, thermal property and crystallization properties were characterized by Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) spectroscopy. The resulting polyurethanes were then used to prepare nanoparticles either by direct dispersion method or dialysis method. Their pH and temperature responsibilities were evaluated by optical transmittance and size measurement in aqueous media. Interestingly, HDI-based and MDI-based polyurethanes exhibited different pH and temperature responsive properties. Nanoparticles based on HDI-HEP and HDI-MDEA were temperature-responsive, while MDI-based biomaterials were not. All of them showed pH-sensitive behavior. The possible responsive mechanism was investigated by 1H NMR spectroscopy. The cytotoxicity of the polyurethanes was evaluated using methylthiazoletetrazolium (MTT) assay in vitro. It was shown that the HDI-based polyurethanes were non-toxic, and could be applied to doxorubicin (DOX) encapsulation. The experimental results indicated that DOX could be efficiently encapsulated into polyurethane nanoparticles and uptaken by Huh-7 cells. The loaded DOX molecules could be released from the drug-loaded polyurethane nanoparticles upon pH and temperature changes, responsively.
Keywords: pH; Temperature; Responsive; Nanoparticle; Polyurethane; Doxorubicin;

The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [3H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [3H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes.
Keywords: Peptide transporters; MDCK cells; Efflux pumps; MDR transfection;

Solubilization of the poorly water soluble drug, telmisartan, using supercritical anti-solvent (SAS) process by Junsung Park; Wonkyung Cho; Kwang-Ho Cha; Junhyun Ahn; Kang Han; Sung-Joo Hwang (50-55).
Telmisartan is a biopharmaceutical classification system (BCS) class II drug that has extremely low water solubility but is freely soluble in highly alkalized solutions. Few organic solvents can dissolve telmisartan. This solubility problem is the main obstacle achieving the desired bioavailability. Because of its unique characteristics, the supercritical anti-solvent (SAS) process was used to BCS class II drug in a variety of ways including micronization, amorphization and solid dispersion. Solid dispersions were prepared using hydroxypropylmethylcellulose/polyvinylpyrrolidone (HPMC/PVP) at 1:0.5, 1:1, and 1:2 weight ratios of drug to polymer, and pure telmisartan was also treated using the SAS process. Processed samples were characterized for morphology, particle size, crystallinity, solubility, dissolution rate and polymorphic stability. After the SAS process, all samples were converted to the amorphous form and were confirmed to be hundreds nm in size. Solubility and dissolution rate were increased compared to the raw material. Solubility tended to increase with increases in the amount of polymer used. However, unlike the solubility results, the dissolution rate decreased with increases in polymer concentration due to gel layer formation of the polymer. Processed pure telmisartan showed the best drug release even though it had lower solubility compared to other solid dispersions; however, because there were no stabilizers in processed pure telmisartan, it recrystallized after 1 month under severe conditions, while the other solid dispersion samples remained amorphous form. We conclude that after controlling the formulation of solid dispersion, the SAS process could be a promising approach for improving the solubility and dissolution rate of telmisartan.
Keywords: Telmisartan; Supercritical anti-solvent process; Solid dispersion; Solubility;

Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems by Sonali Bose; Yuechao Du; Paul Takhistov; Bozena Michniak-Kohn (56-66).
The presence of large amounts of reactive oxygen species (ROS) leads to oxidative stress that can damage cell membranes, lead to DNA breakage and cause inactivation of free radical scavenger enzymes, eventually resulting in skin damage. Quercetin is a natural flavonoid that has been shown to have the highest anti-radical activity, along with the ability to act as a scavenger of free radicals and an inhibitor of lipid peroxidation. In this research work, a solvent-free solid lipid based nanosystem has been developed and evaluated for topical delivery of quercetin. Systematic screening of the formulation and process parameters led to the development of a solid lipid (glyceryl dibehenate) based nanosystem using a probe ultrasonication method. The selected variant demonstrated good physical stability for up to 8 weeks at 2–8 °C. Transmission electron microscopy (TEM) images showed spherical particles in the nanometer range. In vitro release studies showed biphasic release of quercetin from the SLN formulation, with an initial burst release followed by prolonged release for up to 24 h. In vitro permeation studies using full thickness human skin showed higher amounts of quercetin to be localized within the skin compared to a control formulation with particles in the micrometer range. Such accumulation of quercetin in the skin is highly desirable since the efficacy of quercetin in delaying ultra-violet radiation mediated cell damage and eventual necrosis mainly occurs in the epidermis.
Keywords: Sonication; Glyceryl behenate; Solid lipid nanoparticles; Physical stability; Tween 20; Ionic stabilizer;

Rapid determination of the encapsulation efficiency of a liposome formulation using column-switching HPLC by Naozumi Ohnishi; Eiichi Yamamoto; Hiromasa Tomida; Kenji Hyodo; Hiroshi Ishihara; Hiroshi Kikuchi; Kohei Tahara; Hirofumi Takeuchi (67-74).
The feasibility of a rapid automated method for determination of the encapsulation efficiency (EE) of a liposome formulation using a column-switching HPLC system was confirmed by employing several types of liposome formulations containing doxorubicin (DXR). A suspension of DXR liposome was injected directly into an online solid-phase extraction (SPE) system comprising a Diol SPE column and an ODS SPE column connected in series. Free (not encapsulated) DXR was trapped on the Diol SPE column, whereas encapsulated DXR was eluted without interaction. The eluted encapsulated DXR was trapped on the ODS SPE column after being extracted from the inner phase of the liposome by mixing with an organic solvent. Trapped free and encapsulated DXR were eluted sequentially and analyzed separately by gradient HPLC. The time taken by this automated method was only 25 min, whereas conventional methods such as ultracentrifugation are time consuming and labor intensive. Validation results and comparison with ultracentrifugation suggested that our method was sufficiently accurate and sensitive to be used to evaluate EE of a liposome formulation without complicated pretreatment.
Keywords: Doxil®; Encapsulation efficiency; Solid-phase extraction; Centrifugation; Dialysis;

Uniform nano-sized valsartan for dissolution and bioavailability enhancement: Influence of particle size and crystalline state by Qiuping Ma; Hongrui Sun; Erxi Che; Xin Zheng; Tongying Jiang; Changshan Sun; Siling Wang (75-81).
The central purpose of this study was to evaluate the impact of drug particle size and crystalline state on valsartan (VAL) formulations in order to improve its dissolution and bioavailability. VAL microsuspension (mean size 22 μm) and nanosuspension (30–80 nm) were prepared by high speed dispersing and anti-solvent precipitation method and converted into powders through spray drying. Differential scanning calorimetry studies indicated amorphization of VAL in the spray-dried valsartan nanosuspension (SD-VAL-Nano) but recrystallization occurred after 6 months storage at room temperature. The spray-dried valsartan microsuspension (SD-VAL-Micro) conserved the crystalline form. The VAL dissolution rate and extent were markedly enhanced with both SD-VAL-Micro and SD-VAL-Nano as compared to crude VAL crystals over the pH range of 1.2–6.8. Pharmacokinetic studies in rats demonstrated a 2.5-fold increase in oral bioavailability in the case of SD-VAL-Nano compared with the commercial product while the SD-VAL-Micro provided a much less desirable pharmacokinetic profile. In conclusion, reducing particle size to the nano-scale appears to be a worthwhile and promising approach to obtain VAL products with optimum bioavailability. In addition, the impact of crystalline state on the bioavailability of nano-sized VAL might be not as big as that of particle size.
Keywords: Valsartan; Nanosuspension; Particle size; Crystalline state; Dissolution; Bioavailability;

Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes by Sara Movassaghian; Hamid R. Moghimi; Farshad H. Shirazi; Alexander Koshkaryev; Malav S. Trivedi; Vladimir P. Torchilin (82-91).
The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy.Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques.
Keywords: Dendrosome; Liposome; Dendrimer; Cancer gene delivery; Cytotoxicity; Antisense oligodeoxynucleotides;

A prototype ‘Infucon’ device for continuous infusion of microbubbles in vivo by Zuzana Kauerová; Róbert Lukáč; Pavel Kohout; Josef Mašek; Štěpán Koudelka; Jana Plocková; Marta Vašíčková; Michal Vlašín; Jaroslav Turánek (92-98).
A device for continuous infusion of microbubbles (MBs) ‘Infucon’ has been designed, constructed and tested on rabbits. The device prevents MBs from flotation and accumulation in the layer directly below the surface in the syringe injection during i.v. application. Homogenous i.v. application of MBs was tested on 16 male New Zealand White rabbits (average weight about 3.5 kg). Two sorts of MBs were used – a set of commercial SonoVue diagnostic microbubbles (Bracco) and pegylated DPPC microbubbles (PegMBs), which had been prepared in our laboratory. Sulphur hexafluoride was used as a filling gas. The application of MBs by continuous infusion via Infucon prolonged the ultrasound signal period in the heart of the rabbit to 12 min in comparison to about 1 min observed in bolus application. No adverse effects were observed on the tested rabbits after the MB application via Infucon. The principle employed in the prototype device Infucon could be used for development of the device intended for clinical applications.
Keywords: Ultrasonography; Microbubbles; Continuous infusion; Liposome;

The project was aimed to enhance the solubility of ivermectin using surface solid dispersion (SSD), elucidate the mechanism of drug release and to develop its tablet that can draw rapid clinical effects on dogs for controlling Ascaris parasites. Superdisintegrants and adsorbents were used to formulate SSDs by co-evaporation method. Formulation 9th that constituted from drug:aerosil (1:10) was selected after solubility and in vitro dissolution evaluation, and characterized by DSC, XRPD, DRS and SEM analysis. In vitro evaluation and in vivo efficacy of its tablet on dogs (fecal egg and tick counting studies) were compared with marketed tablets. This formulation enhanced the solubility of ivermectin to 456.25 ± 1.70% and released 93.55 ± 0.47% in 60 min. DRS spectral analysis unearthed a novel molecular mechanism of hydrogen bond replacement that drifts the drug into the medium from the surface of the SSD particles. Reduced crystallinity was confirmed by XRPD and supported by SEM. During in vitro and in vivo studies, the formulated tablet showed superiority over marketed tablet with higher parasite inhibition (p  < 0.001). Thus SSD is a promising technique for enhancing IVM solubility at molecular level enabling its rapid oral delivery to control endo- as well as ectoparasites for veterinary purpose.
Keywords: Ivermectin; Surface solid dispersion; Hydrogen bond replacement; Rapid dissolution; Veterinary application;

Synchronized and controlled release of multiple components in silymarin achieved by the osmotic release strategy by Yunchang Xie; Yi Lu; Jianping Qi; Xiaoyang Li; Xi Zhang; Jin Han; Shixiao Jin; Hailong Yuan; Wei Wu (111-120).
The pharmacological activity of herbal medicines is determined by the active components ratio that defines the medicine. Significant alteration of the components ratio will lead to major changes in the pharmacological activities. In designing sustained or controlled release delivery systems of herbal medicines, we developed the concept of synchronized release which was characterized by keeping active components’ initial ratio throughout the whole release process. In this study, this concept was extended by developing a novel synchronized release system based on osmotic release strategy, which was designed as a monolithic osmotic tablet consisting of a tablet core and semi-permeable coating of cellulose acetate with mechanically perforated release orifices. The rationale of synchronized release lies in simultaneous release of silymarin multiple components in a solubilized state through the release orifices. To ensure quick solubilization of multiple components, silymarin was first formulated as fast-release solid dispersion before formulating into the osmotic tablet. The concept of synchronized release was well proved by the results of high similarity of the release profiles of the five active components in silymarin (taxifolin, silychristin, silydianin, silybin and isosilybin). Zero-order release profiles can be maintained up to approximately 20 h. High synchronicity and controlled release can be achieved by adjusting the formulation variables. It is indicated that the osmotic release strategy has great potential to achieve synchronized and controlled release of multiple components in herbal medicines.
Keywords: Synchronized release; Controlled release; Monolithic osmotic tablet; Solid dispersion; Silymarin;

Bitterness prediction of H1-antihistamines and prediction of masking effects of artificial sweeteners using an electronic tongue by Masanori Ito; Kiyoharu Ikehama; Koichi Yoshida; Tamami Haraguchi; Miyako Yoshida; Koichi Wada; Takahiro Uchida (121-127).
The study objective was to quantitatively predict a drug's bitterness and estimate bitterness masking efficiency using an electronic tongue (e-Tongue). To verify the predicted bitterness by e-Tongue, actual bitterness scores were determined by human sensory testing. In the first study, bitterness intensities of eight H1-antihistamines were assessed by comparing the Euclidean distances between the drug and water. The distances seemed not to represent the drug's bitterness, but to be greatly affected by acidic taste. Two sensors were ultimately selected as best suited to bitterness evaluation, and the data obtained from the two sensors depicted the actual taste map of the eight drugs. A bitterness prediction model was established with actual bitterness scores from human sensory testing. Concerning basic bitter substances, such as H1-antihistamines, the predictability of bitterness intensity using e-Tongue was considered to be sufficiently promising.In another study, the bitterness masking efficiency when adding an artificial sweetener was estimated using e-Tongue. Epinastine hydrochloride aqueous solutions containing different levels of acesulfame potassium and aspartame were well discriminated by e-Tongue. The bitterness masking efficiency of epinastine hydrochloride with acesulfame potassium was successfully predicted using e-Tongue by several prediction models employed in the study.
Keywords: Electronic tongue; Taste sensor; Bitterness prediction; Taste masking; H1-antihistamines; Artificial sweetener;

Newly developed surface modification punches treated with alloying techniques reduce sticking during the manufacture of ibuprofen tablets by Takeaki Uchimoto; Yasunori Iwao; Tatsuya Yamamoto; Kazuo Sawaguchi; Toshiaki Moriuchi; Shuji Noguchi; Shigeru Itai (128-134).
Sticking is a serious problem during the manufacturing process of tablets. In order to prevent this, we used alloying techniques to prepare metal hardening (MH) and electron beam processing infinite product (EIP) punches with rougher asperity of surfaces than a hard chrome plated (HCr) punch. This study evaluated the anti-sticking properties of the MH and EIP punches compared to the HCr punch, using quantitative scraper force measurements and visual observation, for the manufacture of ibuprofen (Ibu) tablets. The anti-sticking property mechanism of the MH and EIP punches was also confirmed. The amount of Ibu adhering to the punch surface was 66% lower for the MH and EIP punches than for the HCr punch, suggesting a superior anti-sticking property of the MH and EIP punches. The scraper force of the HCr punch was 2.60–4.28 N, while that for the MH and EIP punches was 0.54–1.64 N and 0.42–1.33 N, respectively. The result of X-ray photoelectron spectroscopy suggested that the anti-sticking property of the EIP punch was attributed by the rough asperity as well as existence of low friction substance carbon fluoride on the punch surface. In conclusion, this study provides new evidence for the mechanisms behind the superior anti-sticking property of the MH and EIP punches.
Keywords: Tableting; Sticking; Scraper force; Ibuprofen; Surface modification;

Detection of component segregation in granules manufactured by high shear granulation with over-granulation conditions using near-infrared chemical imaging by Tatsuo Koide; Takuya Nagato; Yoshiyuki Kanou; Kou Matsui; Susumu Natsuyama; Toru Kawanishi; Yukio Hiyama (135-145).
The objective of this study was to evaluate the high shear granulation process using near-infrared (NIR) chemical imaging technique and to make the findings available for pharmaceutical development. We prepared granules and tablets made under appropriate- and over-granulation conditions with high shear granulation and observed these granules and tablets using NIR chemical imaging system. We found an interesting phenomenon: lactose agglomeration and segregation of ingredients occurred in experimental tablets when over-granulation conditions, including greater impeller rotation speeds and longer granulation times, were employed. Granules prepared using over-granulation conditions were larger and had progressed to the consolidation stage; segregation between ethenzamide and lactose occurred within larger granules. The segregation observed here is not detectable using conventional analytical technologies such as high pressure liquid chromatography (HPLC) because the content of the granules remained uniform despite the segregation. Therefore, granule visualization using NIR chemical imaging is an effective method for investigating and evaluating the granulation process.
Keywords: Image analysis; Near-infrared spectroscopy; High shear granulation; Segregation;

Enhanced transgene expression from chromatinized plasmid DNA in mouse liver by Hiroyuki Kamiya; Shiho Miyamoto; Hitomi Goto; Genki N. Kanda; Miwako Kobayashi; Ichiro Matsuoka; Hideyoshi Harashima (146-150).
Plasmid DNA was chromatinized with core histones (H2A, H2B, H3, and H4) in vitro and was delivered into mouse liver by hydrodynamics-based administration. Transgene expression from the chromatinized plasmid DNA was more efficient than that from plasmid DNA delivered in the naked form. The use of acetylation-enriched histones isolated from cells treated with a histone deacetylase inhibitor (trichostatin A) seemed to be more effective. These results indicated that chromatinized plasmid DNA is useful for efficient transgene expression in vivo.
Keywords: Exogenous DNA; Histone; Nucleosome; Intranuclear disposition; Expression efficiency;

Visualization of ultradeformable liposomes penetration pathways and their skin interaction by confocal laser scanning microscopy by Thirapit Subongkot; Nanthida Wonglertnirant; Pucharee Songprakhon; Theerasak Rojanarata; Praneet Opanasopit; Tanasait Ngawhirunpat (151-161).
The objective of this study was to elucidate the skin penetration pathway of the generated ultradeformable liposomes (ULs) with terpenes for transdermal drug delivery of fluorescein sodium (NaFl). ULs with d-limonene were selected to investigate the penetration pathways and vesicle–skin interaction in terms of release and attachment processes via Confocal Laser Scanning Microscopy (CLSM). A co-localization technique was employed to visualize the skin penetration behavior of UL-labeled red fluorescence (Rh-PE) and fluorescence-entrapped drug (NaFl) through porcine skin. Our results suggested that ULs with entrapped drug might attach to any part of the skin before releasing the entrapped drug into the skin. Most ULs and entrapped drug penetrated through hair follicles more than through the nonfollicular region. In summary, the transfollicular pathway was the major penetration pathway of ULs with d-limonene for transdermal drug delivery of NaFl, whereas the intercellular and transcellular pathways were the minor penetration pathways.
Keywords: Ultradeformable liposomes; Skin penetration pathways; Confocal laser scanning microscopy;

Gastroretentive drug delivery systems with more than 16 h with a swelling ratio over 2 and an adjustable sustained release profileThe aim was to develop gastroretentive drug delivery systems (GRDDSs) by combining floating and swelling. GRDDS tablets formulated with hydroxyethylcellulose (HEC), chitosan (CS) and sodium bicarbonate (SB) for evaluating floating capacity (floating lag time and duration) and swelling characteristics. CS was used because it was swellable in acidic media and biocompatible. Losartan was incorporated into the optimized formulations for sustained release profiling. Results demonstrated that for those formulations at HEC:CS ratio of 5:5 containing CS, both the floating lag time and floating duration were optimal and reached the preferred swelling effect and sustain for 24 h. Adding SB improved the floating capabilities for all ratios of HEC:CS, but reduced the swelling ability for those formulations containing a higher portion of low viscosity grade CS. Sustained release profiles for losartan in those formulations were achievable, using all viscosity grades of CS at all examined HEC:CS ratios; however, it is more adjustable at different HEC:CS ratios when using a lower viscosity grade of CS. Optimized GRDDS formulations for losartan composed of an equivalent ratio of HEC to CS with 20 mg SB resulted in the tablets floating for more than 16 h and an adjustable sustained release profile.
Keywords: Gastroretentive drug delivery system; Hydroxyethylcellulose; Chitosan; Floating; Swelling; Losartan;

Glycopolymer micelles with reducible ionic cores for hepatocytes-targeting delivery of DOX by Yanxia Wang; Xinge Zhang; Peien Yu; Chaoxing Li (170-180).
A novel galactose-decorated cross-linked micelles (cl-micelles) with ionic cores using cystamine (Cys) as a biodegradable cross-linker was prepared by using block ionomer complexes of poly(ethylene glycol)-b-poly(2-acryloxyethyl-galactose)-b-poly(acrylic acid) (PEG-b-PAEG-b-PAA) and Ca2+ (PEG-b-PAEG-b-PAA cl-micelles/Cys). Doxorubicin (DOX) was successfully incorporated into the ionic cores of such micelles via electrostatic interactions. Proton nuclear magnetic resonance spectrum and Fourier transform infrared spectrometer indicated galactose ligands were exposed at the micellar surface. The micelles were spherical in shape, with an average size of 100 nm. The in vitro release studies confirmed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys accomplished rapid drug release under reducing condition. Remarkably, PEG-b-PAEG-b-PAA cl-micelles/Cys efficiently delivered and released DOX into the cell nucleus of HepG2 cells, and the intensity of fluorescence observed in HepG2 cells was stronger than that incubated with the micelles without galactose ligands. In contrast, little fluorescence was observed in NIH3T3 cells after incubation with PEG-b-PAEG-b-PAA cl-micelles/Cys. Interestingly, cytotoxicity assays showed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys retained higher cell inhibition efficiency in HepG2 cells as compared with NIH3T3 cells, and were more potent than the micelles without galactose ligands and the micelles with non degradable cross-links. These results indicate that PEG-b-PAEG-b-PAA cl-micelles/Cys have great potential in liver tumor-targeted chemotherapy.
Keywords: Galactose; Biodegradable; DOX; HepG2 cells;

Polyethylene oxide (Polyox) and carrageenan based solvent cast films have been formulated as dressings for drug delivery to wounds. Films plasticised with glycerol were loaded with streptomycin (30%, w/w) and diclofenac (10%, w/w) for enhanced healing effects in chronic wounds. Blank and drug loaded films were characterised by texture analysis (for mechanical and mucoadhesive properties), scanning electron microscopy, differential scanning calorimetry, X-ray diffraction and Fourier transform infrared spectroscopy. In addition, swelling, in vitro drug release and antibacterial studies were conducted to further characterise the films. Both blank and drug loaded films showed a smooth, homogeneous surface morphology, excellent transparency, high elasticity and acceptable tensile (mechanical) properties. The drug loaded films showed a high capacity to absorb simulated wound fluid and significant mucoadhesion force which is expected to allow effective adherence to and protection of the wound. The films showed controlled release of both streptomycin and diclofenac for 72 h. These drug loaded films produced higher zones of inhibition against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli compared to the individual drugs zones of inhibition. Incorporation of streptomycin can prevent and treat chronic wound infections whereas diclofenac can target the inflammatory phase of wound healing to relieve pain and swelling.
Keywords: Antibacterial; Film dressing; In vitro drug release; Mucoadhesion; Wound healing; Plasticiser;

Influence of niacinamide containing formulations on the molecular and biophysical properties of the stratum corneum by D. Mohammed; J.M. Crowther; P.J. Matts; J. Hadgraft; M.E. Lane (192-201).
Niacinamide-containing moisturisers are known be efficacious in alleviating dry skin conditions and improving stratum corneum (SC) barrier function. However, the mechanisms of action of niacinamide at the molecular level in the SC are still not well understood. Previously, we have reported the development of novel methods to probe SC barrier properties in vivo. The aim of the present study was to characterise changes in Trans Epidermal Water Loss (TEWL), corneocyte surface area and maturity, selected protease activities and SC thickness after repeated application of a simple vehicle containing niacinamide. A commercial formulation was also included as a reference. The left and right mid-volar forearms of 20 healthy volunteers were used as study sites, to which topical formulations were applied twice daily for 28 days. After successive tape-stripping, corneocyte maturity and surface area were assessed. In addition, activity of the desquamatory kallikrein (KLK) protease enzymes KLK5 and KLK7, and tryptase and plasmin (implicated in inflammatory process) were measured using a fluorogenic probe assay. The amount of protein removed and TEWL were also recorded. SC thickness before and after treatment was determined using Confocal Raman Spectroscopy (CRS). Overall (i) corneocyte maturity and surface area decreased with increasing number of tape strips, (ii) activity of both the desquamatory and inflammatory enzymes was highest in the outer layers of the SC and decreased with depth (iii) TEWL increased as more SC layers were removed. Furthermore, areas treated with formulations containing niacinamide were significantly different to pre-treatment baseline and untreated/vehicle-control treated sites, with larger and more mature corneocytes, decreased inflammatory activity, decreased TEWL and increased SC thickness. These data (a) confirm the utility of measures and metrics developed previously for the non-invasive assay of SC barrier function, (b) present an holistic picture of a SC compartment managing barrier function through dynamic optimisation of pathlength and quality of building materials used, and (c) shed new light on niacinamide as a topical formulation adjunct with unique SC barrier-augmentation properties.
Keywords: Niacinamide; TEWL; KLK5; KLK7; Confocal Raman spectroscopy;

Low levels of isoniazid gain access into plasma following oral administration due to its high aqueous solubility, poor permeability and rapid and extensive hepatic metabolism. Further, a small t 1/2 of 1–4 h indicates its short stay in plasma and the need for repetitive or high doses which may subsequently result in hepatotoxicity and neurotoxicity associated with its use. Isoniazid-solid lipid nanoparticles (SLNs) were prepared to achieve improved bioavailability and prolonged effect, thus minimizing pulsatile plasma concentrations (and associated side effects at peak plasma concentrations). Developed SLNs showed high entrapment efficiency (69%) and small size (d 90 48.4 nm) such that they are expected to bypass reticulo-endothelial system (RES) pickup resulting in prolonged circulation times and since liver is the major site of metabolism of isoniazid, RES avoidance will reduce its elimination from the body. Single dose (25 mg/kg BW) oral pharmacokinetic studies were performed in plasma and various tissues of rats. A significant improvement (p  < 0.001) in relative bioavailability in plasma (6 times) and brain (4 times) was observed after administration of isoniazid-SLNs with respect to the free drug solution at the same dose. Insignificant changes in liver concentration coupled with bypass of first pass metabolism and slow release of isoniazid (60%, in 24 h) indicate low incidence of hepatotoxicity. Isoniazid-SLNs showed a 3 times higher LD50.
Keywords: Solid lipid nanoparticles; Isoniazid; Pharmacokinetics; Bioavailability; Tuberculosis;

Slow dissolution behaviour of amorphous capecitabine by Jelte Meulenaar; Jos H. Beijnen; Jan H.M. Schellens; Bastiaan Nuijen (213-217).
In this article, we report the anomalous dissolution behaviour of amorphous capecitabine. In contrast to what is expected from thermodynamic theory, amorphous capecitabine dissolves significantly slower compared to its crystalline counterpart. Our experiments show that this is due to the “gelling” properties of amorphous capecitabine in an aqueous environment. The “gel”, which is immediately formed upon contact with water, entraps the capecitabine and significantly slows down its dissolution. This “gelling” property is hypothesized to be related to the low glass transition temperature (Tg 19 °C) of amorphous capecitabine, resulting in an instant collapse (“gelling”) in an aqueous environment. From IR and DSC analysis it is shown that this collapsed capecitabine is remarkably stable and does not recrystallize upon an increased water content or temperature. This highly reproducible dissolution behaviour can be applied in the development of a sustained release dosage form as substantially less sustained release excipient is required in order to attain the desired release profile. As capecitabine is a high-dosed drug, this is highly favourable in view of the size and thus clinical feasibility of the final dosage form. Currently, we are developing and clinically testing a sustained release formulation making use of amorphous capecitabine and its remarkable dissolution behaviour.
Keywords: Capecitabine; Dissolution; Amorphous; Spray drying; Slow release; Recrystallization;

Pharmaceutical development and optimization of azithromycin suppository for paediatric use by Tina Kauss; Alexandra Gaubert; Chantal Boyer; Boubakar B. Ba; Muriel Manse; Stephane Massip; Jean-Michel Léger; Fawaz Fawaz; Martine Lembege; Jean-Michel Boiron; Xavier Lafarge; Niklas Lindegardh; Nicholas J. White; Piero Olliaro; Pascal Millet; Karen Gaudin (218-226).
Pharmaceutical development and manufacturing process optimization work was undertaken in order to propose a potential paediatric rectal formulation of azithromycin as an alternative to existing oral or injectable formulations. The target product profile was to be easy-to-use, cheap and stable in tropical conditions, with bioavailability comparable to oral forms, rapidly achieving and maintaining bactericidal concentrations. PEG solid solution suppositories were characterized in vitro using visual, HPLC, DSC, FTIR and XRD analyses. In vitro drug release and in vivo bioavailability were assessed; a study in rabbits compared the bioavailability of the optimized solid solution suppository to rectal solution and intra-venous product (as reference) and to the previous, non-optimized formulation (suspended azithromycin suppository). The bioavailability of azithromycin administered as solid solution suppositories relative to intra-venous was 43%, which compared well to the target of 38% (oral product in humans). The results of 3-month preliminary stability and feasibility studies were consistent with industrial production scale-up. This product has potential both as a classical antibiotic and as a product for use in severely ill children in rural areas. Industrial partners for further development are being sought.
Keywords: Azithromycin; Antibiotic; Rectal; Suppository; Pharmaceutical development; Paediatric solid dispersion;

Development and in vivo/in vitro evaluation of novel herpetrione nanosuspension by Jing-jing Guo; Peng-Fei Yue; Jun-lan Lv; Jin Han; Shan-shan Fu; Shi-xiao Jin; Shi-ying Jin; Hai-Long Yuan (227-233).
Herpetrione (HPE), is a new compound extracted from Herpetospermum caudigerum, which is proved to be a novel and potent antiviral agent. However, due to poor water solubility, oral bioavailability of the drug was relatively low. To improve the dissolution and absorption of the drug, formulation of HPE as nanosuspension has been performed in this study. HPE nanosuspension were produced by high pressure homogenization and transformed into dry powder by lyophilization. The nanosuspension was then investigated using photon correlation spectroscopy (PCS), zeta potential measurement, SEM and PXRD. To verify the theoretical hypothesis on the benefit of decreased particle size and increased surface area, in vitro dissolution characterization and in vivo pharmacokinetics were investigated. The inhibitory effect on HBsAg, HBeAg, and HBV-DNA of HPE nanosuspension in 2.2.15 cells was studied. Results showed that a narrow size distributed nanosuspension with a mean particle size of 286 ± 1.3 nm, a polydispersity index of 0.18 ± 0.06 and a zeta potential of −26.9 ± 2.4 mV was obtained. The result of PXRD showed that HPE was amorphous state in both coarse powder and nanosuspension. In the in vitro dissolution test, HPE nanosuspension showed an increased dissolution velocity markedly. In the in vivo evaluation, compared to coarse HPE, nanosuspension exhibited significant increase in AUC0–t , C max and decrease in T max, MRT. The inhibitory effect of HBsAg, HBeAg, and HBV-DNA of 2.2.15 cells treated by HPE nanosuspension were stronger than those of the HPE. The in vitro activity experiments provided evidence for an enhanced efficacy of the HPE nanosuspension formulation compared to HPE coarse suspension. These results revealed that particle size reduction could enhance HPE dissolution rate and absorption in gastrointestinal tract, and nanosuspension might be a good choice for oral delivery of poor bioavailability drug like HPE.
Keywords: Herpetrione; Nanosuspension; Dissolution; Bioavailability; Activity assay;

Cancer cell targeting and imaging with biopolymer-based nanodevices by István Hajdu; Magdolna Bodnár; György Trencsényi; Teréz Márián; György Vámosi; József Kollár; János Borbély (234-241).
We report here the synthesis, in vitro and in vivo investigation of magnetic resonance imaging (MRI) active nanoparticles, which target folate receptor overexpressing tumor cells. Self-assembled nanoparticles with a hydrodynamic size of 50–200 nm were prepared from poly-γ-glutamic acid and chitosan biopolymers with Gd-ions. The nanoparticles are biocompatible, non-toxic and stable for several months in aqueous media. In vitro assays using confocal microscopy, flow cytometry and MR imaging on HeLa human cervix carcinoma tumor cells showed that folic acid targeted nanoparticles were internalized specifically in a folate receptor dependent manner. In vivo study confirmed, that, considerable accumulation of nanosystems was found compared with the control animal represented by the MR images. Relaxometry measurements demonstrated that the nanoparticle-Gd complexes drastically change the signal intensity of the tumor cells. Because of the contrast enhancement, they are attractive candidates as potential contrast agents for a variety of diagnostic applications including early diagnosis of tumors.
Keywords: MRI; Contrast agent; Gadolinium; In vitro; In vivo; Self-assembled nanoparticles;

PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors by Staffan Lindberg; Andrés Muñoz-Alarcón; Henrik Helmfors; Diogo Mosqueira; Daniel Gyllborg; Oana Tudoran; Ülo Langel (242-247).
Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.
Keywords: Cell-penetrating peptide; Drug delivery; Splice correction; MicroRNA; Endosomal escape; Scavenger receptor;

Coated particle assemblies for the concomitant pulmonary administration of budesonide and salbutamol sulphate by Janne Raula; Antti Rahikkala; Tuomas Halkola; Jenni Pessi; Leena Peltonen; Jouni Hirvonen; Kristiina Järvinen; Timo Laaksonen; Esko I. Kauppinen (248-254).
The aims were to prepare stable and well-dispersible pulmonary fine powders composed of combination drugs with different water solubility, to facilitate concomitant release of corticosteroid budesonide and short acting β-agonist salbutamol sulphate and to improve the dissolution of the budesonide. The budesonide nanosuspensions were prepared by a wet milling which were mixed then with salbutamol sulphate, mannitol (bulking material) and leucine (coating material) for the preparation of micron-sized particles by an aerosol flow reactor wherein leucine formed a rough coating layer on particle surface. The stable and intact particle assemblies showed excellent aerosolization performance. The emitted doses from the inhaler, Easyhaler®, were ∼3 mg/dose with a coefficient variation of 0.1, and the fine particle fractions were ∼50%. Complete dissolution of budesonide nanocrystals from the particles took place within 20 min with the same rate as salbutamol sulphate. Combining the two formulation technologies enabled the encapsulation of drugs with different solubility into a single, intact particle. The leucine coating provided excellent aerosolization properties which allowed fine powder delivery from the inhaler without carrier particles. This study showed the feasibility of preparing powders for combination therapy that are utilized, for instance, in inhalation therapy.
Keywords: Nanosuspension; Aerosol; Pulmonary;

A differential scanning fluorimetry (DSF) based high-throughput screening assay with the fluorescent molecular rotor CCVJ (9-(2-carboxy-2-cyanovinyl)julolidine) was developed. CCVJ is mainly sensitive to viscosity and less to polarity in comparison to polarity-sensitive dyes like SYPRO Orange, which was commonly used in DSF measurements. Therefore DSF with CCVJ is a suitable approach for high-throughput screening and stability testing of surfactant-containing protein formulations.Due to the different detection principles of CCVJ and SYPRO Orange, the midpoint of the fluorescence curve of CCVJ, defined as temperature of aggregation (Tagg), was obtained at a higher temperature than the midpoint of the SYPRO Orange fluorescence curve, defined as temperature of hydrophobic exposure (Th). Granulocyte colony stimulating factor (G-CSF) was used as model protein for all measurements.Commonly used surfactants in therapeutic protein formulations (polysorbate 20, polysorbate 80 and poloxamer 188) were investigated by DSF with CCVJ and SYPRO Orange. The fluorescence properties of CCVJ were minimally affected by investigated surfactants at concentrations typically used in pharmaceutical protein formulations. SYPRO Orange however, showed high background fluorescence as it also interacts with hydrophobic groups of surfactants. CCVJ was also capable of detecting thermally induced aggregation in the commercial polysorbate 80-containing product Neupogen®.
Keywords: Differential scanning fluorimetry; Fluorescent molecular rotors; High-throughput screening; Surfactants; Thermal stability; CCVJ;

Delivery of ursolic acid (UA) in polymeric nanoparticles effectively promotes the apoptosis of gastric cancer cells through enhanced inhibition of cyclooxygenase 2 (COX-2) by Hao Zhang; Xiaolin Li; Jing Ding; Huae Xu; Xinzheng Dai; Zhibo Hou; Kai Zhang; Kun Sun; Weihao Sun (261-268).
UA-loaded nanoparticles (UA-NPs) efficiently elicit more accumulation of intracellular drug by endocytosis, leading to more efficient inhibition of COX-2 and activation of caspase-3 and inducing more cell apoptosis, which demonstrates that UA-NPs statistically surpass free UA through COX-2 pathways.It has been demonstrated that ursolic acid (UA) could effectively induces apoptosis of cancer cells by inhibiting the expression of cyclooxygenase 2 (COX-2), which constitutively expresses in gastric cancer. However, the hydrophobicity of UA increases the difficulty in its potential clinical application, which raises the possibility for its application as a novel model drug in nanoparticle-based delivery system. UA-loaded nanoparticles (UA-NPs) were prepared by a nano-precipitation method using amphilic methoxy poly(ethylene glycol)–polycaprolactone (mPEG–PCL) block copolymers as drug carriers. UA was effectively transported into SGC7901 cells by nanoparticles and localized around the nuclei in the cytoplasms. The in vitro cytotoxicity and apoptosis test indicated that UA-NPs significantly elicited more cell death at almost equivalent dose and corresponding incubation time. Moreover, UA-NPs led to more cell apoptosis through stronger inhibition of COX-2 and activation of caspase 3. The most powerful evidence from this report is that the significant differences between the cytotoxicity of free UA and UA-NPs are closely related to the expression levels of COX-2 and caspase-3, which demonstrates the superiority of UA-NPs over free UA through penetrating cell membrane. Therefore, the study offer an effective way to improve the anticancer efficiency of UA through nano-drug delivery system.
Keywords: Ursolic acid; mPEG–PCL; Nanoparticle; COX-2; Gastric cancer;

Food proteins as novel nanosuspension stabilizers for poorly water-soluble drugs by Wei He; Yi Lu; Jianping Qi; Lingyun Chen; Fuqiang Hu; Wei Wu (269-278).
Nanonization of the poorly water-soluble drugs is a promising strategy to improve dissolution and oral bioavailability. To stabilize the drug nanosuspensions, stabilizers are usually used; however, the use of common stabilizers is limited by weak stabilization effect and toxicological concerns for long-term treatment. The present work was to investigate the potential of food proteins as novel safe stabilizers for nanosuspensions using indomethacin as a model drug. The nanosuspensions stabilized by food proteins (soybean protein isolate, whey protein isolate and β-lactoglobulin) were prepared by the nanoprecipitation–ultrasonication method. The particle size could be easily reduced to 100–400 nm with bimodal particle-size distribution through monitoring the preparative variables. The exposure of buried hydrophobic moieties due to heat-denaturation and subsequent adsorption onto the surface of drug particles was assumed to contribute to their efficient stabilization effect. In comparison with conventional stabilizers, food proteins are superior in stabilization efficiency. The dissolution was enhanced significantly owing to particle size reduction. The protein-stabilized nanosuspensions could be easily freeze-fried and reconstituted into nanosuspensions, keeping the original mean particle size and particle-size distribution. It is concluded that the three denatured proteins perform as efficient stabilizers for indomethacin nanosuspensions.
Keywords: Nanosuspensions; Food proteins; Poorly water-soluble drugs; Stabilizers;

The effect of polyethyleneglycol (PEG)-modified liposome as a drug carrier has been demonstrated clinically. We designed and synthesized a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG, DDA-PEG) which had two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposome. DDA-PEG-modified liposomal doxorubicin (DDA-LDOX(7.5)) had the biggest fixed aqueous layer thickness (FALT) compared with other PEG-lipid-modified liposomes even if the added amount was a few. It was thought that FALT was the indication of blood circulation time. In DOX uptake in tumor cells, DDA-LDOX(7.5) group increased the DOX concentration in tumor cells because it had contact ability with tumor cells. Hence, DDA-LDOX(7.5) which has long circulation time in the bloodstream and contact ability with tumor cells, also had a strong antitumor effect on mice bearing M5076 ovarian sarcoma cells which were DOX low sensitive cells according to the expression of multidrug resistance protein. Furthermore, this liposome maintained a high DOX concentration in a tumor for a long time. These results indicated that the useful antitumor effect of DDA-LDOX(7.5) against M5076 ovarian sarcoma cells is a promising DDS carrier for therapies against drug resistant tumors.
Keywords: Liposome; Polyethyleneglycol modification; Long circulation time; Contact ability; Anti-tumor effect;

Intra-articular delivery of liposomal celecoxib–hyaluronate combination for the treatment of osteoarthritis in rabbit model by Ji Dong; Dinghua Jiang; Zhen Wang; Guizhong Wu; Liyan Miao; Lixin Huang (285-290).
Using an incapacitance tester and the histopathological study, it was verified that the liposomal Clx–HA combination are more effective than a single drug in pain control and cartilage protectionOral administration of celecoxib (Clx), which is the traditional treatment for osteoarthritis (OA), is accompanied by a high risk for cardiovascular events, while intra-articular injection of hyaluronate (HA) is a well-documented treatment for knee OA. To improve OA therapy while reducing the adverse effects, we formulate Clx-loaded liposomes embedded in HA gel, then administer the liposomal Clx–HA combination via intra-articular injection. Clx-loaded liposomes showed high efficiency encapsulation (>99%). In vitro release studies demonstrated that the release of Clx from lipsosomes was delayed by the combination of HA with liposomes. We examined the effect of intra-articular injection of liposomal Clx–HA combination on cartilage degeneration in rabbit knee OA model. The rabbits were treated with a single intra-articular injection of a single drug, either Clx liposome or HA, or liposomal Clx–HA combination. Using an incapacitance tester and the histopathological study, it was verified that the liposomal Clx–HA combination was more effective than a single drug in pain control and cartilage protection.
Keywords: Intra-articular injection; Liposome; Celecoxib; Hyaluronate; Combination; Osteoarthritis;

It is well known that combination therapy can significantly enhance the cytotoxicity and bypass some resistance mechanisms. However, the different solubility and pharmacokinetics of drugs limit the applications of combination therapy. In this study, novel glucose-functionalized polymeric micelle nanoparticles containing multidrugs were successfully fabricated and characterized. Two chemotherapeutic agents, cytarabine (Ara-C) and fluorodeoxyuridine (FUDR), were conjugated to a glucose-functionalized amphiphilic random terpolymer to create a novel nanocarrier for the delivery of multiple drugs simultaneously with an identical pharmacokinetic profile. The incorporation of d-glucose markedly increased the dispersity and biocompatibility of the novel polymeric micelles. In vitro drug release studies showed the two anticancer agents could be simultaneously released from multidrug-conjugating nanoparticles. Cellular uptake assay observed by confocal laser scanning microscopy and cytotoxicity tests performed by MTT assay against hepG2 human hepatoma cells indicated that glucose-functionalized multidrug-conjugating nanoparticles could be effectively internalized by HepG2 cells and showed much more effective growth-inhibitory activity than two single-drug-conjugating polymer aggregates or free drugs. This finding, therefore, illustrated that the d-glucose functionalized nanoparticles could be used as a novel potential multidrug delivery vehicle.
Keywords: Glucose-functionalized; Terpolymer; Cytarabine; Fluorodeoxyuridine; Cytotoxicity;

Crystal structure study and investigation of solid-state cyclization for AMG 222, a channel hydrate by Y.-H. Kiang; Karthik Nagapudi; Jodi Liu; Richard J. Staples; Janan Jona (299-306).
In this study, we investigate the solid-state structure and stability of AMG 222 (5-(2-[2-(2-cyano-pyrrolidin-1-yl)-2-oxo-ethylamino]-propyl)-5-(1H-tetrazol-5-yl)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-2,8 dicarboxylic acid bisdimethylamide), a small molecule DPP-IV inhibitor. Crystal structure of AMG 222 has been solved from single crystal X-ray analysis. Crystallographic data are as follows: monoclinic, P21 (no. 4), a  = 9.0327(5) Å, b  = 18.6177(8) Å, c  = 21.4927(10) Å, β  = 90.126(3)°, V  = 3614.4(3) Å3, Z  = 4. Based on single crystal structure, AMG 222 is a pentahydrate with the water molecules sitting in channels formed by the drug framework. There are three distinct crystal structures of AMG 222 between 0 and 95% relative humidity (RH), namely the anhydrate, hemihydrate, and pentahydrate forms. Solid-state stability of the GMP batch showed a high level of cyclized degradation product. It was postulated that the degradation was promoted by increased amorphous content generated as a result of excessive drying that was employed to remove residual crystallization solvent. Material produced using a modified procedure using a humidified nitrogen purge had lower amorphous content and lower levels of cyclic degradation when compared to the GMP batch.
Keywords: Channel hydrate; X-ray powder diffraction; Solid-state stability; Solid-state NMR; Single crystal structure;

Development of a novel histone H1-based recombinant fusion peptide for targeted non-viral gene delivery by Fatemeh Soltani; Mojtaba Sankian; Arash Hatefi; Mohammad Ramezani (307-315).
In this study a new multifunctional recombinant gene delivery system (vector) was developed for targeted gene delivery to ZR-75-1 breast cancer cells. The vector backbone contained multiple domains including: (1) two tandem repeating units of truncated histone H1 to condense pDNA, (2) a model cell targeting peptide to target ZR-75-1 cells, (3) a pH-responsive synthetic fusogenic peptide, KALA, to destabilize endosomal membrane, and (4) a nuclear localization signal from human immunodeficiency virus to enhance translocation of pDNA toward the cell nucleus.The vectors were cloned and expressed in Escherichia coli BL21 (DE3) followed by purification with Ni-NTA affinity chromatography. They were then characterized using physicochemical and in vitro biological methods to evaluate the gene transfer efficiency and vector multifunctionality. The results demonstrated that the recombinant vector bearing all four functional domains had the highest rate of gene transfection efficiency as compared to the vectors which lacked one or more functional motifs. Beside the ability to target, the developed multifunctional vector was able to disrupt endosomal membranes, reach cell nucleus by utilizing microtubules and transfect efficiently while showing no detectable toxicity.
Keywords: Recombinant peptides; Non-viral gene delivery systems; Targeted delivery; Cancer gene therapy;

A mechanistic model for the prediction of in-use moisture uptake by packaged dosage forms by Johan Remmelgas; Anne-Laure Simonutti; Åsa Ronkvist; Lubomir Gradinarsky; Anders Löfgren (316-322).
A mechanistic model for the prediction of in-use moisture uptake of solid dosage forms in bottles is developed. The model considers moisture transport into the bottle and moisture uptake by the dosage form both when the bottle is closed and when it is open. Experiments are carried out by placing tablets and desiccant canisters in bottles and monitoring their moisture content. Each bottle is opened once a day to remove one tablet or desiccant canister. Opening the bottle to remove a tablet or canister also causes some exchange of air between the bottle headspace and the environment. In order to ascertain how this air exchange might depend on the customer, tablets and desiccant canisters are removed from the bottles by either carefully removing only one or by pouring all of the tablets or desiccant canisters out of the bottle, removing one, and pouring the remaining ones back into the bottle. The predictions of the model are found to be in good agreement with experimental data for moisture sorption by desiccant canisters. Moreover, it is found experimentally that the manner in which the tablets or desiccant canisters were removed does not appreciably affect their moisture content.
Keywords: Stability; Moisture; Sorption; Packaging; In-use;

In vitro digestion testing is of practical importance to predict the fate of drugs administered in lipid-based delivery systems. Calcium ions are often added to digestion media to increase the extent of digestion of long-chain triglycerides (LCTs), but the effects they have on phase behaviour of the products of digestion, and consequent drug solubilization, are not well understood. This study investigates the effect of calcium and bile salt concentrations on the rate and extent of in vitro digestion of soybean oil, as well as the solubilizing capacity of the digestion products for two poorly water-soluble drugs, fenofibrate and danazol. In the presence of higher concentrations of calcium ions, the solubilization capacities of the digests were reduced for both drugs. This effect is attributed to the formation of insoluble calcium soaps, visible as precipitates during the digestions. This reduces the availability of liberated fatty acids to form mixed micelles and vesicles, thereby reducing drug solubilization. The use of high calcium concentrations does indeed force in vitro digestion of LCTs but may overestimate the extent of drug precipitation that occurs within the intestinal lumen.
Keywords: Lipid-based drug delivery systems; Pancreatic lipase; Poorly water-soluble drugs; Calcium; Bile salt; Lipolysis;

To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE).HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser® G (2%), FBS (10%) and NuSerum® (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies.Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser® G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (≅2–5-fold for FBS and 2–8-fold for Ultroser® G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1 > PEPT2 > OCT3 > PHT1 > PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [β-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum® compared to Ultroser® G and FBS cultured cells (p  > 0.05).Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser® G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum® as a supplement for growing HNE for transport studies involving SLC transporters.
Keywords: Nasal epithelium; Peptide transporters; Organic cation transporters; OCT1-3; OCTN1; OCTN2; PEPT1; PEPT2;

Chitosan as a pore former in coated beads for colon specific drug delivery of 5-ASA by Wycliffe S. Omwancha; Rama Mallipeddi; Brenda L. Valle; Steven H. Neau (343-351).
A multiparticulate product for colon-specific delivery of a small molecule drug has been developed and characterized. Microcrystalline cellulose core beads containing 5-aminosalicylic acid produced by extrusion-spheronization were coated with chitosan and Aquacoat® ECD mixtures according to a factorial design. Coated beads were characterized in terms of drug release, shape, and friability. The optimum formulation was enteric coated and exposed to media simulating conditions in the stomach, small intestine, and colon. Release studies in simulated intestinal fluid revealed that the drug release rate from the coated beads, which were spherical and rugged, depended on the level of chitosan in the coat and the coat thickness. Enlarged pores observed on the surface of the coated beads exposed to the medium containing rat cecal and colonic enzymes are believed to have caused a significant enhancement of the drug release rate compared to the control exposed only to simulated gastric and intestinal fluids. The release mechanisms involved polymer relaxation and dissolved drug diffusion for simulated intestinal fluid and simulated colonic fluid, respectively. From the facilitated drug release in a colonic environment and the inhibition of drug release under gastric and intestinal conditions, it can be concluded that this multiparticulate system demonstrates the potential for colon-specific drug delivery.
Keywords: Biodegradable polymer; Chitosan; Colonic drug delivery; Controlled delivery; Excipients; Extrusion; Formulation; Site-specific delivery; Spheronization;

Effect of HP-β-cyclodextrin in the diffusion behaviour of hydrocortisone in aqueous solutions at T  = 298.15 K by Ana C.F. Ribeiro; Luís M.P. Veríssimo; Cecilia I.A.V. Santos; Ana M.T.D.P.V. Cabral; Francisco J.B. Veiga; Miguel A. Esteso (352-355).
Binary (D) and ternary (D 11, D 22, D 12 and D 21) mutual diffusion coefficients determined by the Taylor dispersion method are reported for two aqueous systems, pure hydrocortisone (HC/H2O) and HC plus HP-β-cyclodextrin (HC/HP-βCD/H2O), at T  = 298.15 K. From these data, some thermodynamic information as well as conclusions about the influence of that carbohydrate in the diffusion of this drug are given.
Keywords: Hydrocortisone; Cyclodextrins; Diffusion coefficient; Aqueous solutions;

High-throughput 96-well solvent mediated sonic blending synthesis and on-plate solid/solution stability characterization of pharmaceutical cocrystals by Van Luu; Janan Jona; Mary K. Stanton; Matthew L. Peterson; Henry G. Morrison; Karthik Nagapudi; Helming Tan (356-364).
A 96-well high-throughput cocrystal screening workflow has been developed consisting of solvent-mediated sonic blending synthesis and on-plate solid/solution stability characterization by XRPD. A strategy of cocrystallization screening in selected blend solvents including water mixtures is proposed to not only manipulate solubility of the cocrystal components but also differentiate physical stability of the cocrystal products. Caffeine–oxalic acid and theophylline–oxalic acid cocrystals were prepared and evaluated in relation to saturation levels of the cocrystal components and stability of the cocrystal products in anhydrous and hydrous solvents. AMG 517 was screened with a number of coformers, and solid/solution stability of the resulting cocrystals on the 96-well plate was investigated. A stability trend was observed and confirmed that cocrystals comprised of lower aqueous solubility coformers tended to be more stable in water. Furthermore, cocrystals which could be isolated under hydrous solvent blending condition exhibited superior physical stability to those which could only be obtained under anhydrous condition. This integrated HTS workflow provides an efficient route in an API-sparing approach to screen and identify cocrystal candidates with proper solubility and solid/solution stability properties.
Keywords: Cocrystal; Solvent-mediated; Stability; Solubility; XRPD; High-throughput; Automation;

Dexamethasone (Dex)-loaded implants were prepared by poly(d,l-lactic acid) (PLA) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (PEG-PPG-PEG, Pluronic F68) using hot-melt extrusion method. The purpose of this research was to develop a hot-melt extruded implant PLA/F68/Dex for controlling release of Dex at the implant site. Drug loading and encapsulation efficiency were determined by UV spectrophotometer analysis. The maximum value of the drug loading and encapsulation efficiency for the implants was up to 48.9% and 97.9%, respectively. Differential scanning calorimetry was used to evaluate stability and interaction between the implant and drug. We had studied the water uptake of PLA/F68 implants kept constant at about 12% due to the water was absorbed to a large extent. Dex release profile in vitro was studied, and the results showed that the maximum value of the release rate was approximately 20%. The degradation behavior was confirmed by mass loss and scanning electron microscopy. In addition, the in vivo biocompatibility study indicated that the implants had no negative influence as a foreign material in the body response.
Keywords: PLA; PEG-PPG-PEG; Dexamethasone; Hot-melt extrusion; Controlled drug delivery; In vitro study; Biocompatibility;

Chitosan oligosaccharide–arachidic acid-based nanoparticles for anti-cancer drug delivery by Ubonvan Termsarasab; Hyun-Jong Cho; Dong Hwan Kim; Saeho Chong; Suk-Jae Chung; Chang-Koo Shim; Hyun Tae Moon; Dae-Duk Kim (373-380).
Chitosan oligosaccharide–arachidic acid (CSOAA) was synthesized and doxorubicin (DOX)-loaded CSOAA-based self-assembled nanoparticles were prepared. Therapeutic effect of DOX-loaded nanoparticles for head and neck cancer was evaluated.Chitosan oligosaccharide–arachidic acid (CSOAA) conjugate was successfully synthesized and used for the development of self-assembled nanoparticles for doxorubicin (DOX) delivery. The molar substitution of AA on CSO and critical micelle concentration (CMC) of CSOAA were measured. Physicochemical properties of DOX-loaded CSOAA-based nanoparticles, such as particle size, zeta potential and morphology, were also characterized. The DOX-loaded CSOAA-based nanoparticles showed spherical shape with a mean diameter of 130 nm and positive charge. According to the result of in vitro release test, DOX-loaded CSOAA-based nanoparticles exhibited sustained and pH-dependent drug release profiles. The CSOAA showed negligible cytotoxicity in FaDu, human head and neck cancer, cells. Cellular uptake of DOX in FaDu cells was higher in the nanoparticle-treated group compared to the free DOX group. The anti-tumor efficacy of DOX-loaded nanoparticles was also verified in FaDu tumor xenografted mouse model. These results suggested that synthesized amphiphilic CSOAA might be used for the preparation of self-assembled nanoparticles for anti-cancer drug delivery.
Keywords: Arachidic acid; Chitosan oligosaccharide; Doxorubicin; Head and neck cancer; Self-assembly;

Optimizing manufacture of liposomal berberine with evaluation of its antihepatoma effects in a murine xenograft model by Yung-Chang Lin; Jhan-Yen Kuo; Chih-Chieh Hsu; Wen-Che Tsai; Wei-Chu Li; Ming-Chiang Yu; Hsiao-Wei Wen (381-388).
The aim of this work is to establish an optimal process for generating liposomal berberine and assessing its anticancer ability against human hepatic carcinoma in a murine xenograft model. Of various forms of liposomal berberine with different lipid compositions, preparation by various methods, the one that contained 5 mol% polyethenyl glycol (PEG) that was produced by a thin-film hydration/extrusion method exhibited the highest encapsulation efficiency (E.E.) of berberine (14%). Additionally, in vitro studies reveal that this batch of liposomal berberine inhibited the growth of HepG2 cells 2.5 times as effectively as berberine solution since the half maximal inhibitory concentration (IC50) of berberine solution was 4.23 μg berberine/mL while that of liposomal berberine was only 1.67 μg berberine/mL, and this inhibition effect was based on the induction of apoptosis through the caspase/mitochondria-dependent pathway. Additionally, the results of in vivo studies indicate that the liposome effectively reduced the rate of elimination of berberine in both plasma and tissues, and liposomal berberine effectively reduced the size and weight of tumors as compared with the untreated tumor control group. Therefore, this work demonstrates that liposome is a good carrier for berberine to inhibit the tumor growth in HepG2 tumor-bearing mice.
Keywords: Apoptosis; Liposomal berberine; Caspase/mitochondria-dependent pathway; Human hepatic carcinoma;

The effect of solute–membrane interaction on solute permeation under supersaturated conditions by Jingsi Zhang; Mingjing Sun; Aiping Fan; Zheng Wang; Yanjun Zhao (389-394).
The purpose of this work was to investigate the effect of solute–membrane interaction under supersaturated conditions on the transport of model solute (salicylic acid) across poly(dimethylsiloxane) (PDMS) membrane. Supersaturated systems with a degree of saturation (DS) up to 8 were prepared using a molecular form technique with water as the vehicle to minimize the vehicle–membrane interaction. The spectroscopic and thermal analysis revealed the presence of both hydrogen bonding and nonpolar interaction between the solute and PDMS. Upon treatment by supersaturated solutions the degree of solute–membrane interaction increased with increasing DS. This enhanced the barrier property of PDMS and thus led to the flux attenuation compared to that calculated by Higuchi equation. This work highlighted the importance of solute–membrane interaction under supersaturation in the flux reduction, which should be considered when designing, and optimizing supersaturated topical and transdermal drug delivery systems.
Keywords: Supersaturation; Permeation; Poly(dimethylsiloxane); Salicylic acid; Solute–membrane interaction;

Abrogation of the accelerated blood clearance phenomenon by SOXL regimen: Promise for clinical application by Ai Nagao; Amr S. Abu Lila; Tatsuhiro Ishida; Hiroshi Kiwada (395-401).
We investigated the contribution of a repeated administration of l-OHP containing PEGylated liposomes in a SOXL regimen, a combination regimen of S-1 and l-OHP containing PEGylated liposomes, in the induction of the ABC phenomenon in both normal and tumor bearing mice. We demonstrated that SOXL regimen attenuates the ABC phenomenon in both normal mice and tumor-bearing mice. The abrogation of the ABC phenomenon restored the intratumor accumulation of subsequently injected PEGylated liposomes.We recently proposed an S-1 combined with oxaliplatin (SOXL) regimen, a combination treatment consisting of oral metronomic S-1 dosing and intravenous administration of oxaliplatin (l-OHP) containing PEGylated liposomes, which showed potent antitumor activity in vivo. PEGylated liposomes induce what is referred to as the “accelerated blood clearance (ABC) phenomenon” upon repeated administration and consequently lose their long-circulating characteristics. This phenomenon seems to pose an impediment for the clinical application and use of PEGylated liposomal formulations. In the present study, l-OHP-containing PEGylated liposomes in the SOXL regimen significantly attenuated the ABC phenomenon in a dose-dependent manner through suppression of the anti-PEG IgM response, which allowed an enhanced hepatic uptake of subsequently injected test PEGylated liposomes. In tumor-bearing mice, the abrogation of the ABC phenomenon restored intratumor accumulation of subsequently injected PEGylated liposomes. Consequently, the therapeutic efficacy of the SOXL regimen over the combination of the free form of the drugs was credited not only with the selective delivery of drugs to the tumor tissue but also with ensuring an adequate accumulation of subsequent doses within the tumor tissue. The SOXL regimen we proposed may hold promise as a safe and effective treatment regimen for advanced colorectal cancer.
Keywords: Accelerated blood clearance (ABC) phenomenon; PEGylated liposome; Polyethylene glycol (PEG); Oxaliplatin; S-1;

Real-time monitoring of lubrication properties of magnesium stearate using NIR spectrometer and thermal effusivity sensor by Hiroshi Nakagawa; Manabu Kano; Shinji Hasebe; Tatsuya Suzuki; Naoki Wakiyama (402-413).
Real-time monitoring techniques based on an NIR spectrometer and a thermal effusivity sensor for lubrication properties of magnesium stearate (Mg-St) are proposed. The lubrication properties of Mg-St are defined by its concentration distribution and flatting state in a mixture. The concentration distribution of Mg-St significantly affects the absorbance in the NIR wavelength region between 1128 nm and 1240 nm. Thus, the absorbance area in this region after baseline correction was selected as a monitoring index (MI). In laboratory-scale experiments, the difference of Mg-St concentration distribution could be detected by the proposed MI with high sensitivity. In addition, experimental results using spherical mannitol granules confirmed that the changes of the flatting state of Mg-St could also be detected by the proposed MI. Similar experiments with spherical mannitol granules and the thermal effusivity sensor confirmed that effusivity could also be used to detect the changes of the flatting state of Mg-St. The applicability of these monitoring techniques was verified using a 2000 L commercial-scale blender equipped with the NIR spectrometer and the thermal effusivity sensor. The results showed that both lubrication properties could be monitored by the proposed MI, and that the flatting state of Mg-St could be monitored more sensitively by using the effusivity.
Keywords: Near infrared; Effusivity; Magnesium stearate; Blend uniformity; Lubrication property; Process analytical technology;

Polysaccharide-anchored fatty acid liposome by Hsiao Wei Tan; Misni Misran (414-423).
In this study, the preparation of N-pamitoyl chitosan (ChP) anchored oleic acid (OA) liposome was demonstrated. Two different types of water-soluble ChPs with different degrees of acylation (DA) were selected for this study. The presence of ChPs on the surface of OA liposome was confirmed with their micrographs and physicochemical properties. The “peeling off” effect on the surface of the ChP-anchored OA (OAChP) liposomes was observed on the atomic force microscope micrographs and confirmed the presence of the ChPs layer on the liposome surface. The surface tension of the OAChPs liposome solution was found to be higher than that of the OA liposome solution. This result indicated the removal of OA monomer by ChPs from the air–water interface. The increase in the minimum area per headgroup (A min ) of the OA with the presence of ChPs has further proved the interaction between OA monomer and the hydrophobic moieties of the ChPs. The ChPs anchored onto the OA monolayer increased the curvature of the OAChP liposomes monolayer and reduced the liposome size. The size of the OAChP liposomes was reduced by 30 nm as compared with the unmodified OA liposome. Results revealed that the anchored ChPs can improve the integrity and rigidity of the OA liposome.
Keywords: Oleic acid vesicle; Surface tension; Atomic force microscope; Drug delivery; Chitosan;

Fabrication and evaluation of pH-modulated solid dispersion for telmisartan by spray-drying technique by Nirmal Marasini; Tuan Hiep Tran; Bijay Kumar Poudel; Hyuk Jun Cho; Young Keun Choi; Sang-Cheol Chi; Han-Gon Choi; Chul Soon Yong; Jong Oh Kim (424-432).
The present study was undertaken to overcome the problems associated with solubility, dissolution and oral bioavailability of a poorly water-soluble ionizable drug, telmisartan (TMS). For these purposes, a solubility test was carried to select the appropriate formulation composition from various carriers and alkalizers. Solid dispersions (SDs) of TMS were prepared at different drug-to-carrier ratios by the spray-drying technique, and were characterized by dissolution and aqueous solubility studies. The optimum formulation was investigated by dissolution studies at different pH and water media and its solid state characterisations were performed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies. In solubility and dissolution tests, all TMS-loaded pH-modulated SDs (pHM-SDs) exhibited marked improvement in the dissolution behavior when compared with crystalline TMS powder. The optimum formulation of pHM-SD consisted of TMS/PVP (polyvinylpyrrolidone) K30/Na2CO3 at a weight ratio of 2/0.5/3 and showed significant improvement in the aqueous solubility and dissolution rate by approximately 40,000- and 3-fold, respectively, compared to TMS powder. Solid-state characterization revealed the changed in crystallinity of TMS into amorphous state. Furthermore, area under the drug concentration time-curve (AUC) of TMS from the pHM-SD increased by 13.4- and 2.1-fold, compared with TMS powder and commercial product, respectively. According to these observations, taken together with dissolution and pharmacokinetic behaviors, pH-modulated SD in the presence of an alkalizer for a poorly water-soluble ionizable drug, TMS, appeared to be efficacious for enhancing its bioavailability.
Keywords: Solid dispersion; Telmisartan; Alkalizer; Spray-drying; Bioavailability;

A miniaturized flow-through cell to evaluate skin permeation of endoxifen by Choon Siong Mah; Jaspreet Singh Kochhar; Pei Shi Ong; Lifeng Kang (433-440).
Endoxifen, an anti-estrogenic agent, has been recently implicated in the use of breast cancer. Its physicochemical properties make it a good candidate for transdermal delivery. However, as an investigative drug, its limited supply makes it difficult to conduct extensive pre-formulation studies. To address this issue, a miniaturized flow-through diffusion cell has been fabricated that utilized minimal amounts of the drug for in vitro skin permeation studies. The novel flow-through cells have been validated against horizontal diffusion cells and shown to cause no noticeable damage to the applied skin, as observed by histological sectioning. The cells were also demonstrated to be useful in search of suitable enhancers for endoxifen. Endoxifen permeation using permeation enhancers was tested by using this new device and limonene was found to achieve highest flux, attaining the requirement for clinical applications. The fabricated cells can thus be useful in carrying out pre-formulation studies for expensive, new drug entities, both in industrial as well as academic research.
Keywords: Flow-through diffusion cell; Transdermal drug delivery; Breast cancer; Endoxifen; Polydimethylsiloxane;

Relationship between surface properties determined by inverse gas chromatography and ibuprofen release from hybrid materials based on fumed silica by Joanna Kołodziejek; Eliza Główka; Kinga Hyla; Adam Voelkel; Janina Lulek; Kasylda Milczewska (441-448).
The ability of organic–inorganic hybrid materials to act as drug release-modifying agents was examined. In this study, ibuprofen, a non-steroidal anti-inflammatory drug was used as a model active pharmaceutical ingredient. The physicochemical properties of individual components of the hybrids, as well as these for two- and three-component systems were examined by inverse gas chromatography. The dispersive component of the free surface energy ( γ S D ), K A and K D parameters describing acidity and basicity of hybrid materials, respectively, as well as Flory–Huggins parameters were determined. χ 12 ∞ and χ ′ 23 parameters characterize the interactions between the hybrids and a test solute, or interactions between the drug and inorganic–organic materials, respectively. Additionally, Brunauer–Emmett–Teller (BET) method was used to characterize adsorption activity of the studied materials. The prepared hybrid materials were also characterized by Fourier transform infra-red (FTIR) spectroscopy. The release profiles of ibuprofen for the created hybrid materials were determined. Relationship between the physicochemical activity of hybrid materials and ibuprofen release was presented and discussed.
Keywords: Hybrid materials; Inverse gas chromatography; Ibuprofen; Sustained release;

The aim of this study was to prepare solid lipid microspheres (SLM) with incorporated Cyclosporine A (Cs), suitable for ocular application. For this purpose, SLM were formulated by using different lipids and three different nonionic surfactants. The SLM were produced using a hot emulsification method. The SLM dispersions contained 10, 20 or 30% of lipid (w/w) and up to 2% (w/w) of Cs. The size of the microspheres with Cs ranged from 1 to 15 μm. Physically stable SLM with Cs were prepared using Compritol, as a lipid matrix, and Tween 80, as a surfactant. In contrast, dispersion with Precirol alone, formed semi-solid gels during storage, while in formulations with Precirol and Miglyol, crystals of Cs were observed. In vitro release profile of Compritol formulations showed that 40% of Cs is released within 1 h, while the release of the following 40% takes more time, depending on lipid content in the formulations. The large part of Cs, added to SLM formulations (from 45 to 80%), was found on the surface of microparticles, but no drug crystallization occurred during a long-term storage.
Keywords: Cyclosporine; Solid lipid microspheres; Phase distribution; Stability; Solubility;

Permutation Test (PT) and Tolerated Difference Test (TDT): Two new, robust and powerful nonparametric tests for statistical comparison of dissolution profiles by José-David Gómez-Mantilla; Vicente Germán Casabó; Ulrich F. Schaefer; Claus-Michael Lehr (458-467).
The most popular way of comparing oral solid forms of drug formulations from different batches or manufacturers is through dissolution profile comparison. Usually, a similarity factor known as (f2) is employed; However, the level of confidence associated with this method is uncertain and its statistical power is low. In addition, f2 lacks the flexibility needed to perform in special scenarios. In this study two new statistical tests based on nonparametrical Permutation Test theory are described, the Permutation Test (PT), which is very restrictive to confer similarity, and the Tolerated Difference Test (TDT), which has flexible restrictedness to confer similarity, are described and compared to f2. The statistical power and robustness of the tests were analyzed by simulation using the Higuchi, Korsmayer, Peppas and Weibull dissolution models. Several batches of oral solid forms were simulated while varying the velocity of dissolution (from 30 min to 300 min to dissolve 85% of the total content) and the variability within each batch (CV 2–30%). For levels of variability below 10% the new tests exhibited better statistical power than f2 and equal or better robustness than f2. TDT can also be modified to distinguish different levels of similarity and can be employed to obtain customized comparisons for specific drugs. In conclusion, two new methods, more versatile and with a stronger statistical basis than f2, are described and proposed as viable alternatives to that method. Additionally, an optimized time sampling strategy and an experimental design-driven strategy for performing dissolution profile comparisons are described.
Keywords: Dissolution profile comparisons; Similarity factor; Dissolution models; Non-parametric tests; Statistical robustness; Statistical power;

Selection of PLA polymers for the development of injectable prilocaine controlled release microparticles: Usefulness of thermal analysis by Marco Bragagni; Cristina Beneitez; Cristina Martín; Dolores Hernán Pérez de la Ossa; Paola Angela Mura; María Esther Gil-Alegre (468-475).
The use of injectable local anaesthetics for the treatment of severe postoperative pain is limited by the short duration of the painkilling effect. Pre-formulation studies were carried out for the development of an injectable microparticle formulation for controlled release of prilocaine, an amino-amide type local anaesthetic suitable for intravenous, subcutaneous and intramuscular administration. To the best of our knowledge, the encapsulation of prilocaine into microparticles has not been investigated yet. Three different poly-lactic-acid (PLA) polymers were separately employed for the preparation of the microparticles. Thermal analyses by differential scanning calorimetry (DSC) were carried out for the characterization of the raw materials, to assess the drug–polymer compatibility and miscibility, to investigate the effects of the production process on the components. Empty and prilocaine loaded microparticles were prepared by double emulsion method. All formulations were fully characterized in terms of drug content, morphology, size and in vitro drug release. The preliminary value of PRL solubility in the polymer material determined by DSC was evaluated and discussed as a predictive value for encapsulation efficiency and controlled release. DSC analysis turned out to be a usefulness tool for a fast polymer selection. Microparticles prepared with PLA R202 and R203S showed desirable characteristics for subcutaneous administration and could represent two promising formulations for the development of innovative pharmacological tools in the treatment of postoperative pain.
Keywords: DSC; Drug–polymer miscibility; Compatibility; Highly water-soluble drug; Local anaesthesia;

In a previous study, we reported on the efficient delivery of an antigen to the cytosol and a specific-antigen presentation on MHC class I in dendritic cells by rationally controlling the intracellular trafficking of ovalbumin (OVA), a model antigen, with stearylated octaarginine-modified liposomes (R8-Lip/OVA). However, no significant difference in antitumor effects against E.G7-OVA, OVA expressed lymphoma, was observed between R8-Lip/OVA and an electrostatic complex of R8 and OVA (R8/OVA-Com). In this study, we hypothesized that use of adjuvants clarified the difference in immune responses between R8-Lip/OVA and R8/OVA-Com, and selected polyinosine–polycytidylic acid (polyI:C) as an adjuvant. Cytotoxic T lymphocyte (CTL) activity of the polyI:C and OVA encapsulated R8-Lip (R8-Lip/PIC/OVA) was drastically enhanced compared to R8-Lip/OVA and complete Freund's adjuvant with OVA. Moreover, the incorporation of polyI:C clearly was critical for the difference in antitumor effects and CTL activities between R8-Lip/OVA and R8/OVA-Com. These findings suggest that the carriers that are incorporated polyI:C has a great influence on the induction of cellular immunity in vivo.
Keywords: Liposome; Cell-penetrating peptide; Delivery system; Adjuvant;

Optimization of thermosensitive chitosan hydrogels for the sustained delivery of venlafaxine hydrochloride by Ying Peng; Jie Li; Jing Li; Yin Fei; Jiangnan Dong; Weisan Pan (482-490).
Delivery of venlafaxine hydrochloride with thermosensitive chitosan hydrogels system: diffusion controlled release and kinetic gelation mechanism is nucleation and growth.Chitosan/glycerophosphate disodium (GP) thermosensitive hydrogels were prepared for the sustained delivery of venlafaxine hydrochloride (VH) and optimization of this formulation was mainly studied. Release mechanism was investigated by applying various mathematical models to the in vitro release profiles. Overall, drug release from the hydrogels showed best fit in first-order model and drug release mechanism was diffusion-controlled release. Optimization of VH chitosan/GP thermosensitive hydrogels was conducted by using a three-level three-factorial Box–Behnken experimental design to evaluate the effects of considered variables, the strength of the formulation, chitosan concentration and GP amount, on the selected responses: cumulative percentage drug release in 1 h, 24 h and the rate constant. It presented that higher strength and GP concentration resulted in higher initial release and rate constant, which supported the hypothesis that the kinetic gelation mechanism of this system was nucleation and growth. Drug release profiles illustrated that controlled drug delivery could be obtained over 24 h, which confirmed the validity of optimization. In vivo pharmacokinetic study was investigated and it demonstrated that compared with VH solution, chitosan/GP thermosensitive hydrogels had a better sustained delivery of VH.
Keywords: Chitosan; Glycerophosphate disodium; Thermosensitive hydrogel; Box–Behnken design; Release mechanism; Venlafaxine;

Preparation and characterization of CK2 inhibitor-loaded cyclodextrin nanoparticles for drug delivery by Florent Perret; Christelle Marminon; Wael Zeinyeh; Pascal Nebois; Andre Bollacke; Joachim Jose; Hélène Parrot-Lopez; Marc Le Borgne (491-498).
Casein Kinase 2 (CK2) is a ubiquitous kinase protein currently targeted for the treatment of some cancers. Recently, the series of indeno[1,2-b]indoles has revealed great interest as potent and selective CK2 ATP-competitive inhibitors. Among them, 1-amino-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (CM1) was selected for an encapsulation study in order to improve its biodisponibility. Its complexation was evaluated at the molecular scale, with a series of fluorinated or hydrocarbonated amphiphilic cyclodextrins (CDs). Then the encapsulation of CM1 within CD nanoparticles at the supramolecular level was achieved. Nanoparticles formed between CM1 and hexakis[6-deoxy-6-(3-perfluorohexylpropanethio)-2,3-di-O-methyl]-α-cyclodextrin, a fluorinated amphiphilic α-cyclodextrin, gave the best results in terms of encapsulation rate, stability and drug release. These nanospheres showed an encapsulation efficiency of 65% and a sustained release of the entrapped drug over 3 h. Based on these results, encapsulation within fluorinated amphiphilic CD nanoparticles could be considered as a potential drug delivery system for indenoindole-type CK2 inhibitors, allowing better biodisponibility and offering perspectives for tumor targeting development.
Keywords: CK2 inhibitor; Indeno[1,2-b]indole derivative; Amphiphilic cyclodextrins; Drug delivery; Nanoparticles; Encapsulation;

In this work, we evaluated combinations of doxorubicin with INNO-206, a (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) that is currently undergoing two phase II clinical trials, in a primarily chemoresistant tumor indication, i.e. pancreatic cancer. Thus, we compared the antitumor efficacy and tolerability of the following weekly intravenous treatments in the MIA PaCa-2 xenograft model: 3 × 6 mg doxorubicin (MTD), 3 × 24 mg/kg DOXO-EMCH (doxorubicin equivalents, MTD), 3 × 3 mg/kg doxorubicin followed 6 h later by 3 × 12 mg/kg DOXO-EMCH, and 3 × 12 mg/kg DOXO-EMCH followed 6 h later by 3 × 3 mg/kg doxorubicin. Whereas therapy with doxorubicin only produced a moderate tumor inhibition, all other therapy arms induced complete and partial remissions up to the end of the experiment on day 43. Although the total amount of doxorubicin equivalents is 72 mg/kg when DOXO-EMCH is administered alone, but only 45 mg/kg doxorubicin equivalents are administered in the combination regimens, the antitumor efficacy in all treated groups was essentially identical, a surprising finding of this study. However, there were significant differences in the tolerability as assessed by the body weight changes: whereas therapy at the MTD of DOXO-EMCH (3 × 24 mg/kg) produced a body weight loss of −16% including one death, therapy with 3 × 12 mg/kg DOXO-EMCH followed 6 h later by 3 × 3 mg/kg doxorubicin produced −7% body weight loss, and 3 × 3 mg/kg doxorubicin followed 6 h later by 3 × 12 mg/kg DOXO-EMCH produced a body weight gain of +2% as a clear indication of minimal systemic toxicity. In addition, cell culture experiments revealed additive to synergistic effects when MIA PaCa-2 cells were exposed to doxorubicin followed 6 h later to exposure of the albumin-bound form of DOXO-EMCH spanning a ratio of 1:5 to 5:1 (analyzed for synergistic, additive or antagonistic effects using the software program CalcuSyn®). This animal study demonstrates that the time-dependent schedule of an albumin-binding prodrug and a free drug has a critical influence on the overall tolerability. A combination of doxorubicin and DOXO-EMCH is currently being investigated in a phase Ib study.
Keywords: Doxorubicin; (6-Maleimidocaproyl)hydrazone of doxorubicin; DOXO-EMCH; INNO-206; Prodrugs; Human serum albumin; Combination therapy; Pancreatic cancer;

Dexamethasone eye drops containing γ-cyclodextrin-based nanogels by Maria D. Moya-Ortega; Tiago F.G. Alves; Carmen Alvarez-Lorenzo; Angel Concheiro; Einar Stefánsson; Margrét Thorsteinsdóttir; Thorsteinn Loftsson (507-515).
Sustained release aqueous eye drops of dexamethasone, based on cyclodextrin (CD) nanogels, were designed and tested in vivo. γCD units were cross-linked in the form of nanogels by means of an emulsification/solvent evaporation process. The composition of the nanogels was optimized with regard to drug loading and release rate. The eye drops consisted of an aqueous solution of dexamethasone in 2-hydroxypropyl-γ-cyclodextrin (HPγCD) medium containing γCD nanogels. The nanogel eye drops (containing 25 mg dexamethasone per ml) were tested in rabbits and compared to the commercially available product Maxidex® (suspension with 1 mg dexamethasone per ml). One drop administration of the nanogel eye drops resulted in nearly constant dexamethasone concentration for at least 6 h in the tear fluid (mean concentration ± SD = 295 ± 59 μg/ml) whereas the concentration after administration of Maxidex® fell rapidly from 9.72 ± 3.45 μg/ml 1 h after application to 3.76 ± 3.26 μg/ml 3 h after application. The maximum dexamethasone concentration in the aqueous humor (2 h after application) was 136 ± 24 mg/ml after application of the nanogel eye drops, and only 44.4 ± 7.8 μg/ml after application of Maxidex®. The dexamethasone nanogel eye drops were well tolerated with no macroscopic signs of irritation, redness or other toxic effects.
Keywords: Cyclodextrin; Nanogels; Dexamethasone; Eye drop; Ocular drug delivery;

Thermo-sensitive gels containing lorazepam microspheres for intranasal brain targeting by S. Jose; C.R. Ansa; T.A. Cinu; A.J. Chacko; N.A. Aleykutty; S.V. Ferreira; E.B. Souto (516-526).
Scanning electron microscopy analysis of chitosan microspheres and the percentage of cumulative in vitro lorazepam release from control sample 1 (C1), control sample 2 (C2), and from test sample (S). The chitosan microspheres embedded in the hydrogel show a retarding effect on the release of lorazepam, compared with the chitosan microspheres alone. The in vitro release rate of lorazepam from microspheres suspended in thermo-sensitive gels was assessed for 24 h and was found to be prolonged.Thermo-sensitive gels containing lorazepam microspheres were developed and characterized for intranasal brain targeting. Pluronics (PF-127 and PF-68) have been selected since they are thermo-reversible polymers with the property of forming a solution at low temperatures (4–5 °C), and a gel at body temperature (37 °C). This property makes them an interesting material to work with, especially in case of controlled release formulations. The present study focuses on the development of an intranasal formulation for lorazepam, as an alternative route of drug delivery to the brain. Direct transport of drugs to the brain circumventing the brain barrier, following intranasal administration, provides a unique feature and better option to target brain. The presence of mucoadhesive microspheres in the gel vehicle via nasal route can achieve a dual purpose of prolonged drug release and enhanced bioavailability. To optimise the microsphere formulation, Box Behnken design was employed by investigating the effect of three factors, polymer concentration (chitosan), emulsifier concentration (Span 80) and cross-linking agent (glutaraldehyde) on the response variable which is the mean particle size. The concentration of 21% PF-127 and 1% PF-68 were found to be promising gel vehicles. The results showed that the release rate followed a prolonged profile dispersion of the microspheres in the viscous media, in comparison to the microspheres alone. Histopathological studies proved that the optimised formulation does not produce any toxic effect on the microscopic structure of nasal mucosa.
Keywords: Lorazepam; Thermo-sensitive gels; Microspheres; Pluronics; Brain targeting; Brain delivery; Intranasal delivery;

Flow and compaction behaviour of ultrafine coated ibuprofen by Parth K. More; Kailas S. Khomane; Arvind K. Bansal (527-534).
Good flow and compaction properties are prerequisites for successful compaction process. Apart from initial profile, mechanical properties of pharmaceutical powders can get modified during unit processes like milling. Milled powders can exhibit a wide range of particle size distribution. Further downstream processing steps like compaction can be affected by this differential particle size distribution. This has greatest implications for formulations like high dose drugs wherein the active pharmaceutical ingredient (API) contributes the maximum bulk in the final formulation. The present study assesses the impact of dry coating with ultrafine particles of same material, on the flow and compaction properties of the core material. Ibuprofen was selected as model drug as it has been reported to have poor mechanical properties. Ultrafine ibuprofen (average size 1.75 μm) was generated by Dyno® milling and was dry coated onto the core ibuprofen particles (average size 180 μm). Compaction studies were performed using a fully instrumented rotary tablet press. Compaction data was analyzed for compressibility, tabletability, compactibility profiles and Heckel plot. Dry coating of the ibuprofen exhibited greater compressibility and tabletability, at lower compaction pressure. However, at compaction pressure above 220 MPa, compressibility and tabletability of coated as well as uncoated materials were found to be similar. Heckel analysis also supported the above findings, as P y value of uncoated ibuprofen was found to be 229.49 MPa and for 2.0% ultrafine coated ibuprofen was found to be 158.53 MPa. Lower P y value of ultrafine coated ibuprofen indicated ease of plastic deformation. Superior compressibility and deformation behaviour of ultrafine coated ibuprofen attributed to increased interparticulate bonding area. This strategy can also be explored for improving tabletability of high dose poorly compressible drugs.
Keywords: Compaction; Dry coating; Ibuprofen; Tableting; Mechanical properties; Super plasticity; Compression; Crushing strength;

Biotin uptake by T47D breast cancer cells: Functional and molecular evidence of sodium-dependent multivitamin transporter (SMVT) by Aswani Dutt Vadlapudi; Ramya Krishna Vadlapatla; Dhananjay Pal; Ashim K. Mitra (535-543).
The objective of this study was to investigate functional and molecular evidence of carrier mediated system responsible for biotin uptake in breast cancer (T47D) cells and to delineate mechanism of intracellular regulation of this transporter. Cellular accumulation of [3H] biotin was studied in T47D and normal mammary epithelial (MCF-12A) cells. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the molecular expression of sodium dependent multivitamin transporter (SMVT) in T47D cells. Quantitative real time PCR analysis was also performed to compare the relative expression of SMVT in T47D and MCF-12A cells. [3H] biotin uptake by T47D cells was found to be concentration dependent with K m of 9.24 μM and V max of 27.34 pmol/mg protein/min. Uptake of [3H] biotin on MCF-12A cells was also found to be concentration dependent and saturable, but with a relatively higher K m (53.10 μM) indicating a decrease in affinity of biotin uptake in normal breast cells compared to breast cancer cells. [3H] biotin uptake appears to be time-, temperature-, pH- and sodium ion-dependent but independent of energy and chloride ions. [3H] biotin uptake was significantly inhibited in the presence of biotin, its structural analog desthiobiotin, pantothenic acid and lipoic acid. Concentration dependent inhibition of biotin uptake was evident in the presence of valeric acid which possesses free carboxyl group and biocytin and NHS biotin which are devoid of free carboxyl group. No significant inhibition was observed in the presence of structurally unrelated vitamins (ascorbic acid, folic acid, nicotinic acid, thiamine, pyridoxine and riboflavin). Modulators of PTK, PKC and PKA mediated pathways had no effect, but uptake in presence of calmidazolium (calcium-calmodulin inhibitor) was significantly inhibited. [3H] biotin uptake in the presence of calmidazolium was found to be saturable with a K m and V max values of 13.49 μM and 11.20 pmol/mg protein/min, respectively. A band of SMVT mRNA at 774 bp was identified by RT-PCR analysis. Quantitative real time PCR confirmed higher expression of SMVT in T47D cells relative to MCF-12A cells. All these studies demonstrated for the first time the functional and molecular expression of sodium dependent multivitamin transporter (SMVT), a specific carrier-mediated system for biotin uptake, in human derived breast cancer (T47D) cells. The present study also indicated that cancer cells could import more vitamin compared to normal breast cells possibly for maintaining high proliferative status. We investigated the likelihood of selecting this cell line (T47D) as an in vitro cell culture model to study biotin-conjugated anti-cancer drugs/drug delivery systems.
Keywords: Biotin; SMVT; Expression; Activity; T47D and MCF-12A; In vitro cell culture models;

Photodynamic therapy has a great potential in the treatment of cervical cancer. The aim of this study was to develop bioadhesive pellets containing hexylaminolevulinate (HAL), a precursor of the photoactive substance PpIX, with a fast release for vaginal drug delivery. Pellets were produced by extrusion/spheronization, and Carbopol® 934 was used to obtain bioadhesive properties. A 22-factorial design with center point investigating the HAL content (1 and 10%, w/w) and Carbopol® 934 content (1 and 8%, w/w) was set up. The most suitable formulations were mechanically stable and showed bioadhesive properties toward vaginal tissue. The drug load was released within 20 min in phosphate buffer pH 4 and 6.8 in the in vitro dissolution test. The stability of HAL in the pellet formulations varied, but the most stable formulation showed 96–97% HAL remaining in the formulation after 6–7 weeks of storage at accelerated temperature conditions (40 °C). The investigated formulations seem promising for vaginal delivery of HAL.
Keywords: Hexylaminolevulinate (HAL); Pellets; Carbopol; Vaginal drug delivery; Photodynamic therapy (PDT); Cervical cancer;

Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate–chitosan–silica biocomposite by Paul Cǎtǎlin Balaure; Ecaterina Andronescu; Alexandru Mihai Grumezescu; Anton Ficai; Keng-Shiang Huang; Chih-Hui Yang; Carmen Mariana Chifiriuc; Yung-Sheng Lin (555-561).
This work is focused on the fabrication of a new drug delivery system based on polyanionic matrix (e.g. sodium alginate), polycationic matrix (e.g. chitosan) and silica network. The FT-IR, SEM, DTA-TG, eukaryotic cell cycle and viability, and in vitro assay of the influence of the biocomposite on the efficacy of antibiotic drugs were investigated. The obtained results demonstrated the biocompatibility and the ability of the fabricated biocomposite to maintain or improve the efficacy of the following antibiotics: piperacillin-tazobactam, cefepime, piperacillin, imipenem, gentamicin, ceftazidime against Pseudomonas aeruginosa ATCC 27853 and cefazolin, cefaclor, cefuroxime, ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole against Escherichia coli ATCC 25922 reference strains.
Keywords: Chitosan; Sodium alginate; Silica network; Eukaryotic cell; Prokaryotic cell; Biocomposite;

Chitosan microspheres as an alveolar macrophage delivery system of ofloxacin via pulmonary inhalation by Ju-Hwan Park; Hyo-Eon Jin; Dae-Duk Kim; Suk-Jae Chung; Won-Sik Shim; Chang-Koo Shim (562-569).
Because Mycobacterium tuberculosis, which causes tuberculosis, survives mainly in the alveolar macrophages, the remedial efficiency of anti-tuberculosis drugs such as ofloxacin may be improved by their direct delivery to the lungs via pulmonary inhalation. For this purpose, ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-in-oil emulsification method. The particle size of the OCMs was around 1–6 μm, and the content of ofloxacin was 27% (w/w). A twin-stage impinger (TSI) study revealed that the device-removal efficiency of the drug from the capsule and the arrival rate of the drug to stage II of the apparatus were substantially improved for OCMs compared to ofloxacin itself (i.e., 81 vs. 98% and 13 vs. 45%, respectively). Also, the in vitro uptake of ofloxacin from the OCMs to alveolar macrophages (NR8383) was substantially accelerated: the cellular ofloxacin concentrations at 4 and 24 h after the application were >3.5-fold greater than those for free ofloxacin. The above results indicate that pulmonary inhalation of OCMs might improve the delivery efficiency of ofloxacin to the alveolar macrophages, thereby shortening the length of time that is required to cure tuberculosis with the drug—usually at least 6 months when administered orally.
Keywords: Ofloxacin; Chitosan; Microspheres; Delivery; Alveolar macrophage; Inhalation;

Performance and characteristics evaluation of a sodium hyaluronate-based microneedle patch for a transcutaneous drug delivery system by Yasuhiro Hiraishi; Takeshi Nakagawa; Ying-Shu Quan; Fumio Kamiyama; Sachiko Hirobe; Naoki Okada; Shinsaku Nakagawa (570-579).
The MicroHyala® microneedle (MN) patch was developed to provide a simple, safe, and effective drug delivery system. In this study, we examined the performance and characteristics of our fabricated MN patch to identify potential quality issues with future commercial application. Mechanical failure force analysis identified that the strength of the MN patch was affected by environmental humidity, because higher moisture levels weakened the strength of the MN. Incorporation of all-trans retinoic acid (ATRA) or ovalbumin (OVA) into the MN patch decreased the mechanical failure force by almost 50% of the strength of placebo (without drug) patches. ATRA-loaded MN patches displayed good stability after storage at 4 °C, with more than 90% and 85% of the drug remaining in the patch after 8 and 24 weeks of storage, respectively. Tetanus toxoid- and diphtheria toxoid-loaded MN patches stored for 12 months induced robust antigen-specific immune responses similar to the responses by freshly prepared MN patches. Fluorescence imaging findings suggested that prolonged antigen deposition was induced by MN-mediated fluorescein isothiocyanate-labeled (FITC)–OVA vaccination. Overall, although the strength of MN requires improvement, our developed MN patch appears to be an effective pharmaceutical product providing a simple, safe, and relatively painless approach.
Keywords: MicroHyala®; Microneedle patch; Transcutaneous drug delivery; All-trans retinoic acid;

Particle size distribution of particles prepared with myristic acid and PEG of different molecular weights.The production of microparticles using a supercritical carbon dioxide based PGSS technique (CriticalMix™) has been exploited to develop blended systems targeted at pulmonary delivery. Hence, PEG based polymers of different molecular weights (1000–6000 Da) were blended in situ with fatty acids (stearic, palmitic or myristic acid) or with commercially available PEG-stearates. The effect of the different thermodynamic properties of the polymers was evaluated by characterising the microparticles produced in terms of their melting temperature by conventional DSC and in the presence of high pressure CO2 using a high pressure variable volume view cell. The microparticles produced were also assessed by SEM and particle size distribution. It is well known that as the molecular weight of the PEG chains increases, so does the viscosity of the melt and this leads to an increase in the particle size. In the paper we show that blending with myristic acid provides optimal control of particle size when the blend is sprayed from scCO2 leading to high yields in the optimal aerodynamic size range of 2–5 μm for the deep lung delivery. The highest yield and smallest particles (∼5 μm) were produced with a blend of PEG 3000 and myristic acid (1:1) whereas the batches containing palmitic acid and stearic acid showed lower yields and larger particle sizes.
Keywords: Fatty acids; PEG; PEG-stearates; Polymer blends; Supercritical fluids;

Injectable PLGA/hydrocortisone formulation produced by continuous supercritical emulsion extraction by Nunzia Falco; Ernesto Reverchon; Giovanna Della Porta (589-597).
Schematic representation of SEE-C process layout and example of HA release profile from PLGA microspheres with mean size of 2 μm and charged with 23 mg/g of HA.The objective of the present study was to develop an anti-inflammatory prolonged action formulation for local injection in prefilled syringes. Hydrocortisone acetate (HA) was selected as a model corticosteroid drug to be incorporated in poly(lactic-co-glycolic) (PLGA) microspheres. The formulation was obtained by supercritical emulsion extraction in continuous operation layout (SEE-C) to test the process robustness for a continuous industrial production. PLGA/HA microspheres with mean sizes between 1 μm (SD ± 0.20) and 5 μm (SD ± 1.45) were obtained when operating at 80 bar and 38 °C with a L/G ratio of 0.1 in the counter-current tower. The produced microdevices showed excellent encapsulation efficiencies between 75% and 80%, depending on the emulsion formulations tested, and different sustained release in the range of 6–15 days. In dependence of the different emulsion (single or double) processed by SEE-C, different products can be obtained according to the therapeutic requests. SEE-C confirms to be an innovative and flexible technology for biopolymer microdevices production, coupling the efficiency of continuous operation to the easy process scalability.
Keywords: Supercritical fluids; Microspheres; PLGA; Corticosteroids; Sustained release; Emulsion;

Detoxifying emulsion for overdosed aspirin intoxication by Wenjun Zhang; Moncef Stambouli; Dominique Pareau (598-602).
Aspirin overdose could lead to intoxication, with the clinical manifestations of vomit, pulmonary edema and severe dyspnea. Stomach washing, emetics and activated charcoal are the common treatments with a limited efficiency for the intoxication. In this study, an active emulsion for aspirin intoxication was prepared with the detoxifying efficiency of 100% in less than 15 min, with the conditions of dodecane used as the oil phase, 8% Abil EM90 as the surfactant and 0.1 mol/L sodium hydroxide as the inner aqueous phase in a volume ratio of 2 between internal aqueous phase and the oil phase.
Keywords: Aspirin; Drug overdose; Detoxifying emulsion;

The use of acoustic spectroscopy in the characterisation of ternary phase diagrams by Giulia Bonacucina; Marco Cespi; Giovanna Mencarelli; Luca Casettari; Giovanni F. Palmieri (603-610).
This study shows novel and interesting applications of acoustic spectroscopy for characterisation of ternary systems such as isopropylmiristate (IPM)/polysorbate 80 (T)/water (W). Particle size and microrheological extensional moduli (i.e. G′ and G″) of different systems were determined by means of acoustic parameters such as sound attenuation and speed. Electric conductivity was also measured using the same instrument. The ultrasonic profile in terms of attenuation and sound speed in the megahertz frequency range, allowed the characterisation of the different zones of the ternary diagram such as microemulsion, emulsion and gel zones, as well as the evaluation of water state and particle size.This last parameter is a very effective tool in quantifying the phase transitions of systems and understanding which system is formed in any phase diagram zone. In fact, it is possible to analyse samples without dilution and despite their degree of turbidity, allowing complete characterisation of both properties and structure.
Keywords: Ultrasound; Acoustic spectroscopy; Rheology; Ternary phase diagram; Particle size;

Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers by Hesham M. Tawfeek; Andrew R. Evans; Abid Iftikhar; Afzal R. Mohammed; Anjum Shabir; Satyanarayana Somavarapu; Gillian A. Hutcheon; Imran Y. Saleem (611-619).
This study investigated optimizing the formulation parameters for encapsulation of a model mucinolytic enzyme, α-chymotrypsin (α-CH), within a novel polymer; poly(ethylene glycol)-co-poly(glycerol adipate-co-ω-pentadecalactone), PEG-co-(PGA-co-PDL) which were then applied to the formulation of DNase I. α-CH or DNase I loaded microparticles were prepared via spray drying from double emulsion (w1/o/w2) utilizing chloroform (CHF) as the organic solvent, l-leucine as a dispersibility enhancer and an internal aqueous phase (w1) containing PEG4500 or Pluronic® F-68 (PLF68). α-CH released from microparticles was investigated for bioactivity using the azocasein assay and the mucinolytic activity was assessed utilizing the degradation of mucin suspension assay. The chemical structure of PEG-co-(PGA-co-PDL) was characterized by 1H NMR and FT-IR with both analyses confirming PEG incorporated into the polymer backbone, and any unreacted units removed. Optimum formulation α-CH-CHF/PLF68, 1% produced the highest bioactivity, enzyme encapsulation (20.08 ± 3.91%), loading (22.31 ± 4.34 μg/mg), FPF (fine particle fraction) (37.63 ± 0.97%); FPD (fine particle dose) (179.88 ± 9.43 μg), MMAD (mass median aerodynamic diameter) (2.95 ± 1.61 μm), and the mucinolytic activity was equal to the native non-encapsulated enzyme up to 5 h. DNase I-CHF/PLF68, 1% resulted in enzyme encapsulation (17.44 ± 3.11%), loading (19.31 ± 3.27 μg/mg) and activity (81.9 ± 2.7%). The results indicate PEG-co-(PGA-co-PDL) can be considered as a potential biodegradable polymer carrier for dry powder inhalation of macromolecules for treatment of local pulmonary diseases.
Keywords: Microparticles; Pulmonary drug delivery; Macromolecules; Polyester polymer; Dry powder inhalation;

Ionic liquids as ingredients in topical drug delivery systems by Dorota Dobler; Thomas Schmidts; Ines Klingenhöfer; Frank Runkel (620-627).
Because of their properties, ionic liquids (ILs) (Ranke et al.) offer many advantages in topical drug delivery systems. For example, ionic liquids can be used to increase the solubility of sparingly soluble drugs and to enhance their topical and transdermal delivery. Furthermore, ILs can be used either to synthesize active pharmaceutical ingredients or as antimicrobial ingredients. In the present work, the conventional oil-in-water (O/W) and water-in-oil (W/O) emulsions containing the hydrophilic IL [HMIM] [Cl] and the hydrophobic IL [BMIM] [PF6] were prepared, and the influence of the ILs on emulsion properties was evaluated. It was found that ILs could be successfully incorporated into the emulsion structure, resulting in stable formulations. The antimicrobial activity of ILs in the formulations was estimated, and their application as preservatives was confirmed by performing preservative efficacy tests. Evaluation of the in vitro cytotoxicity of the emulsions containing hydrophilic or hydrophobic ILs showed the low cytotoxicity of the carriers. Finally, penetration enhancement of a fluorescent dye as a model drug in the presence of ionic liquids was shown.
Keywords: Ionic liquids; Emulsion stability; Solubility; Antimicrobial activity; Penetration enhancement;

Development of a percutaneous penetration predictive model by SR-FTIR by E. Jungman; C. Laugel; D.N. Rutledge; P. Dumas; A. Baillet-Guffroy (628-635).
This work focused on developing a new evaluation criterion of percutaneous penetration, in complement to Log Pow and MW and based on high spatial resolution Fourier transformed infrared (FTIR) microspectroscopy with a synchrotron source (SR-FTIR). Classic Franz cell experiments were run and after 22 h molecule distribution in skin was determined either by HPLC or by SR-FTIR. HPLC data served as reference. HPLC and SR-FTIR results were compared and a new predictive criterion based from SR-FTIR results, named S index, was determined using a multi-block data analysis technique (ComDim). A predictive cartography of the distribution of molecules in the skin was built and compared to OECD predictive cartography. This new criterion S index and the cartography using SR-FTIR/HPLC results provides relevant information for risk analysis regarding prediction of percutaneous penetration and could be used to build a new mathematical model.
Keywords: FTIR microscpectroscopy; Synchrotron; Percutaneous penetration; Predictive model;

Carrier mediated delivery of vaccines along with adjuvants can possibly address the issue related to oral vaccines like inadequate immune potentiation. In this study, chitosan functionalized gold nanoparticles (CsAuNPs) were used as a carrier for the model antigen tetanus toxoid (TT) along with immunostimulant Quillaja saponaria extract (QS). Physicochemical properties (size, zeta potential, pH value) of formulation were investigated as stability indicating parameters. The synthesized CsAuNPs were spherical in shape, around 40 nm in size, positively charged (around +35 mV) and had TT and QS payload of 65% and 0.01%, respectively. Formulation parameters did not alter the secondary structure of TT, as determined by FTIR, fluorescence and CD spectroscopy. Antigen specificity, determined by an ELISA, was also not compromised. The CsAuNPs conferred protection to TT against gastric hydrolysis as studied in vitro. TT–QS–CsAuNPs induced up to 28-fold immune responses compared to control formulations (TT, TT–QS) after oral administration of formulations in BALB/c mice. The immune responses were quantified by measuring the TT-specific IgG and IgA titers using ELISA. Findings herein demonstrate that co-delivery of TT and QS with functionalized CsAuNPs promotes better systemic and local immune responses and hence can be considered as a sound approach for oral vaccine delivery.
Keywords: Gold nanoparticle; Mucosal vaccine; Adjuvant synergy; Formulation stability;

In line monitoring of the preparation of water-in-oil-in-water (W/O/W) type multiple emulsions via dielectric spectroscopy by Sebastian Beer; Dorota Dobler; Alexander Gross; Martin Ost; Christiane Elseberg; Ulf Maeder; Thomas Michael Schmidts; Michael Keusgen; Martin Fiebich; Frank Runkel (643-647).
Multiple emulsions offer various applications in a wide range of fields such as pharmaceutical, cosmetics and food technology. Two features are known to yield a great influence on multiple emulsion quality and utility as encapsulation efficiency and prolonged stability. To achieve a prolonged stability, the production of the emulsions has to be observed and controlled, preferably in line. In line measurements provide available parameters in a short time frame without the need for the sample to be removed from the process stream, thereby enabling continuous process control. In this study, information about the physical state of multiple emulsions obtained from dielectric spectroscopy (DS) is evaluated for this purpose. Results from dielectric measurements performed in line during the production cycle are compared to theoretically expected results and to well established off line measurements. Thus, a first step to include the production of multiple emulsions into the process analytical technology (PAT) guidelines of the Food and Drug Administration (FDA) is achieved. DS proved to be beneficial in determining the crucial stopping criterion, which is essential in the production of multiple emulsions. The stopping of the process at a less-than-ideal point can severely lower the encapsulation efficiency and the stability, thereby lowering the quality of the emulsion. DS is also expected to provide further information about the multiple emulsion like encapsulation efficiency.
Keywords: Dielectric spectroscopy; Multiple emulsion; In line monitoring;

The interest in the preparation and application of polymeric blends is growing since they can exhibit properties of great industrial interest. The current study focuses on the preparation of polymeric blends of varying compositions of eudragit and chitosan and their miscibility studies. The preparation was carried out by using ethanol and 1% acetic acid in water. FT-IR spectra reveal the possibilities of chemical interactions between eudragit/chitosan. The miscibility of polymeric blend at different composition has been investigated by viscosity, ultrasonic velocity, density, refractive index and adiabatic compressibility values measured at two different temperatures 30 °C and 40 °C. The interaction parameters ΔB, μ and α, were determined from viscosity data. From the values observed, it is found that the blend is miscible in all compositions at 30 °C whereas at 40 °C, it seems to be immiscible in certain compositions. It is found that the blend is miscible, when the chitosan concentration is more than 70% (v/v) at both the temperatures and also observed that variation of temperature has no effect on the miscibility of eudragit/chitosan blend.
Keywords: Eudragit; Chitosan; Blend; Miscibility; Viscosity; Ultrasonic velocity;

Glycyrrhetinic acid-graft-hyaluronic acid (HGA) conjugate was synthesized as a carrier for intravenous administration of paclitaxel (PTX), which combined hyaluronic acid (HA) and glycyrrhetinic acid (GA) as the active targeting ligands to liver tumor. In the present study, physicochemical characteristics, cellular uptake efficiency, and in vivo fates of HGA conjugates were investigated. HGA nanoparticles could readily load PTX with high efficiency up to 31.16 wt.% and entrapment efficiency to 92.02%. Moreover, PTX-loaded HGA nanoparticles exhibited more significant cytotoxicity to HepG2 cells than B16F10 cells due to simultaneously over-expressing HA and GA receptors. Meanwhile, the cellular uptake of nanoparticles was clearly enhanced in HepG2 and B16F10 cells compared to a normal fibroblast cell (HELF cells). In particular, more HGA nanoparticles were taken up by HepG2 cells than by B16F10 cells, which might be attributed to the affinity of multiple ligands of HA and GA to HepG2 cells. Furthermore, liver and tumor targeting activity of HGA nanoparticles was also confirmed by in vivo imaging analysis. The fluorescence signals of DiR-labeled HGA nanoparticles in tumor and liver were 2.88 and 1.83 folds stronger than that of the control, respectively. These results indicate HGA nanoparticles can be a potential drug carrier with “double target sites” for liver cancer therapy.
Keywords: Conjugates; Hyaluronic acid; Glycyrrhetinic acid; Synergistic targeted delivery; Liver tumor;

Recent advances in drug eluting stents by Amey S. Puranik; Eileen R. Dawson; Nicholas A. Peppas (665-679).
One of the most common medical interventions to reopen an occluded vessel is the implantation of a coronary stent. While this method of treatment is effective initially, restenosis, or the re-narrowing of the artery frequently occurs largely due to neointimal hyperplasia of smooth muscle cells. Drug eluting stents were developed in order to provide local, site-specific, controlled release of drugs that can inhibit neointima formation. By implementing a controlled release delivery system it may be possible to control the time release of the pharmacological factors and thus be able to bypass some of the critical events associated with stent hyperplasia and prevent the need for subsequent intervention. However, since the advent of first-generation drug eluting stents, long-term adverse effects have raised concerns regarding their safety. These limitations in safety and efficacy have triggered considerable research in developing biodegradable stents and more potent drug delivery systems. In this review, we shed light on the current state-of-the-art in drug eluting stents, problems related to them and highlight some of the ongoing research in this area.
Keywords: Drug eluting stent; Stent; Controlled release; Restenosis; Sirolimus;

Formulation and in vitro absorption analysis of Rhizoma paridis steroidal saponins by Zhen Liu; Jieyin Wang; Wenyuan Gao; Shuli Man; Huimin Guo; Jingze Zhang; Changxiao Liu (680-686).
Rhizoma paridis steroidal saponins (RPS) have been prepared and identified as the active compounds for antitumor activity in our previous study. However, the low oral bioavailability of the steroidal saponins restricted its using. In the present research, solid dispersion (SD) and phytosome (PHY) formulation of RPS were prepared, and the physicochemical parameters as well as the intestinal absorption in rat everted gut sac model were investigated. Seven agents were selected as the carriers of SD, and poloxamer 407 (P 407) was the most suitable one. SD reduced the particle size of saponins in the water solution, enhanced the solubility of the saponins by about 3.5 folds, and significantly improved the absorption transport of saponins from 48 to 104 μg in everted gut sac of the rat system. PHY significantly enhanced the hydrophilic of saponins but showed little effect on the absorption in small intestine. Jejunum and ileum part absorbed more absolute contents of total saponins than duodenum parts. Six saponins, the main contents of RPS, used as the index of comparing the three forms, were also further investigated in the physico-chemical properties and the absorption tests. n-Octanol/water partition coefficients of the six saponins ordered in RPS, SD and PHY were Chonglouoside H > Dioscin > Polyphyllin D > Gracillin > Paris-VII > Formosanin C. All the saponins possessed the higher absorptive characteristics in SD formulation. The absorption rate of diosgenyl saponins in intestine was more than the pennogenyl saponins.
Keywords: Steroidal saponins; Solid dispersion; Phytosome; Absorption; Everted rat gut sac;

Gap junction protein connexin43 (Cx43) specific antisense oligodeoxynucleotides (AsODN) have been shown to improve a number of inflammatory conditions and may therefore offer a novel strategy for persistent pain management. However, for such molecules to be clinically effective, delivery challenges owing to the molecules’ high molecular weight, negative charge and hydrophilicity have to be overcome. In this study, the effect of various chemical penetration enhancers and cathodal iontophoresis on transdermal delivery was evaluated. Initial skin permeation studies revealed only a slight increase in the passive flux of the model anionic drug sodium fluorescein using limonene/ethanol. Applying cathodal iontophoresis, the amount of the model drug permeated through untreated skin was tripled, while a combination of chemical and physical penetration enhancement resulted in a fourfold increase in the fluorescein amount permeated. However, even the synergistic effect of limonene/ethanol and iontophoresis was insufficient to achieve complete permeation of Cy3-labeled Cx43 AsODN across the entire skin thickness. Instead, molecules were trapped in the epidermis or permeated deeply into the hair follicles. These results suggest that the synergistic effect of chemical and physical penetration enhancement increases intradermal delivery of oligonucleotides but is insufficient to deliver such large molecules across intact skin.
Keywords: Iontophoresis; Penetration enhancers; Antisense oligodeoxynucleotides; Transdermal delivery; Transappendageal transport; Confocal laser scanning microscopy;

Integrity and stability of oral liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media by Shunwen Hu; Mengmeng Niu; Fuqiang Hu; Yi Lu; Jianping Qi; Zongning Yin; Wei Wu (693-700).
The objective of this study was to investigate the integrtity and stability of oral liposomes containing glycocholate (SGC-Lip) in simulated gastrointestinal (GI) media and ex vivo GI media from rats in comparison with conventional liposomes (CH-Lip) composed of soybean phosphatidylcholine and cholesterol. Membrane integrity of liposomes was evaluated by monitoring calcein release, particle size and distribution in different simulated GI media. The stability of liposomes encapsulating insulin was investigated in simulated GI fluids containing pepsin or pancreatin and ex vivo GI enzyme fluids. Simulated GI media with low pH or physiological bile salts resulted in significant increase in calcein release, but dynamic laser scattering data showed that the size and distribution were generally stable. SGC-Lip retained the major amount of the initially encapsulated insulin as compared with CH-Lip in simulated GI fluids (SGF, FaSSGF, SIF and FeSSIF-V2). SGC-Lip retained respectively 17.1% and 20.5% of the initially encapsulated insulin in ex vivo GI fluid, which were also significantly more than CH-Lip. These results suggested that SGC-Lip could protect insulin from degradation to some degree during their transit through the gastrointestinal tract and contributed to enhanced oral absorption.
Keywords: Liposomes; Glycocholate; Membrane integrity; Stability; Particle size; Entrapment efficiency; Oral; Insulin;

Delivery of paclitaxel across cellular barriers using a dendrimer-based nanocarrier by Huey Minn Teow; Zhengyuan Zhou; Mohammad Najlah; Siti R. Yusof; N. Joan Abbott; Antony D’Emanuele (701-711).
The aim of this study was to investigate the ability of a third-generation (G3) polyamidoamine (PAMAM) dendrimer-based carrier to enhance the permeability of paclitaxel (pac) and to overcome cellular barriers. G3 dendrimers were surface modified with lauryl chains (L) and conjugated with paclitaxel (pac) via a glutaric anhydride (glu) linker, followed by labeling with FITC. Biological evaluation of the dendrimer and conjugates was conducted using the human colon adenocarcinoma cell line (Caco-2) and primary cultured porcine brain endothelial cells (PBECs). LDH assay was used to evaluate the cytotoxicity of the dendrimer and conjugates. Cytotoxicity studies showed that the conjugation of lauryl chains and paclitaxel on G3 dendrimer significantly (p  < 0.05) increased the cytotoxicity against both cell types. Permeability studies of dendrimer–drug conjugates demonstrated an increase in the apparent permeability coefficient (P app) in both apical to basolateral A → B and basolateral to apical B → A directions across both cell monolayers compared to unmodified G3 and free drug. The B → A P app of paclitaxel was significantly (p  < 0.05) higher than the A → B P app, indicating active function of P-gp efflux transporter system in both cell models. L6-G3-glu-pac conjugate had approximately 12-fold greater permeability across both cell monolayers than that of paclitaxel alone.
Keywords: Paclitaxel, Dendrimer prodrugs; Caco-2 cells; Blood–brain barrier; Nano carrier; Drug delivery;

Large scale preparation of midkine antisense oligonucleotides nanoliposomes by a cross-flow injection technique combined with ultrafiltration and high-pressure extrusion procedures by Jing Zhong; Xing Yao; Dong Li Li; Li Qin Li; Lin Fu Zhou; Hui Lian Huang; Li Shan Min; Jing Li; Fang Fang Fu; Li Cheng Dai (712-720).
The midkine antisense oligonucleotide (MK-ASODN, 5′-CCC CGG GCC GCC CTT CTT CA-3′) nanoliposomes have been identified to suppress hepatocellular carcinoma (HCC) growth effectively, and have a great potential to be an effective target drug for HCC. In this study, a facile and reproducible method for large-scale preparation of MK-ASODN nanoliposomes followed by lyophilization has been developed successfully. Meanwhile, the MK-ASODN nanoliposomes characteristics, storage stability and their antitumor efficiency were studied. The mean particle size of MK-ASODN nanoliposomes were 229.43 ± 15.11 nm, and the zeta potential were 29.7 ± 1.1 mV. High entrapment efficiency values were achieved around 90%. Transmission electron microscopy images revealed spherical shaped nanoliposomes. Nanoliposomes allowed sustained MK-ASODN release for as long as 14 days. During 180 days of storage, freeze-dried nanoliposomes showed no significant change in the mean size, zeta potential, entrapment efficiency and drug release ratio. Regarding their antitumor efficiency, the in vitro proliferation of human liver cancer cells were significantly inhibited by the MK-ASODN nanoliposomes. Furthermore, the MK-ASOND nanoliposomes also significantly inhibited the growth of HCC in the mouse model. In summary, the results confirmed that this large-scale preparation of MK-ASOND nanoliposomes was facile and reproducible, and potentially, could speed up the application process of our MK-ASOND nanoliposomes for HCC therapy.
Keywords: Midkine; Antisense oligonucleotide; Liposome; Large-scale preparation; HCC therapy;

Nanogels fabricated by lysozyme and sodium carboxymethyl cellulose for 5-fluorouracil controlled release by Kunkun Zhu; Ting Ye; Jinjin Liu; Zheng Peng; Shasha Xu; Jieqiong Lei; Hongbing Deng; Bin Li (721-727).
Lysozyme (Ly) and sodium carboxymethyl cellulose (CMC) were used to fabricate nanogels by a convenient method without using any chemical treatment except simple heating to achieve the denaturation temperature of Ly. The prepared nanogels were characterized by dynamic laser scattering (DLS), rheological analysis, transmission electron microscopy (TEM), field emission scanning electron microscope (FE-SEM) and X-ray photoelectron spectroscopy (XPS). The nanogels are of spherical shape with average hydrodynamic diameter of 241 nm and the swelling ratio of nanogels is about 5. Then 5-fluorouracil was used as a model drug to investigate the entrapment efficiency and release ability in nanogels. It turned out to be that the release in simulated gastric fluid (SGF) was more slowly compared with that in simulated intestinal fluid (SIF), which could protect the 5-Fu in stomach and ensure it released in intestines.
Keywords: Lysozyme; CMC; Electrostatic attraction; Nanogels; Drug delivery;

In vitro and in vivo anticancer activity of a novel puerarin nanosuspension against colon cancer, with high efficacy and low toxicity by Yancai Wang; Yingying Ma; Ying Zheng; Ju Song; Xiao Yang; Chao Bi; Dianrui Zhang; Qiang Zhang (728-735).
Puerarin nanosuspensions were prepared by the high pressure homogenization (HPH) method. While improving the ratio of early apoptosis, the puerarin nanosuspensions also enhanced growth suppression, and induced apoptosis of HT-29 cells. In vivo studies puerarion nanosuspensions showed higher antitumor efficacy as measured by reduced tumor volume and tumor weight, as well as lower toxicity in HT-29 bearing mice compared to free puerarion solution.The present study aims to evaluate the anticancer activity of puerarin nanosuspensions in human colon cancer HT-29 cell line in vitro and in vivo. Puerarin nanosuspensions were prepared by the high-pressure homogenization (HPH) technique. The HT-29 cells were incubated with increasing concentrations of puerarin solution and nanosuspensions for indicated times. MTT evaluated cellular viability and investigated the effect of puerarin on cell proliferation of HT-29. Annexin V-FITC/PI staining method was conducted to determine the influences of the puerarin nanosuspensions on cell cycle and apoptosis. The in vivo anticancer activity of the puerarin nanosuspensions was observed in HT-29 cancer bearing mice. The puerarin nanosuspensions were well re-dispersed in aqueous media a mean diameter about 400–500 nm. Cytotoxicity assay, observation of morphological changes and early apoptosis revealed that the puerarin nanosuspensions could significantly enhance the in vitro anti-proliferation against HT-29 cells compared to the puerarin free solution. The prepared puerarin nanosuspensions in vivo evaluation showed higher anticancer efficacy and lower toxicity compared to the free solution, as shown by changes in tumor volumes, body weights, and survival rates. Based on these data, the potential of the puerarin nanosuspensions to serve as a cancer chemotherapeutic agent for colon cancer could be suggested.
Keywords: Puerarin; Nanosuspensions; In vivo; HT-29 cells; Apoptosis;

Characterization of reducible peptide oligomers as carriers for gene delivery by Anton Kiselev; Anna Egorova; Antti Laukkanen; Vladislav Baranov; Arto Urtti (736-747).
The stability of DNA-polyplexes and intracellular DNA release are important features of gene delivery systems. To study these features, we have evaluated reducible cysteine-flanked linear lysine and arginine-rich peptides, modified with histidine residues. The reducible disulfide bonds in cysteine flanked peptides and histidine residues should augment DNA release from the peptide–DNA complexes upon disintegration of the reducible bonds. Template polymerization and oxidative polycondensation were applied to obtain peptide oligomers used for DNA-polyplex preparation. The peptides and DNA–peptide complexes were investigated with physical, chemical and transfection measurements. Physicochemical and transfection properties of DNA-polyplexes depended on the amino acid sequence of the peptidic polymers and type of the polymerization. MALDI-TOF analysis of oxidatively polycondensed products revealed several forms of peptide oligomers corresponding to 5–8 amino acid monomers. DNA–peptide particles based on template-polymerized complexes were more resistant to relaxation by negatively charged heparan sulfate than polyplexes formed with oxidatively condensed peptides. Complexes of DNA with the polycations prepared by oxidative polycondensation exhibited a 100–1000-fold higher level of gene expression compared to DNA/template-polymerized peptide complexes. The most efficient transgene expression was shown with arginine-rich polyplexes. Transfection efficacy of the arginine-rich polyplexes was even 10-fold better than that of DNA/PEI complexes. On average, polyplexes based on cysteine-flanked peptide oligomers showed lower cytotoxicity than non-reducible high molecular weight polylysine/DNA particles. We conclude that reducible peptide oligomers provide efficient DNA transfection and have the potential as vehicles for gene delivery.
Keywords: Polyplex; Peptide; Cross-linking peptide; Nanoparticle; DNA release; Reducible bonds; Cellular delivery;

Preparation and evaluation of lidocaine hydrochloride-loaded TAT-conjugated polymeric liposomes for transdermal delivery by Yue Wang; Wenya Su; Qin Li; Changyi Li; Hanjie Wang; Yuchen Li; Yan Cao; Jin Chang; Lianyun Zhang (748-756).
Transactivation transcriptional activator (TAT) peptides were conjugated on the octadecyl-quaternized, lysine-modified chitosan to form polymeric liposomes (TAT-PLs) with cholesterol for improving transdermal delivery of local anesthetic lidocaine hydrochloride (LID). In this study, the LID loaded TAT-conjugated polymeric liposomes (LID-TAT-PLs) have been successfully prepared. LID-TAT-PLs were characterized by determination of their particle size, polydispersity, morphology, drug encapsulation efficiency, drug release behavior in vitro, and storage-stability. The skin permeation of LID-TAT-PLs was examined using a Franz diffusion cell mounted with depilated mouse skin in vitro, and penetration of TAT-PLs was visualized by confocal laser scanning microscopy (CLSM). The results showed that LID-TAT-PLs were spherical in solution, with substantially smaller mean diameter (154.7 ± 10.7 nm), higher encapsulation efficiency (80.05 ± 2.64%) and better stability in contrast to conventional liposomes (CLs). From the in vitro skin permeation results, transdermal flux of LID-TAT-PLs was approximately 4.17 and 1.75 times higher than that of LID solution and LID CLs (P  < 0.05). CLSM studies also confirmed that TAT-PLs reached viable layers of the skin. Hence, the results indicate that LID-TAT-PLs are effective and potential alternative for the LID transdermal formulation.
Keywords: Polymeric liposomes; TAT; Transdermal delivery; Lidocaine hydrochloride;

Self-microemulsifying drug delivery system (SEDDS) cored-polymeric nanocapsules (NC) were fabricated using emulsion diffusion method for the controlled oral absorption of the poorly water soluble drug, cyclosporine. Poly-dl-lactide (PDLLA) was used as the shell-forming polymer. The NCs in different polymer/oil ratios (from 25/125 to 125/125) were prepared following a solvent-diffusion method. Especially, the SEDDS oil-core compositions, which can form microemulsions on dispersion, were selected based on a pseudo-phase diagram study and further optimized based on the solubility and permeability studies. The prepared NCs were with a mean diameter of 150–220 nm and 9.4–4.5% w/w drug loading. In vivo study in rats showed that the optimized NC(50/125) and NC(100/125) released the drug in controlled way as well as enhanced the bioavailability significantly with AUC0–24 h values of 14880.3 ± 1470.6 and 12657.8 ± 754.5 ng h/ml, respectively, compared to that of SEDDS-core solution (9878.9 ± 409.6 ng h/ml). Moreover it was observed that the NCs maintained blood concentration of cyclosporine (>500 ng/ml) for 14–20 h but in the case of control formulation it was only 7.33 h. Our results suggest that the prepared NCs could be a potential carrier for the oral controlled release formulation of cyclosporine.
Keywords: Nanocapsules; SEDDS oil-core; Polymer shell; Poly-dl-lactide; Cyclosporine; Oral absorption;

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution.
Keywords: Cornea; Drug absorption; Cell culture; In vitro model; ABC transporter;

Fabrication of new drug imprinting polymer beads for selective extraction of naproxen in human urine and pharmaceutical samples by Homayon Ahmad Panahi; Alireza Feizbakhsh; Sardar Khaledi; Elham Moniri (776-780).
A drug imprinting polymer based on suspension polymerization was prepared with N,N-dimethylacrylamide and 1-(N,N-bis-carboxymethyl) amino-3-allylglycerol as functional monomers, N,N methylene diacrylamid as the cross-linker, naproxen as the template and 2,2′-azobis (2-methylbutyronitrile) as the initiator. The drug imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermogravimetric analysis and transmission electron microscopy. The imprinted polymer of agglomerated micro-particles with multi-pores was used for solid phase extraction. The drug imprinted polymer sorbent was selective for naproxen. The profile of the naproxen uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. In addition, the equilibrium adsorption data of naproxen by imprinted polymer were analyzed by Langmuir isotherm models. The developed method was utilized for determination of naproxen in pharmaceutical and human urine samples by high performance liquid chromatography with satisfactory results.
Keywords: Naproxen; Molecularly imprinted polymer; Solid phase extraction; Pharmaceutical samples; Human urine;

A novel sensing technique for measurement of magnitude and polarity of electrostatic charge distribution across individual particles by Tariq Hussain; Waseem Kaialy; Tong Deng; Mike S.A. Bradley; Ali Nokhodchi; David Armour-Chélu (781-789).
Electrostatic charge is generated during powder handling due to particle–particle and particle–wall collisions, rubbing, sliding, and rolling. In case of bipolar charge generation, the electrostatic forces may significantly change the inner forces and increase powder adhesion and cause a serious problem in material handling process. Therefore, the knowledge of distribution of charge across the individual particles is helpful to identify the role of triboelectrification and the effects of various relevant variables especially change in the contact materials, environmental conditions during processing, etc. A novel approach based on inductive sensor has been developed to detect the either polarity of charged particle and to characterise the bipolar charge distribution in the population of particulate material. To achieve this, an amplification unit configured as a pure integrator and signal processing techniques has been used to de-noise and correct the baseline of signal and MATLAB algorithm developed for peak detection. The polarity of charged particles obtained by this method is calibrated with Faraday pail method and the results are promising. Experimental study has been carried out by using two distinct populations of oppositely charged particles (glass beads-PVC, olivine sand, and silica sand). The obtained results indicate that the method is able to detect the distribution of polarities of charged particles.
Keywords: Electrostatic charge; Powder; Blending; Inductive sensor; Bipolar charge; Particulate material;

A new methodology for high drug loading wet granulation formulation development by Lixia Cai; Leon Farber; Dina Zhang; Feng Li; Julianne Farabaugh (790-800).
A new methodology that enables efficient and rapid development of high drug load (>85%) high shear wet granulation formulations is proposed and tested in this work. Correlations between the API properties, the binder types, the granulation fluid levels, and the product attributes revealed in course of the study are discussed. The key feature of the methodology is that an excipient is added to the formulation only when it is demonstrated that it is required to improve processability or performance of the formulation. To evaluate this approach, three compounds: simvastatin, etoricoxib, and metformin hydrochloride were selected as model drug substances. These compounds differ significantly with respect to their particle size distributions, wettability, and solubility. The compounds were granulated using a range of granulation fluid levels, with or without a polymeric binder. Granule size distribution, strength, flowability, dissolution, and compactibility were characterized. Select granulations were further compressed into tablets, both “as-is” and with addition of extragranular excipients. One formulation was also investigated in an in vivo dog PK study. The study demonstrated that using the methodology, high drug load formulations with satisfactory attributes could be successfully developed for all three model compounds. Applicability and benefits of the proposed methodology are discussed.
Keywords: High shear wet granulation; High drug loading; Polymeric binders;

Mucosa-plate for direct evaluation of mucoadhesion of drug carriers by Amornset Tachaprutinun; Porntip Pan-In; Supason Wanichwecharungruang (801-808).
The method to prepare mucosa-plates, glass slides covalently coated with mucin, is demonstrated. The use of the plate to evaluate mucoadhesion of nanocarriers made from different four polymeric materials, N-succinylchitosan (NS-chitosan), alginate (ALG), ethylcellulose (EC), and a blend of EC and methylcellulose (EC/MC), was demonstrated. While different mucoadhesion of the four carriers could be detected using mucosa-plate, the conventional viscosity measurement could not differentiate their mucin-binding ability. ALG and NS-chitosan nanospheres showed the best attachment to the mucosa-plate compared to the EC/MC and EC spheres. Capsaicin, a model hydrophobic drug, was loaded into the carriers and the ability of the different polymeric carriers to retain capsaicin at the stomach tissue was compared using an ex vivo fresh porcine stomach assay. Ability to retain capsaicin at the stomach tissue correlated well with binding affinity toward the mucosa-plate and the loading capacity of the carriers.
Keywords: Mucoadhesion; Mucin; Nanocarrier; Stomach;

Microchannels based microfluidic systems are able to obtain monodispersed microparticles but are limited by cost, time and channel clogging. We succeeded in on the fly encapsulation of high ketoprofen contents in acrylate-based copolymer microbeads by environment friendly UV induced free radical polymerization in off-the-shelf co-axial microfluidic device. FTIR shows complete polymerization of acrylate monomers and interaction between carboxylic group of ketoprofen and ester group of monomers. DSC and XRD confirm amorphous nature of drug in microbeads. Different comonomer content formulations show limited drug release at low pH, a helpful properties to avoid gastric irritating effect of ketoprofen associated with conventional dosage forms. At pH 6.8 microbeads release higher content of drug by a non-Fickian diffusion mechanism. Their drug release rate depends upon the weight content of ethyl acrylate in the formulation as well as their size, increasing by increasing the former and decreasing the later.
Keywords: Microfluidics; Ketoprofen; Microbeads; Ethyl acrylate; Drug delivery;

Drug-printing by flexographic printing technology—A new manufacturing process for orodispersible films by Eva Maria Janßen; Ralf Schliephacke; Armin Breitenbach; Jörg Breitkreutz (818-825).
Orodispersible films (ODFs) are intended to disintegrate within seconds when placed onto the tongue. The common way of manufacturing is the solvent casting method. Flexographic printing on drug-free ODFs is introduced as a highly flexible and cost-effective alternative manufacturing method in this study. Rasagiline mesylate and tadalafil were used as model drugs. Printing of rasagiline solutions and tadalafil suspensions was feasible. Up to four printing cycles were performed. The possibility to employ several printing cycles enables a continuous, highly flexible manufacturing process, for example for individualised medicine. The obtained ODFs were characterised regarding their mechanical properties, their disintegration time, API crystallinity and homogeneity. Rasagiline mesylate did not recrystallise after the printing process. Relevant film properties were not affected by printing. Results were comparable to the results of ODFs manufactured with the common solvent casting technique, but the APIs are less stressed through mixing, solvent evaporation and heat. Further, loss of material due to cutting jumbo and daughter rolls can be reduced. Therefore, a versatile new manufacturing technology particularly for processing high-potent low-dose or heat sensitive drugs is introduced in this study.
Keywords: Orodispersible films; Drug printing; Individualised medicine; Rasagiline; Tadalafil; Flexographic printing;

Predicting drug release from HPMC/lactose tablets by J. Siepmann; Y. Karrout; M. Gehrke; F.K. Penz; F. Siepmann (826-834).
Three mathematical models were applied to quantify drug release from HPMC/lactose-based matrix tablets loaded with varying amounts of theophylline: (i) a numerical model considering drug diffusion in axial and radial direction in cylinders as well as limited drug solubility effects, (ii) an analytical solution of Fick's second law of diffusion considering axial and radial mass transport in a cylinder, but neglecting limited drug solubility effects, and (iii) a simple early time approximation of the analytical solution, considering only radial mass transport and neglecting axial diffusion as well as limited drug solubility effects. The three models were fitted to experimentally determined drug release kinetics from various types of tablets in 0.1 M HCl and phosphate buffer pH 7.4. Interestingly, the agreement between fitted theories and experimental data was similar in all cases. However, the determined system specific model parameters (apparent diffusion coefficients of theophylline in the polymeric matrices) were significantly biased when using simplified theories. Nevertheless, the reliability of theoretical predictions was similar for all three models, since the determined apparent diffusivities are partially “lumped” parameters. Thus, from a practical point of view, very simple equations can be used during product optimization, allowing estimating the effects of formulation parameters on drug release.
Keywords: Mathematical modeling; Drug release mechanism; HPMC; Lactose; Theophylline; Diffusion;