International Journal of Pharmaceutics (v.352, #1-2)

Tretinoin-loaded nanocapsules: Preparation, physicochemical characterization, and photostability study by A.F. Ourique; A.R. Pohlmann; S.S. Guterres; R.C.R. Beck (1-4).
The aim of this study was to prepare and characterize tretinoin-loaded nanocapsules as well as to evaluate the influence of this nanoencapsulation on tretinoin photostability. Tretinoin-loaded nanocapsules (0.5 mg ml−1) were prepared by interfacial deposition of preformed polymer (poly-ɛ-caprolactone) using two different oily phases: capric/caprylic triglycerides and sunflower seed oil. Tretinoin-loaded nanocapsules presented drug content close to the theoretical value, encapsulation efficiencies higher than 99.9%, nanometric mean size with a polydispersity index below 0.25, and pH values between 5.0 and 7.0. Regarding photodegradation studies, tretinoin methanolic solution showed a half-life time around 40 min according to a first order equation, whereas tretinoin nanocapsule suspensions showed a half-life between 85 and 100 min (twofold higher than in methanolic solution) according to a zero order equation. Tretinoin-loaded nanocapsules improved tretinoin photostability, independently on the type of oily phase used in this study, and represent a potential system to be incorporated in topical or systemic dosage forms containing tretinoin.
Keywords: Nanocapsules; Nanoparticles; Photostability; Retinoic acid; Tretinoin;

Inclusion complexation of danazol with β-cyclodextrin (BCD) in aqueous solution, in solid state and in silico state was investigated to examine the interactions of danazol with BCD. The study also explored the potential application of danazol–β-cyclodextrin complex as an oral antiovulatory agent. Phase solubility analysis suggested formation of first-order soluble complex with stability constant 972.03 M−1 while Job's plot affirmed 1:1 stoichiometry. Solution state complexation in water was studied by ultra violet absorption, circular dichroism and nuclear magnetic resonance (1H NMR) spectroscopy. The solid state complexes were evaluated by differential scanning calorimetry, powder X-ray diffractometry, fourier transform infrared spectroscopy and scanning electron microscopy. Thermodynamic studies in water indicated exothermic nature of inclusion complexation. Molecular modeling was used to help establish the mode of interaction of BCD with danazol. 1H NMR analysis suggested that the protons of steroidal skeleton of danazol are preferably involved in the complexation with BCD, which was confirmed by molecular dynamic simulations. An inclusion complex model has been established for explaining the observed enhancement of solubility of danazol in water by BCD. Moreover, in mouse model, danazol–β-cyclodextrin complex at 51.2 mg/kg (equivalent to 400 mg human dose) showed 100% anovulation when given orally.
Keywords: Danazol; β-Cyclodextrin; Solubility; Molecular modeling; Antiovulatory activity;

The purpose of this work was to study the effect of pH on the liposomal encapsulation of a model camptothecin anti-tumor agent, DB-67, by considering the state of ionization and bilayer membrane/water partitioning of the drug as a function of pH. A novel fluorescence method was developed to monitor intravesicular pH in liposomal formulations containing entrapped DB-67 by using the drug itself as a pH indicator. Fluorescence spectra were recorded in aqueous buffers and liposomes and used to estimate the ionization constant of the A-ring phenol of DB-67 ( p K a 2 ) and shifts in ionization constants ( p K a 1 and p K a 2 ) due to membrane binding. Bilayer/water partitioning studies by equilibrium dialysis were employed to show that DB-67 is highly membrane bound over the entire pH range examined though binding decreases with an increase in pH. The observed ionization constants of membrane-bound DB-67 obtained from the equilibrium dialysis experiments were consistent with observations from fluorescence measurements and previous permeability results. The pH dependence of DB-67 loading using a passive loading technique was found to reflect the pH dependence of membrane binding of the drug. This results in poor encapsulation efficiency of DB-67 at high pH, necessitating further development of formulation strategies to improve loading efficiency.
Keywords: Liposomes; Nanoparticles; Intraliposomal pH; Membrane binding; Lipid bilayers; Camptothecins; Tumor drug delivery; Fluorescence; Drug encapsulation;

Voriconazole into PLGA nanoparticles: Improving agglomeration and antifungal efficacy by Hai-sheng Peng; Xi-jun Liu; Gui-xiang Lv; Bo Sun; Qing-fei Kong; Dong-xu Zhai; Qi Wang; Wei Zhao; Guang-you Wang; Dan-dan Wang; Hu-lun Li; Lian-hong Jin; Nikolaos Kostulas (29-35).
This study is concerned with preparing PLGA nanoparticles loaded with voriconazole (PNLV), investigating the burst release and agglomeration of PNLV, and also evaluating antifungal efficacy of PNLV compared with voriconazole (VRC). The emulsion–solvent evaporation technique for nanoparticles and tests against fungi were completed. The amount of VRC in PNLV with sodium hexametaphosphate was 2.01 ± 0.27%, and burst release of PNLV was reduced by about 33% using 20% ethanol solution (n  = 3). The mean D 50 of PNLV with or without this salt was 132.8 nm and 6.3 μm, respectively (n  = 5). In vitro; the fungal numbers treated with PNLV (3.5 mg/ml, equal amount calculated by VRC) and VRC (70 μg/ml) in tubes at the day 7 were 5.74 log10 and 6.72 log10, respectively (P  < 0.05). In vivo; the fungal burden treated with PNLV and VRC in tissue from mice kidneys at day 7 after administration was 0.64 log10 and 2.61 log10, respectively (5 mg/kg, P  < 0.001). The hematoxylin–eosin stain in mice kidney showed that the pathological lesions treated with PNLV were relieved in contrast with those with VRC. These results suggest that the emulsion–solvent evaporation process is feasible in preparing PNLV. Moreover, ethanol solution decreased burst release and Na-HMP inhibited agglomeration. PNLV could improve the VRC antifungal efficacy.
Keywords: Voriconazole; Agglomeration; PNLV; Burst release; Candida albicans;

Evaluation of in vivo dissolution behavior and GI transit of griseofulvin, a BCS class II drug by Yoshitsugu Fujioka; Yukiko Metsugi; Ken-ichi Ogawara; Kazutaka Higaki; Toshikiro Kimura (36-43).
Mean plasma concentration–time profile of griseofulvin, a BCS class II drug, orally administered as powders into rats, was predicted based on GITA model. However, it was very difficult to predict the individual plasma profile because of large inter-individual difference. As the absorption of griseofulvin would be rate-limited by the dissolution process, we tried to analyze the in vivo dissolution kinetics of griseofulvin by focusing on gastric emptying and intestinal transit as physiological factors influencing the in vivo dissolution kinetics. After oral administration of griseofulvin, theophylline and sulfasalazine into rats, gastric emptying and intestinal transit were simultaneously estimated by analyzing the absorption kinetics of theophylline and observing the appearance of sulfapyridine in plasma, respectively. Gastric emptying kinetics was not significantly correlated with absorption or dissolution behavior of griseofulvin. On the other hand, the cecum-arriving time reflecting the intestinal transit was significantly correlated with both AUC and total dissolved amount of griseofulvin. T max of griseofulvin also increased with the increase of cecum-arriving time. These results clearly indicate that the longer residence time could lead to the higher dissolution and absorption of griseofulvin and that the variance of intestinal transit could be responsible for the inter-individual difference of the in vivo absorption behavior.
Keywords: Gastric emptying; Cecum-arriving time; Intestinal transit; In vivo dissolution; Griseofulvin; Biopharmaceutics classification system class II;

Fluoropolymers as low-surface-energy tooth coatings for oral care by David Churchley; Gareth D. Rees; Eugen Barbu; Thomas G. Nevell; John Tsibouklis (44-49).
A range of low-surface-energy fluoropolymers has been synthesised and their effectiveness as dental-care coatings for plaque, stain and erosion prevention has been evaluated using a series of oral care models employing pressed discs of calcium hydroxyapatite or sections of human teeth. Since the blocking of dentinal tubules is a key mechanistic strategy in the treatment of dentine hypersensitivity, the capability of these non-permanent fluoropolymer coatings to occlude the pore structure of human dentine and to reduce the outward flow of simulated dentinal fluid has also been investigated. Several of the fluoropolymer coatings have been found to inhibit bacterial adhesion but no correlation has been established between anti-adhesion efficacy and fluorine content or surface energy. All the fluoropolymers have been seen to reduce stain uptake by pellicle-coated HA discs, with homopolymers being considerably more effective than copolymers. Some fluoropolymer coatings have also been shown to inhibit the acid demineralisation of hydroxyapatite discs and to reduce dentine permeability. Coatings of the 2:1 copolymer of 1H,1H,2H,2H-perfluorodecyl acrylate and 2-hydroxyethyl acrylate are most promising, exhibiting significant anti-adhesion and anti-erosion efficacy and reducing dentine permeability to a level that is comparable with that achieved with the standard treatment employed in commercial anti-sensitivity formulations.
Keywords: Fluoropolymer; Hydroxyapatite; Demineralisation; Bacterial adhesion; Dentine hypersensitivity;

PLGA/PEG-derivative polymeric matrix for drug delivery system applications: Characterization and cell viability studies by A. Fernández-Carballido; P. Pastoriza; E. Barcia; C. Montejo; S. Negro (50-57).
The incorporation of additives such as polyoxyethylated oleic acid glycerides (PEG-derivative) can modify the release of drugs from microparticles. PEG-derivative decreases the release rate of drugs that are dissolved in PLGA matrices but if un-dissolved the initial release rate slightly increases. To clarify this behaviour the influence of adding PEG-derivative in the preparation of microspheres was investigated by scanning electron microscopy, differential scanning calorimetry, gel permeation chromatography, nuclear magnetic resonance and infrared spectroscopy. Cytotoxicity of this resulting PLGA/PEG-derivative matrix was evaluated in cell lines (fibroblasts) which are more reproducible but less specific and in primary cell cultures (splenocytes and human leucocytes) which have the advantage of their specificity.Scanning electron microscopy revealed that PLGA/PEG-derivative microspheres exhibited small surface concavities with a highly porous polymeric matrix. The incorporation of PEG-derivative caused a slight reduction in the T g values of PLGA. In vitro degradation studies showed that PEG-derivative remains within the microspheres as long as the matrix does. This PLGA/PEG-derivative matrix was well tolerated exhibiting cell viabilities similar to PLGA microspheres and can be used to modulate the release of drugs from microparticulate systems destined for parenteral administration.
Keywords: PLGA/PEG-derivative; Microspheres; Cell viability;

The purpose of this study was to design injectable controlled release polymer formulations for growth hormone using triblock copolymer PLGA–PEG–PLGA (MW 1400–1000–1400). Porcine growth hormone (pGH) formulations were prepared by adding pGH into 30% (w/v) aqueous solution of triblock copolymer. pGH concentrations in the released samples were determined using a standard MicroBCA method. In vitro release studies demonstrated that there were no initial burst of pGH from both formulations containing a low dose (0.12%, w/v) and a high dose (0.42%, w/v) of pGH. In vivo absorption study of pGH in rabbits showed that constant serum levels of exogenous pGH (3–7 ng/mL from high dose and 2–4 ng/mL from low dose) were detected for nearly 4 weeks from delivery systems upon single subcutaneous injection. The absolute bioavailability of pGH enhanced from the thermosensitive polymer-based systems, which was ∼5–15-fold those of subcutaneous aqueous solution. MTT assay and light microscopy were used to investigate the in vitro and in vivo biocompatibility of thermosensitive polymer delivery systems, respectively. Both in vitro and in vivo results support the biocompatible nature of these polymer delivery systems. Thus, the triblock copolymer used in this study was able to control the release of incorporated pGH in vitro and in vivo for longer duration and the delivery system was biocompatible.
Keywords: Controlled release; Protein; Thermosensitive polymer; Biocompatible; Growth hormone; Subcutaneous;

Evaluation of poly(styrene-alt-maleic anhydride)–ethanol as enteric coating material by Xiaolin Lai; Chengdong Sun; Hua Tian; Wenjun Zhao; Lin Gao (66-73).
This study aims at evaluating the potential of SMA–ethanol as enteric coating polymer for erythromycin tablets. SMA–ethanol was synthesized and characterized for physicochemical properties, molecular weight and thermal analysis. Free films were prepared by adding different kinds and amounts of plasticizers, the film surface topography was determined by a SEM, the tensile strength, water vapor transmission rate and moisture absorption were also tested to choose the most promising film. DBP was proved to be the most suitable plasticizer with a best using amount of 20%, such polymer films had low vapor transmission rate and low moisture absorption which were very important to an enteric coating material. The polymer was further characterized for film coating by evaluating the release of erythromycin tablets in vitro, tablets coated with SMA–ethanol can satisfy the drug release requests of USP when the film weight gains were between 4 and 6%; tablets coated with both a subcoat and the polymer showed excellent gastro-resistance, less than 0.2% drug release occurred even with weight gains as less as 2% after 2 h exposure to acid (pH 1), while over 90% drug release occurred in pH 6.8 sodium phosphate buffer within 45 min, regardless of weight gains of coating material, moreover, we confirmed that the application of a subcoat could decrease the amount of required coating polymer. In conclusion, the potential use of SMA–ethanol as enteric coating material was demonstrated.
Keywords: SMA–ethanol; Enteric coating; Erythromycin tablets;

In order to utilize the psyllium husk, a medicinally important natural polysaccharide, to develop the hydrogels meant for the drug delivery, we have prepared psyllium 2-hydroxylethylmethacrylate (HEMA) and acrylamide (AAm)-based polymeric networks by using N,N′-methylenebisacrylamide (N,N′-MBAAm) as crosslinker and ammonium persulfate (APS) as initiator. The polymeric networks thus formed [psy-cl-poly(HEMA-co-AAm)] were characterized with FTIR and swelling studies which were carried out as a function of crosslinker concentration, time, pH and [NaCl] of the swelling medium. The swelling kinetics of the hydrogels and in vitro release dynamics of model drug (tetracycline hydrochloride) from these hydrogels has been studied for the evaluation of swelling mechanism and drug release mechanism from the hydrogels. The values of the diffusion exponent ‘n’ have been obtained 0.5 for both swelling kinetics and drug release dynamics. This value shows that the Fickian type diffusion mechanism has occurred for the swelling of the polymers and for the release of drug from the polymers in different release mediums. The values of the initial diffusion coefficients (10.6 × 10−4, 13.1 × 10−4, 14.0 × 10−4) cm2/min, average diffusion coefficients (22.2 × 10−4, 25.7 × 10−4, 27.0 × 10−4) cm2/min and late diffusion coefficients (1.68 × 10−4, 2.15 × 10−4, 2.28 × 10−4) cm2/min for the release of tetracycline HCl respectively in distilled water, pH 2.2 buffer and pH 7.4 buffer from the drug loaded samples shows that in the initial stages, the rate of release of drug from the hydrogels is slow and rate of diffusion of drug increases with time.
Keywords: Drug delivery devices; Hydrogels; Psyllium; Release dynamics; Swelling kinetics;

Release profile comparison and stability of diltiazem–resin microcapsules in sustained release suspensions by Varaporn Buraphacheep Junyaprasert; Greepol Manwiwattanakul (81-91).
A sustained release suspension of diltiazem, a short half-life calcium channel blocker, was developed to reduce frequency of drug administration, ease of dose adjustment and improve patient compliance. In this study, the sustained release of diltiazem was obtained by complexing the drug with Dowex® 50W×4 and Dowex® 50W×8, strong cationic exchange resins with 4% and 8% degree of cross-linking, respectively. The diltiazem–Dowex® 50W×4 complexes provided the highest drug release and were subsequently used to prepare the microcapsules by emulsion–solvent evaporation method, using 0.75–5.00% cellulose acetate butyrate (CAB) in methylene chloride as a coating solution. As the concentration of CAB increased, the size of microcapsule increased and the drug release from the microcapsule was retarded. From release profile comparison using f 1 and f 2 factors, it was found that the microcapsules coated with 1.75% CAB provided a release profile equivalent to the commercial product of diltiazem sustained release capsule, Herbesser® 90SR. Furthermore, sustained release suspensions of the diltiazem microcapsules were formulated with the use of 0.8% sodium carboxymethylcellulose or 0.4% xanthan gum as a suspending agent. The suspension of 0.4% xanthan gum showed superior in physical appearance after 120-day storage at 30 and 45 °C. In addition, all sustained release suspensions possessed good stability with low drug leaching and their release profiles were unchanged when compared with the dried microcapsules for 120 days at 30 and 45 °C.
Keywords: Diltiazem; Ion-exchange resin; Emulsion–solvent evaporation; Sustained release suspension;

Effect of O-acylmenthol on transdermal delivery of drugs with different lipophilicity by Ligang Zhao; Liang Fang; Yongnan Xu; Yanyan Zhao; Zhonggui He (92-103).
To develop more effective compounds as enhancers, O-acylmenthol derivatives which were expected to be enzymatically hydrolyzed into nontoxic metabolites by esterases in the living epidermis were synthesized from l-menthol and pharmaceutical excipient acids (lactic acid, cinnamic acid, salicylic acid and oleic acid) in this study. Their promoting activity on the percutaneous absorption of five model drugs, 5-fluorouracil (5-FU), isosorbide dinitrate (ISDN), lidocaine (LD), ketoprofen (KP), and indomethacin (IM), which were selected based on their lipophilicity represented by log  K O/W, were tested in vitro across full thickness rat skin with each of the evaluated drugs in saturated donor solution. 2-Isopropyl-5-methylcyclohexyl 2-hydroxypanoate (M-LA) provided the highest increase of accumulation of 5-FU (3.74-fold) and LD (4.19-fold) in the receptor phase while 2-isopropyl-5-methylcyclohexyl cinnamate (M-CA) was ineffective for most of the drugs; Both 2-isopropyl-5-methylcyclohexyl 2-hydroxybenzoate (M-SA) and (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate (M-OA) had better promoting effects on the drugs with low water-solubility. The four O-acylmenthol enhancers produced parabolic relationship between the lipophilicity (log  K O/W) of the model drugs (5-FU, ISDN, KP, IM) and their enhancement ratio of the permeation coefficient (ERP), indicating that the lipophilicity of the penetrants has significant effect on the permeation results, r  = 0.989 (P  = 0.144) for M-LA, r  = 0.965 (P  = 0.216) for M-CA, r  = 0.786 (P  = 0.630) for M-SA, and r  = 0.996 (P  = 0.088) for M-OA.
Keywords: O-Acylmenthol derivatives; Pharmaceutical excipient acids; Percutaneous absorption; Drug lipophilicity;

In vitro characterization of α-tocopherol SEDDS formulations was performed by (1) lipolysis in bio-relevant media, and (2) physical assessment by dissolution, particle size, and turbidity analyses. Both methods were statistically correlated using a 25-run, five-factor multiple-level d-optimal mixture design. Independent variables were SEDDS composition [vitamin E (12.5–25%), Tween® 80 (10–40%), labrasol (0–10%), alcohol (0–10%), and captex 355 (20–50%)]. Measured responses were percent lipolysis, percent vitamin E retained in the aqueous layer of the digestion medium, and percent vitamin E dissolved in the dissolution medium. Percent lipolysis ranged from 0% to 66.3%. Percent vitamin E retrieved in the aqueous layer of the digestion and dissolution media ranged from 3% to 29.3% and from 25.9% to 101.7%, respectively. Turbidity ranged from 28 to 403 JTU and the average droplet size was >1.0 μm. All formulation ingredients had significant (p  < 0.05) effect on percent lipolysis. Only two factors, Tween® and vitamin E had significant effect on vitamin retention in the aqueous layer post-lipolysis. Tween®, labrasol, and captex 355 had significant effect on vitamin E dissolution. Poor correlation was observed between the responses. Formulation ingredients influenced each response differently; and therefore, each method can only reveal distinctive characteristics of the SEDDS formulation and may not be used interchangeably.
Keywords: Lipolysis; SEDDS; Griseofulvin; Dissolution; Optimization;

Cryo-scanning electron microscopy (cryo-SEM) as a tool for studying the ultrastructure during bead formation by ionotropic gelation of calcium pectinate by Pornsak Sriamornsak; Nartaya Thirawong; Kamonrak Cheewatanakornkool; Kanokporn Burapapadh; Witoon Sae-Ngow (115-122).
Understanding the gel ultrastructure is of great importance for process and product development having great effects on the product characteristics. The samples containing high amount of water could not be directly observed using scanning electron microscope (SEM) without removing of water. However, cryo-SEM can be used to study the ultrastructure of hydrated samples. In this study, ultrastructural information of internal structure was obtained by imaging the cryo-fractured beads in a cryo-SEM. This technique was found to be excellent for studying the detailed morphology of structural development and showed better images than normal SEM procedures using freeze-drying for sample preparation. Also, the studies illustrated a morphological change, e.g. from net-like structure to membranous structure caused by syneresis, accompanied by a significant increase in mechanical properties, when the beads are formed by ionotropic gelation. The gelation time of 20 min was found to be the minimum for a complete bead formation, based on the mechanical and SEM data. The results demonstrate the advantageous of cryo-SEM for examining the ultrastructure during bead formation of calcium pectinate.
Keywords: Scanning electron microscopy (SEM); Cryo-scanning electron microscopy (cryo-SEM); Morphology; Bead formation; Gelation kinetics; Calcium pectinate; Pectin;

The chemical stability of tretinoin (RA) and isotretinoin (13RA) in ethanol and dermatological cream preparations exposed to solar simulated light (SSL), UVA, and visible light has been studied. Photostability was monitored by an HPLC method that allowed simultaneous analysis of RA and 13RA, thus allowing photodegradation due to isomerization to other retinoids and photolysis to non-retinoid products to be monitored. Both retinoids undergo both isomerization and photolysis following SSL, UVA and visible light exposure but RA is more sensitive to photodegradation than 13RA. Degradation of both retinoids by photolysis is considerably greater in cream formulations than in ethanol and the photodegradation follows second order kinetics. Rate constants and half-lives for degradation of RA and 13RA in ethanol solution and cream preparations subjected to different light sources are reported. The UVA component of SSL is the major contributor to photodegradation. Since UVA penetrates deeply into skin, our results suggest that photodegradation of RA may contribute to the photosensitivity associated with RA therapy. Our studies suggest that development of improved formulations and the use of effective UVA sunscreens may reduce the side effects of RA therapy.
Keywords: Retinoic acid; Photostability; UVA; Photoisomerization; Photolysis;

Synergistic enhancement of skin permeability by N-lauroylsarcosine and ethanol by Yeu-Chun Kim; Jung-Hwan Park; Peter J. Ludovice; Mark R. Prausnitz (129-138).
To develop formulations for transdermal drug delivery, this study tested the hypothesis that the anionic surfactant, N-lauroylsarcosine (NLS), and ethanol synergistically increase skin permeability by increasing the fluidity of stratum corneum lipid structure. Skin permeability experiments showed that transdermal delivery of fluorescein across human cadaver epidermis was increased by up to 47-fold using formulations containing NLS in aqueous ethanol solutions. Skin permeability was increased by increasing NLS concentration in combination with 25–50% ethanol solutions. Skin permeability was shown to correlate with skin electrical conductivity measurements, changes in differential scanning calorimetry lipid transition peak temperature, and Fourier transform infrared spectroscopy C―H stretching peak shifts indicative of stratum corneum lipid fluidization and changes in protein conformation. Evidence for lipid extraction was also evident, but did not appear to be responsible for the observed increases in skin permeability. We conclude that NLS in aqueous ethanol formulations can dramatically increase skin permeability by a mechanism involving synergistic lipid-fluidization activity in the stratum corneum.
Keywords: Differential scanning calorimetry; Fourier transform infrared spectroscopy; N-Lauroylsarcosine; Ethanol; Skin penetration enhancer; Skin resistance;

Simulated neonatal exposure to DEHP and MEHP from PVC enteral nutrition products by Satoshi Takatori; You Okamoto; Yoko Kitagawa; Shinjiro Hori; Shun-Ichiro Izumi; Tsunehisa Makino; Hiroyuki Nakazawa (139-145).
The leaching of di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) from medical products made of polyvinyl-chloride (PVC) to enteral nutrition (EN) for neonatal patients was determined in a simulated study. The study simulated a typical case of EN administration to a neonatal patient (body weight, 3 kg) in a neonatal care unit (temperature, 25 °C); the medical products used were an irrigator and catheter containing DEHP (9.1–31.8%, w/w) as a plasticizer. The worst-case daily exposures of the neonatal patient to DEHP and MEHP by the administration of EN were estimated to be 148 and 3.72 μg/(kg day), respectively, as assessed from the levels of these compounds leaching from the medical products to the EN. The use of DEHP-free medical products reduced the exposure of DEHP and MEHP to the minimum levels contained in the EN at preparation. A transition to DEHP-free medical products for neonatal patients would be effective in reducing the exposure of neonatal patients to DEHP via EN administration.
Keywords: Di(2-ethylhexyl)phthalate; Mono(2-ethylhexyl)phthalate; Polyvinyl-chloride; Enteral nutrition; Medical product; Neonate;

Physicochemical properties of tamoxifen hemicitrate sesquihydrate by Takashi Kojima; Fumie Katoh; Yoshihisa Matsuda; Reiko Teraoka; Shuji Kitagawa (146-151).
A novel modification of tamoxifen [(Z)-2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethylamine] citrate, tamoxifen hemicitrate hydrate was prepared. The crystalline form was identified and characterized by powder and single crystal X-ray diffractometries, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and hot-stage microscopy, and its physicochemical stability was also evaluated. The results of an elemental analysis, a single crystal X-ray analysis, and the TGA suggested that the molar ratio of tamoxifen:citric acid:water was 2:1:3 indicating it to be tamoxifen hemicitrate sesquihydrate. Simultaneous XRD–DSC measurements also indicated that two hydrates, sesquihydrate and hemihydrate, and an anhydrous form would exist during heating. The physicochemical stability of tamoxifen citrate forms A and B suspended in water and of form A during kneading and drying suggested that tamoxifen citrate was transformed into tamoxifen hemicitrate hydrate in water within 24 h, whereas tamoxifen citrate in a mixture with microcrystalline cellulose was quite stable during kneading. These results suggested that water and a mixture of water and organic solvent should be used for the manufacturing process with special attention paid to the transformation to tamoxifen hemicitrate sesquihydrate, because it showed a different stoichiometry from the active ingredient, tamoxifen citrate.
Keywords: Tamoxifen; Hydrate; Physicochemical characterization; Physicochemical stability;

In a polymer–water matrix, freezable water is depressed due to either porosity confinement or interaction. The aim of the study was to examine water crystallization/melting depression by sub-ambient differential scanning calorimetry. The selected starch- and cellulose-based polymers including pre-gelatinized starch (PS), sodium alginate, sodium starch glycolate, hydroxypropylmethyl cellulose (HPMC), sodium carboxymethyl cellulose, and croscarmellose sodium were employed. The pre-treated with ambient humidity (85–100% relative humidity, at 30.0 ± 0.2 °C for 10 days) and with excess water (hydrogels) samples were subjected to between 25 and −150 °C cooling–heating cycle at 5.00 °C/min rate. The volume fractions of hydrogels were measured by light scattering technique. It was observed that all polymers but PS and HPMC with ambient humidity presented freezable water in two distinct fractions namely bound water where crystallizing/melting temperature was depressed and bulk water. The water transition in samples with various contents exhibited the pattern as a polymer solution, thus rather than confinement, the depression was due to interaction. The volume fraction-melting temperature data derived from endotherms of hydrogels were successfully fitted to Flory's model (r 2: 0.934–0.999). The Flory's interaction parameters (χ 1) were found to vary between 0.520 and 0.847. In addition, the smaller the value of χ 1, the larger melting was depressed, i.e., stronger affinity for water.
Keywords: Freezable water; Water–polymer interaction; Flory's interaction parameter (χ 1); Melting point depression; Hydrogel;

A novel system for three-pulse drug release based on “tablets in capsule” device by Bin Li; JiaBi Zhu; ChunLi Zheng; Wen Gong (159-164).
The objective of the present study was to obtain programmed drug delivery from a novel system, which contains a water-soluble cap, impermeable capsule body, and two multi-layered tablets. Types of materials for the modulating barrier and its weight can significantly affect the lag time (defined as the time when drug released 8% of the single pulse dosage). We chose sodium alginate and hydroxy-propyl methyl cellulose (HPMC E5) as the candidate modulating barrier material. Through adjusting ratio of sodium alginate and lactose, lag time was controllable between the first two pulsatile release. Linear relationship was observed between the ratio and the lag time. Through adjusting the ratio of HPMC E5/lactose, lag time between the second and the third pulse can be successfully modulated. In further studies, drug release rate of the second pulsatile dose can be improved by adding a separating layer between the third and the modulating barrier layer in the three-layered tablet. To evaluate contribution of bulking agent to drug release rate, lactose, sodium chloride, and effervescent blend were investigated. No superiority was found using sodium chloride and effervescent blend. However, lactose favored it. The results reveal that programmed drug delivery to achieve pulsatile drug release for three times daily can be obtained from these tablets in capsule system by systemic formulation approach.
Keywords: Pulsatile; Sodium alginate; Multi-layered tablets;

Methacholine dry powder inhaler as a new tool for bronchial challenge test by G. Colombo; C. Terzano; P. Colombo; A. Petroianni; A. Ricci; F. Buttini (165-171).
The methacholine (MCH) challenge test is performed to detect bronchial hyperresponsiveness in subjects suffering from asthma. It is conducted by inhaling spasmogen substances at increasing doses and measuring FEV1-PD20 variation following the bronchoconstriction evoked.This paper describes a new method for MCH challenge test using pre-metered respirable powders of MCH at different doses for facilitating test execution. The availability of a series of pre-metered doses gives higher control over aerosolized dose and fine particle fraction (respirable dose), improving the accuracy and repeatability of the test. Dosimetric tests with MCH solution and pre-dosed powder challenge tests were clinically compared.The inhalation powders were prepared by spray drying of solutions of methacholine, mannitol and hydroxypropylmethylcellulose in which different concentrations of MCH were included. The methacholine powders prepared were carefully characterized in terms of aerodynamic properties.Inhalation powders containing methacholine from 12.5 to 200 μg per metered dose, having a fine particle fraction between 40 and 60%, were prepared using mannitol and cellulose polymer. Eighteen subjects (12 hyperresponsive and six normal) were subjected to both the MCH solution and powder tests in random sequence. No significant differences in FEV1 and PD20 values were found between the challenge tests performed with liquid and powder formulations of methacholine.Powders of MCH having high respirability of the delivered doses can be prepared by spray drying. They allow for the performance of a challenge test using a dry powder inhaler. The powder dose series can be an alternative to the current dosimetric test with MCH solutions.
Keywords: Respirable particles; Hyperresponsiveness; Bronchoconstriction; Dry powder inhaler; Methacholine; Challenge test;

Development of a new photocrosslinkable biodegradable bioadhesive by P. Ferreira; J.F.J. Coelho; M.H. Gil (172-181).
Adhesives provide a needle-free method of wound closure and do not require local anaesthetics. Polymeric adhesives have been used for about 3 decades for joining several tissues of the organism. Also, they can accomplish other tasks, such as haemostasis and the ability to seal air leakages and have the potential to serve as delivery systems.PCL was modified with 2-isocyanatoethylmethacrylate to form a macromer that was crosslinked via UV irradiation using Irgacure 2959 by CIBA® as the photoinitiating agent. The characterization of the materials was accomplished by: attenuated total reflectance–Fourier transform infrared (ATR-FTIR), swelling capacity determination, evaluation of adhesive capacity (by reaction with aminated substrates) and determination of surface energy by contact angle measurement. Thermal characterization of the adhesive was performed by dynamical mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA). The morphology of PCL networks was observed using scanning electron microscopy (SEM) both after crosslinking process and following biodegradation in human plasma.The haemocompatibility of the membranes was also evaluated by thrombosis and haemolysis tests.
Keywords: Polycaprolactone; Tissue adhesive; Photocrosslinking; Thermal characterization; Haemocompatibility;

Frog intestinal sac as an in vitro method for the assessment of intestinal permeability in humans: Application to carrier transported drugs by Massimo Franco; Angela Lopedota; Adriana Trapani; Annalisa Cutrignelli; Daniela Meleleo; Silvia Micelli; Giuseppe Trapani (182-188).
The aim of this study was to investigate the presence of pharmaceutically relevant drug transporters in frog intestine which has been proposed as model for intestinal permeability screening assays of passively absorbed drugs in humans [Trapani, G., Franco, M., Trapani, A., Lopedota, A., Latrofa, A., Gallucci, E., Micelli, S., Liso, G., 2004. Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability. J. Pharm. Sci. 93, 2909–2919]. The expression of transporters in frog intestine was supported by the following observations: (i) the involvement of purine nucleobase transport system was deduced by inhibition of acyclovir transport in the presence of adenine; (ii) baclofen or l-dopa transport was inhibited by the digitalis steroid ouabain and it may be related to the Na+ electrochemical potential difference, presumably involving amino acid transporters; (iii) the presence of proton-dependent peptide transporters was argued evaluating the effect of the pH change (from pH 5.9 to pH 7.4) on the transport of glutathione; (iv) the possible expression in the frog intestine of an efflux system distinct from P-glycoprotein (Pgp) in the benzylpenicillin transport was deduced using a glucose enriched frog Ringer with or without the known Pgp inhibitor verapamil; (v) the contribution of Pgp-mediated efflux system in determining the frog intestinal absorption of drugs was supported by the specific inhibition of cimetidine or nadolol transport in the presence of verapamil. These results indicate that pharmaceutically relevant drug transporters should be also expressed in frog intestine.In this work, an attempt was also made to compare the measured P app values in the frog intestinal model for the aforementioned series of actively/effluxed transported drugs in humans to the corresponding literature values for the fraction absorbed. The P app values used in these comparisons were obtained at high concentrations of drugs at which probably saturation of the carrier occurs. Interestingly, it was found that drugs that are completely absorbed had P app values >3 × 10−6  cm/s, while drugs absorbed <90% had P app values lower than 1 × 10−6  cm/s. In these cases, indeed, a borderline region characterized by the apparent permeability coefficient P app value between 1 × 10−6   and 3 × 10−6  cm/s should be considered for which the prediction of the absorbed fraction after oral administration in humans become more uncertain by the frog intestinal sac system.
Keywords: Frog intestinal sac; Permeability; Carrier transported drugs; Transporter systems;

Evaluation of in vivo efficacy of topical formulations containing soybean extract by Sandra. R. Georgetti; Rúbia Casagrande; Waldiceu. A. Verri; Renata F.V. Lopez; Maria J.V. Fonseca (189-196).
In the present study it was evaluated the: (i) functional stability of the soybean extract as a raw material and dispersed in two different topical formulations, (ii) skin retention using modified Franz diffusion cells, and (iii) in vivo activity of these formulations to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced hydrogen peroxide (H2O2) and malondialdehyde (MDA) increases in the skin of hairless mice. The physico-chemical stability was evaluated by pH, globule size and centrifugation test. Furthermore, functional stability was also evaluated by antilipoperoxidative activity. The two topical formulations were stored at 4 °C, 30 °C/60% RH and 40 °C/70% RH for 6 months. The evaluation of the antiperoxidative stability of soybean extract itself and incorporated in formulations did not demonstrate loss of activity by storage at 4 °C/6 months. During 6 months of the study in different storage conditions the formulations 1 and 2 added or not with soybean extract were stable to physico-chemical tests. The effect of antioxidant compounds detected by the inhibition of MDA formation was time-dependent for formulation 2 as detected in the skin retention study. Pretreatment with formulation 1 or 2 significantly diminished TPA-induced H2O2 and MDA generation. In conclusion, the present results suggest for the first time that formulations containing soybean extract may be a topical source of antioxidant compounds that decrease oxidative damages of the skin.
Keywords: Antioxidant; Isoflavonoid; In vitro retention; Stability test; Skin; Topical formulation;

Mechanism of the photochemical degradation of amlodipine by Elisa Fasani; Angelo Albini; Silvia Gemme (197-201).
A mechanistic investigation on the photodegradation of amlodipine, the corresponding besylate and a simple analogue lacking the β-aminoethoxy group has been carried out in water and in organic solvents. Irradiation leads to aromatization to the corresponding pyridines through an oxygen-independent process. The quantum yield for amlodipine base is Φ  ≅ 0.001 under UV-A light, about one order of magnitude larger than that for the model bearing no amino group, supporting intramolecular assistance by that group. The value of Φ increases up to ca. 0.01 at shorter wavelength. The photolability of this drug according to ICH criteria is justified by the high absorptivity in the UV-A range (ɛ UV-A), despite the low quantum yield. Some comments are added about the fact that product Φ  ×  ɛ UV-A is more significative than Φ alone for the photolability (in solution) and about the lacking possibility to quench the photoreactivity where, as in the present case, this involves only short-lived intermediates.
Keywords: Photochemistry; Amlodipine; Mechanism; Quantum yield; Dihydropyridine; Photolability;

This study describes application of a newly developed method to the fluidized bed granulation. The method is based on predicting suitable amounts of water to be added to multi-component formulations using the corresponding values of components prior to granulation trials. The range of appropriate amount of water for each component in a model formulation was estimated in our previous studies with a refractive near-infrared (NIR) moisture sensor. Using those values, we calculated the range of suitable amount of water to add for the model pharmaceutical formulation. In this study, we examined the relationship between the amount of water added to the model formulation and the NIR sensor output value. Then, we performed fluidized bed granulation of the model formulation at steady-state moisture content levels under monitoring with NIR sensor, within and beyond the suitable range of added water that was calculated from the corresponding range of each component. For the model formulation, we found that the predicted values for suitable amounts of added water well corresponded to those in the granulation trials, suggesting that this predictive method may be useful in estimating suitable amounts of water to be added to formulations before fluidized bed granulation trials.
Keywords: NIR; Moisture content; Pharmaceutical formulation; Fluidized bed granulation; Liquid bridge;

Characterization and aerodynamic evaluation of spray dried recombinant human growth hormone using protein stabilizing agents by Monireh Jalalipour; Kambiz Gilani; Hosnieh Tajerzadeh; Abdolhossien Rouholamini Najafabadi; Mohammadali Barghi (209-216).
The effect of the protein stabilizers on the stability and aerosol performance of spray dried recombinant human growth hormone (SD rhGH) was investigated. rhGH solution was spray dried alone, with polysorbate 20 (at three concentrations of 0.05%, 0.01%, and 0.005%), Zn2+ (by Zn2+:rhGH molar ratio of 2:1 and 4:1), and/or lactose (by lactose:rhGH weight ratio of 2:1). Size exclusion chromatography (SEC) analysis of spray dried powders demonstrated that of all the potential protein stabilizers, the combination of polysorbate 20 (0.05%), Zn2+ (Zn2+:rhGH molar ratio of 2:1) and lactose (lactose:rhGH weight ratio of 2:1) was the most effective at protecting rhGH against aggregation during spray drying. The results of circular dichroism (CD) analysis revealed that using of polysorbate 20 (in all concentrations) and Zn2+ (by Zn2+:rhGH molar ratio of 2:1) together in the formulations would preserve rhGH conformational stability during the process. The particle size distribution data obtained by laser diffraction method showed all SD rhGH formulations had volume median diameter and mean diameter below 5 μm. The characterization of the aerosol performance of the spray dried powders by Andersen cascade impactor (ACI) showed that by increasing the concentration of polysorbate 20 in the formulations the aerodynamic efficiency of the resultant particles was reduced. In conclusion, the optimum amounts of polysorbate 20, Zn2+ and lactose satisfied both physical stability during spray drying process (2.37% aggregation) and good aerosol performance (fine particle fraction; FPF = 38.52%).
Keywords: Spray drying; Dry powder inhaler; Fine particle fraction; Recombinant human growth hormone; Protein stabilizers;

This study was aimed at investigating the pH-dependent solubility and in vitro transmucosal permeability of sildenafil, an amphoteric compound with limited aqueous solubility, across parallel artificial membrane. The aqueous solubility and permeability of sildenafil as a function of solution pH were theoretically derived from the individual contributions of all species (cationic, neutral and anionic). The stability, octanol–water distribution coefficient (log  D), and solubility of sildenafil were then determined at various pHs, the permeability study was also performed at different pHs using parallel artificial membrane. The pH-solubility and -permeability profiles were then fitted to theoretical equations using non-linear regression.The experimental pH-solubility profile was fitted very well to the theoretical equations (R 2  = 0.9996). The in vitro permeability of saturated sildenafil solution at different pH values also showed similar trend as the predicted one (R 2  = 0.7829). The two optimum pH (pHmax) values were found to be 4.50 and 10.24, where the maximum solubility of either cationic or neutral species, or anionic and neutral species is simultaneously obtained, and the maximal transmucosal fluxes (J ss) are achieved. The above method can be applied to optimize the transmucosal delivery of other amphoteric drugs with low aqueous solubility.
Keywords: Sildenafil; Solubility; Sublingual; Permeability; pH; Parallel artificial membrane;

In this paper, a bilayer-core osmotic pump tablet (OPT) which does not require laser drilling to form the drug delivery orifice is described. The bilayer-core consisted of two layers: (a) push layer and (b) drug layer, and was made with a modified upper tablet punch, which produced an indentation at the center of the drug layer surface. The indented tablets were coated by using a conventional pan-coating process. Although the bottom of the indentation could be coated, the side face of the indentation was scarcely sprayed by the coating solution and this part of the tablet remained at least partly uncoated leaving an aperture from which drug release could occur. Nifedipine was selected as the model drug. Sodium chloride was used as osmotic agent, polyvinylpyrrolidone as suspending agent and croscarmellose sodium as expanding agent. The indented core tablet was coated by ethyl cellulose as semipermeable membrane containing polyethylene glycol 400 for controlling the membrane permeability. The formulation of core tablet was optimized by orthogonal design and the release profiles of various formulations were evaluated by similarity factor (f 2 ). It was found that the optimal OPT was able to deliver nifedipine at an approximate zero-order up to 24 h, independent on both release media and agitation rates. The preparation of bilayer-core OPT was simplified by coating the indented core tablet, by which sophisticated technology of the drug layer identification and laser drilling could be eliminated. It might be promising in the field of preparation of bilayer-core OPT.
Keywords: Bilayer-core osmotic pump tablet; Indented core tablet; Orthogonal design; Similarity factor; Nifedipine;

In the present study the effect of type and concentration of a cosurfactant and oil on the ability of nonionic surfactant PEG-8 caprylic/capric glycerides (Labrasol®) to solubilize both oil and water phases was evaluated. Seven different cosurfactants (polyglyceryl-6 dioleate (Plurol Oleique®) (PO), polyglyceryl-6 isostearate (Plurol Isostearique®) (PI), polyglyceryl-4 isostearate (Isolan® GI 34) (IGI34), octoxynol-12 (and) polysorbate 20 (Solubilisant gamma® 2421) (SG2421), octoxynol-12 (and) polysorbate 20 (and) PEG-40 hydrogenated castor oil (Solubilisant gamma® 2429) (SG2429), PEG-40 hydrogenated castor oil (Cremophor® RH 40) (CRH40) and diethyleneglycol monoethyl ether (Transcutol®)) and six oils (isopropyl myristate, ethyl oleate, decyl oleate, medium chain triglycerides, mineral oil and olive oil) were used in phase behaviour studies of a quaternary system Labrasol®/cosurfactant/oil/water. The amount of surfactant required to completely homogenize equal masses of oil and water to form a single phase microemulsion (termed as balanced microemulsion) (Smin, %w/w), the minimal concentration of the surfactant/cosurfactant blend required to produce a balanced microemulsion (SCoSmin, %w/w) as well as the maximum concentration of water solubilized in investigated surfactant/oil and surfactant/cosurfactant/oil mixtures (W max, %w/w) were determined. The obtained results indicated that Labrasol® showed a good efficiency in the presence of lower molecular volume fatty acid esters with a preferred chemical structure such as isopropyl myristate (Smin 56.14% (w/w); W max 12.28% (w/w)). Oils with high molecular volume (olive oil and mineral oil) do not result in microemulsion formation. Transcutol® decreased the capacity of Labrasol® for solubilization of oil and water phases. The tendency of Labrasol® to solubilize both, water and oil phases, was favoured by polyglycerol-6 ester type of cosurfactants (PO and PI) while the influence of the polyglycerol-4 ester (IGI34) as well as of polyoxyethylene type of cosurfactants (CRH40, SG2421 and SG2429) on the surfactant efficiency, was not significant. Furthermore, the results revealed the significant influence of the surfactant/cosurfactant mass ratio (K m) on synergistic effect between polyglyceryl-6 esters and Labrasol® in the formation of microemulsions using isopropyl myristate as oil phase. Optimized microemulsion systems were stabilized with Labrasol®/polyglyceryl-6 esters blend at K m 5:5 (SCoSmin, 27.5% (w/w) and W max, 71.43% (w/w) for PI; SCoSmin, 29.18% (w/w) and W max, 65.00% (w/w) for PO) and the electrical conductivity measurement results for optimized balanced microemulsions showed that their structures were highly conductive indicating a bicontinuous microstructure.
Keywords: Microemulsions; SMEDDS; The efficiency of a surfactant; Labrasol®; Polyglycerol esters; Polyoxyethylene surfactants;

A biodegradable drug delivery system for the treatment of postoperative inflammation by S. Eperon; L. Bossy-Nobs; I.K. Petropoulos; R. Gurny; Y. Guex-Crosier (240-247).
Cataract surgery is often performed in patients suffering from associated pathologies. Our goal is to develop a biodegradable drug delivery system (DDS) combined with the artificial intraocular lens (IOL). DDS were manufactured using poly(d,l-lactide-co-glycolide), or PLGA, and were loaded with triamcinolone acetonide (TA). The loading capacity was approximately 1050 μg of TA per DDS. The higher the molecular weight of PLGA (34,000, 48,000 and 80,000 Da), the slower was the release of TA in vitro. Cataract surgery was performed on the right eye of rabbits. IOL was inserted with (i) no DDS, (ii) unloaded DDS PLGA48000, (iii) one loaded DDS PLGA48000, (iv) two loaded DDS. The number of inflammatory cells and the protein concentration were measured in the aqueous humor (AH). Unloaded DDS showed good ocular biocompatibility. One DDS PLGA48000 loaded with TA significantly reduced postoperative ocular inflammation. Two loaded DDS PLGA48000 was even more effective in inhibiting such inflammation. On long-term observation (days 63 and 84), reduction of inflammation could be obtained by insertion of one DDS PLGA48000 and a second DDS PLGA80000. Therefore, our “all in one” system is very promising since it could replace oral treatment and reduce the number of intraocular injections.
Keywords: Biodegradable polymer; Drug delivery; Intraocular lens; Rabbit; Triamcinolone;

Characteristics of niosomes prepared by supercritical carbon dioxide (scCO2) fluid by Aranya Manosroi; Romchat Chutoprapat; Masahiko Abe; Jiradej Manosroi (248-255).
Characteristics of niosomes prepared by a novel supercritical carbon dioxide fluid (scCO2) technique have been investigated. Niosomes were composed of Tween61/cholesterol at 1:0, 3:1, 1:1, 1:3 and 0:1 molar ratios and entrapped with d-(+)-glucose by the scCO2 method without and with ethanol at 5, 10 and 15 % (w/w) as a co-solvent, and the conventional chloroform film method with sonication. Tween61/cholesterol at 1:1 molar ratio niosomes prepared by all methods exhibited the best physical stability. Niosomes by the scCO2 method with 10 % (w/w) ethanol gave higher trapping efficiency (12.22 ± 0.26%) than those by the conventional chloroform film method with sonication (10.85 ± 0.24%) and the scCO2 method without ethanol (8.40 ± 1.60%). Niosomes by the scCO2 method with and without ethanol were large unilamellar structure under TEM with the average sizes of 271.9 ± 159.6 and 202.5 ± 136.7 nm, respectively, whereas those by the conventional chloroform film method with sonication were multilamellar and unilamellar structure with the average size of 58.4 ± 74.6 nm. However, the dispersibility of niosomes by the conventional chloroform film method with sonication was better than that by the scCO2 either with or without ethanol, because of smaller particle size. This present study has demonstrated the trapping efficiency enhancement of water-soluble compounds in niosomes by the scCO2 method with 10 % (w/w) of ethanol.
Keywords: Niosomes; Supercritical carbon dioxide; Conventional method; Water-soluble compounds; Trapping efficiency;

Distribution of nobiletin chitosan-based microemulsions in brain following i.v. injection in mice by Jing Yao; Jian Ping Zhou; Qi Neng Ping; Yun Lu; Liang Chen (256-262).
The purpose of this study was to characterize the in vitro properties of a number of chitosan-based microemulsions containing nobiletin and determine its distribution in mice brain following i.v. administration. The phase behavior and properties of chitosan-based microemulsions were investigated in a pseudo-ternary system composed of polyoxyethylene 35 castor oil/benzyl alcohol/medium-chain triglyceride/tea oil/water with the chitosan. The droplet sizes were found to be smaller than 25 nm by photo correlation spectrometer. The nobiletin-loaded hyaluronic acid chitosan-based microemulsion (HAC-ME) carried negative charge and nobiletin-loaded hydrochlorate chitosan-based microemulsion (HCC-ME) carried positive charge. The concentrations of nobiletin in tissues were determined by HPLC after i.v. administration of HAC-ME, nobiletin-loaded microemulsion (ME), HCC-ME and nobiletin solution. Based on AUC0–t , MRT and C max, HAC-ME delivered more nobiletin to the brain compared to nobiletin solution, ME and HCC-ME. The long-circulation effect might contribute to the higher AUC0–t for HAC-ME in brain. On the other hand, the AUC0–t in plasma and brain after i.v. administration of HCC-ME were not significantly increased relative to ME. These results indicate that HAC-ME may be presented as potential candidates for delivering more drugs into the brain.
Keywords: Chitosan-based microemulsions; Nobiletin; Hyaluronic acid chitosan; Hydrochlorate chitosan; Brain;

Effect of bicellar systems on skin properties by L. Barbosa-Barros; C. Barba; M. Cócera; L. Coderch; C. López-Iglesias; A. de la Maza; O. López (263-272).
Bicelles are discoidal aggregates formed by a flat dimyristoyl-glycero-phosphocholine (DMPC) bilayer, stabilized by a rim of dihexanoyl-glycero-phosphocholine (DHPC) in water. Given the structure, composition and the dimensions of these aggregates around 10–50 nm diameter, their use for topical applications is a promising strategy. This work evaluates the effect of DMPC/DHPC bicelles with molar ratio (2/1) on intact skin. Biophysical properties of the skin, such as transepidermal water loss (TEWL), elasticity, skin capacitance and irritation were measured in healthy skin in vivo. To study the effect of the bicellar systems on the microstructure of the stratum corneum (SC) in vitro, pieces of native tissue were treated with the aforementioned bicellar system and evaluated by freeze substitution applied to transmission electron microscopy (FSTEM). Our results show that bicelles increase the TEWL, the skin elastic parameters and, decrease skin hydration without promoting local signs of irritation and without affecting the SC lipid microstructure. Thus, a permeabilizing effect of bicelles on the skin takes place possibly due to the changes in the phase behaviour of the SC lipids by effect of phospholipids from bicelles.
Keywords: Freeze-substitution transmission electron microscopy; Tewl; Skin hydration; Biophysical properties of skin; Stratum corneum permeability; Stratum corneum microstructure;

This work systematically studied the intravenous injection formulation of solid lipid nanoparticles (SLNs) loaded with actarit, a poor water soluble anti-rheumatic drug. The goal of this study was to design passive targeting nanoparticles which could improve therapeutic efficacy and reduce side-effects such as nephrotoxicity and gastrointestinal disorders commonly associated with oral formulations of actarit. Based on the optimized results of single-factor and orthogonal design, actarit-loaded SLNs were prepared by a modified solvent diffusion–evaporation method. The formulated SLNs were found to be relatively uniform in size (241 ± 23 nm) with a negative zeta potential (−17.14 ± 1.6 mV). The average drug entrapment efficiency and loading were (50.87 ± 0.25)% and (8.48 ± 0.14)%, respectively. The actarit-loaded SLNs exhibited a longer mean retention time in vivo (t 1/2(β), 9.373 h; MRT, 13.53 h) compared with the actarit 50% propylene glycol solution (t 1/2(ke), 0.917 h; MRT, 1.323 h) after intravenous injection to New Zealand rabbits. The area under curve of plasma concentration–time (AUC) of actarit-loaded SLNs was 1.88 times greater than that of the actarit in 50% propylene glycol solution. The overall targeting efficiency (TE C ) of the actarit-loaded SLNs was enhanced from 6.31% to 16.29% in spleen while the renal distribution of actarit was significantly reduced as compared to that of the actarit solution after intravenous administration to mice. These results indicated that injectable actarit-loaded solid lipid nanoparticles were promising passive targeting therapeutic agents for rheumatoid arthritis.
Keywords: Passive target; Solid lipid nanoparticles; Actarit; Rheumatoid arthritis;

Effect of cationic lipid composition on properties of oligonucleotide/emulsion complexes: Physico-chemical and release studies by Érico Martini; Elias Fattal; Mônica Cristina de Oliveira; Helder Teixeira (280-286).
This paper describes the influence of cationic lipid composition on physico-chemical properties of complexes formed between oligonucleotides (ON) and cationic emulsions. Formulations containing medium chain triglycerides, egg lecithin, increasing amounts of either oleylamine (OA) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and water were prepared by a spontaneous emulsification procedure. ON adsorption on emulsions was evidenced by the inversion of the ζ-potential, the increase in droplet size, and the morphology of the oil droplet examined through transmission electron microscopy. Adsorption isotherms showed a higher amount of ON adsorbed on emulsions containing DOTAP when compared to emulsions containing OA. In a final step, the role of the main parameters, which may in fact influence the ON release rate from emulsions, was investigated. ON were progressively released from emulsions with an increase in dilution ratio and remained quite similar for both OA and DOTAP emulsions over time. Conversely, the effect of the cationic lipid composition was observed upon increasing the charge ratio of complexes. ON release at a same charge ratio was lower from emulsions containing DOTAP (bearing dioleyl chains) than from those containing OA (bearing monoleyl chain).
Keywords: Oligonucleotides; Cationic emulsions; DOTAP; Oleylamine; Release; Adsorption isotherms;

Loading of curcumin into macrophages using lipid-based nanoparticles by Keitaro Sou; Shunsuke Inenaga; Shinji Takeoka; Eishun Tsuchida (287-293).
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Cm) is a natural compound which possesses antioxidant, anti-inflammatory and anti-tumor ability. Here, phospholipid vesicles or lipid-nanospheres embedding Cm (CmVe or CmLn) were formulated to deliver Cm into tissue macrophages through intravenous injection. Cm could be solubilized in hydrophobic regions of these particles to form nanoparticle dispersions, and these formulations showed ability to scavenge reactive oxygen species as antioxidants in dispersions. At 6 h after intravenous injection in rats via the tail vein (2 mg Cm/kg bw), confocal microscopic observations of tissue sections showed that Cm was massively distributed in cells assumed as macrophages into the bone marrow and spleen. Taken together, these results indicate that the lipid-based nanoparticulates provide improved intravenous delivery of Cm to tissues macrophages, specifically bone marrow and splenic macrophages in present formulation, which has therapeutic potential as an antioxidant and anti-inflammatory.
Keywords: Nanoparticles; Liposomes; Bone marrow; Macrophage; Antioxidant; Drug delivery;

Core-modified chitosan-based polymeric micelles for controlled release of doxorubicin by Yi-Qing Ye; Feng-Liang Yang; Fu-Qiang Hu; Yong-Zhong Du; Hong Yuan; He-Yong Yu (294-301).
Amphiphilic stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles have been shown a good drug delivery system by incorporating hydrophobic drugs into the core of the micelles. One of the problems associated with the use of CSO-SA micelles is disassociation or the initial burst drug release during the dilution of drug delivery system by body fluid. Herein, the core of CSO-SA micelles was modified by the physical solubilization of stearic acid (SA) to reduce the burst drug release and enhance the physical stability of CSO-SA micelles. The CSO-SA micelles had 27.4 ± 2.4 nm number average diameter, and indicated pH-sensitive properties. The micelle size and drug release rate from micelles increased with the decrease of pH value. After the incorporation of SA into CSO-SA micelles, the micelle size was increased, and the zeta potential was decreased. The extents of the increase in micelle size and the decrease of zeta potential related with the incorporated amount of SA. The in vitro drug release tests displayed the incorporation of SA into CSO-SA micelles could reduce the drug release from the micelles due to the enhanced hydrophobic interaction among SA, hydrophobic drug and hydrophobic segments of CSO-SA.
Keywords: Chitosan oligosaccharide; Stearic acid; Micelles; Doxorubicin; Core modification; Controlled drug release;

Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene by Aijun Zuo; Pei Sun; Dongchun Liang; Wenguang Liu; Ronglan Zhao; Gang Guo; Nan Cheng; Jingyu Zhang; Kangde Yao (302-308).
Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatus csn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by β-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.
Keywords: Chitosan; Chitosanase gene; Co-transfection;

Formation mechanism of colloidal nanoparticles obtained from probucol/PVP/SDS ternary ground mixture by Adchara Pongpeerapat; Chalermphon Wanawongthai; Yuichi Tozuka; Kunikazu Moribe; Keiji Yamamoto (309-316).
The purpose of this study was to investigate the formation mechanism of colloidal nanoparticles after dispersion of probucol/polyvinylpyrrolidone (PVP)/sodium dodecyl sulphate (SDS) ternary ground mixture (GM) into water. Probucol, PVP and SDS were mixed at a weight ratio of 1:3:1 and ground for 30 min with a vibrational rod mill. The morphology and physicochemical properties were investigated through high resolution scanning electron microscopy (SEM), environmental SEM, dynamic light scattering, 13C NMR and zeta potential measurements. SEM images confirmed the presence of 20 nm size primary particles in the GM powder of probucol/PVP K17/SDS. Spherical nanoparticles with a size of around 100 nm, formed after dispersion of the GM into water, suggested an agglomeration of the primary particles. A further agglomeration of around 160 nm was observed with the stability experiment. Zeta potential and particle size measurements using latex beads revealed that PVPK 17/SDS complex was adsorbed on the probucol particle surface forming a layered structure. A similar agglomeration behavior was observed using the GM of probucol/PVP K12/SDS, though the molecular state of the PVPK 12/SDS complex at the particle surface was different from that of the PVPK 17/SDS complex. 13C NMR results suggested that intermolecular interactions between PVP K12 and SDS did not reach the same level as the interactions between PVP K17 and SDS. This study proposed a formation mechanism of colloidal nanoparticles.
Keywords: Nanoparticle; Grinding; Probucol; Polyvinylpyrrolidone; Sodium dodecyl sulphate; Environmental scanning electron microscopy;

Ciprofloxacin-encapsulated poly(dl-lactide-co-glycolide) nanoparticles and its antibacterial activity by Young-Il Jeong; Hee-Sam Na; Dong-Hyuk Seo; Dong-Gon Kim; Hyun-Chul Lee; Mi-Kyeong Jang; Sang-Kwon Na; Sung-Hee Roh; Sun-Il Kim; Jae-Woon Nah (317-323).
The aim of this study was to prepare ciprofloxacin HCl (CIP)-encapsulated poly(dl-lactide-co-glycolide) (PLGA) copolymer nanoparticles and its antibacterial potential was evaluated with pathogenic bacteria, Escherichia coli (E. coli), in vitro and in vivo. CIP-encapsulated nanoparticles of PLGA were prepared by multiple emulsion solvent evaporation method. PLGA nanoparticles showed spherical shapes with particle sizes around 100–300 nm. Loading efficiency was lower than 50% (w/w) because of water-solubility properties of CIP. At drug release study, CIP showed initial burst effect for 12 h and then continuously released for 2 weeks. At in vitro antibacterial activity test, CIP-encapsulated nanoparticles showed relatively lower antibacterial activity compared to free CIP due to the sustained release characteristics of nanoparticles. However, CIP-encapsulated PLGA nanoparticles (doses: 25 mg CIP/kg of mice) effectively inhibited the growth of bacteria due to the sustained release characteristics of nanoparticles, while free CIP was less effective on the inhibition of bacterial growth. These results indicated that CIP-encapsulated PLGA nanoparticles have superior effectiveness to inhibit the growth of bacteria in vivo.
Keywords: Ciprofloxacin HCl; Sustained release; Poly(dl-lactide-co-glycolide); Nanoparticles; Animal infection model;

Noticeboard (324).