International Journal of Pharmaceutics (v.414, #1-2)

Functionalization of lactose as a biological carrier for bovine serum albumin by electrospraying by M.A. Tavares Cardoso; M. Talebi; P.A.M.H. Soares; C.U. Yurteri; J.R. van Ommen (1-5).
Electrohydrodynamic atomization (EHDA) is an attractive technique to make new types of composite particles for pharmaceutical use. The aim of this work is to prove that EHDA can be successfully used to attach nano/micro-particles of protein to lactose, the commonly used excipient for pulmonary delivery, keeping all the biological properties of the protein after dissolution of the complex. Bovine serum albumin (BSA) was used as a model protein. The atomization of BSA was tested with two different solvents, dimethyl sulfoxide (DMSO) and ethanol. The process using DMSO resulted in the formation of a thin layer of protein while the tests using ethanol resulted in the formation of spherical particles with mean diameters around 700 nm. Ethanol as solvent was also used to produce a composite formed by BSA adsorbed at the surface of lactose by electrostatic forces. No denaturation or significant conformational changes of the protein were observed, although an increase in the exposition of the lactose to the jet of the solution decreases the reproducibility of the method. Due to the absence of denaturation in the model protein, this new approach can be tested for the production of new formulations for dry powders for drug delivery systems.
Keywords: Electrospray; Bovine serum albumin; Lactose; Dry powder; Drug delivery systems (DDS); Pharmaceutical formulation;

This study described the gelation process of acidic chitosan solution with NaHCO3, that sol–gel transition was controlled from a gradual neutralization of chitosan accompanying with CO2 emitting.In situ gelling systems are attractive as injectable vehicles for drug delivery. The present work described a novel gelation process of acidic chitosan solution in the presence of sodium bicarbonate (NaHCO3). The NaHCO3 concentration played an important role in this gelling system. When it came within the appropriate range, the chitosan/NaHCO3 system would stay at sol state in certain condition and showed sol–gel transition from the top to the bottom after heating. The rheological properties of the gelling system, as well as the morphology and erosion behavior of the formed chitosan hydrogels were evaluated as a function of the NaHCO3 concentration in sols. The hydrogels showed porous morphologies with some diversification depending on the NaHCO3 concentration, which also affected their erosion behaviors and drug release rates. Moreover, the gelation mechanism of such chitosan/NaHCO3 system was studied and proposed as the formation of three-dimensional chitosan network with physical junctions thanks to the deprotonation of –NH3 + in chitosan accompanying with the gradual neutralization between HCO3 and acid. In vivo gelation test was also performed by the dorsal subcutaneous injection of chitosan/NaHCO3 solution in rat. The formation of in situ gels suggested such system promising applications in injectable drug delivery system.
Keywords: Gelation; Chitosan; In situ gel; Drug delivery;

Use of remote film loading methodology to entrap sirolimus into liposomes: Preparation, characterization and in vivo efficacy for treatment of restenosis by Azadeh Haeri; Saeed Sadeghian; Shahram Rabbani; Maryam Sotoudeh Anvari; Mohammad Ali Boroumand; Simin Dadashzadeh (16-27).
Neointimal area and %stenosis are markedly reduced in vessel segments treated with sirolimus (SIR)-loaded nanoliposomes.The main objective of this study was to formulate an effective controlled-release liposomal drug delivery system for sirolimus (SIR), a potent antiproliferative and anti-inflammatory drug, to be used for the treatment of restenosis following local vascular delivery. Liposomes were prepared using remote film loading method and characterized with regard to entrapment efficiency (EE), size distribution and zeta potential. The effects of key formulation and proceeding variables on both EE and drug release were studied using a fractional factorial design. By means of this entrapment technique, 98% SIR incorporation was achieved. Nanoliposomes were found to have average size of 110 nm and zeta potential of −9 mV. Developed formulations were found to have prolonged drug release for up to 3 weeks in vitro; this was best fitted by the Higuchi model. Other scopes of this work were to determine the applicability of sirolimus-loaded nanoliposomes (SIR-L) as drug carriers for the treatment of restenosis and to evaluate the effect of the presence of rigid lipids on the in vivo efficacy of the liposomal carrier of SIR. In vivo studies in balloon injured rat carotid arteries revealed the potential of SIR-loaded liposomes as efficient local and controlled drug delivery systems to reduce restenosis.
Keywords: Sirolimus; Nanoliposomes; Remote film loading; Restenosis; Local delivery; Sustained release;

Zinc-pectinate beads as an in vivo self-assembling system for pulsatile drug delivery by Claire Dhalleine; Ali Assifaoui; Brice Moulari; Yann Pellequer; Philippe Cayot; Alf Lamprecht; Odile Chambin (28-34).
Zinc-pectinate beads are interesting drug carriers for oral delivery. In order to investigate their in vitro and in vivo release behaviour, ionotropic gelation was used to entrap theophylline into calcium- or zinc-pectinate beads. Beads were investigated in vitro for their particle properties, especially the release kinetic in different media, and their in vivo pharmacokinetic parameters were tested in rats. Particle size varied between 1.8 and 2.8 mm and encapsulation rates between 27 and 30% for Ca- and Zn-pectinate beads, respectively. While Ca-pectinate beads revealed a relative fast disintegration, drug release profiles from Zn-pectinate beads were very much release medium-dependent. Especially, in the presence of phosphate ions, the release from Zn-pectinate beads was blocked at 20% and 40% of the total drug load when tested in phosphate buffer or simulated colonic medium. In vivo Zn-pectinate beads (t max: 12.0 ± 0.1 h) led to a significant lag time for the theophylline absorption compared to Ca-pectinate (t max: 6.0 ± 2.8 h) or free theophylline (t max: 2.5 ± 2.1 h). This delayed release was attributed to the formation of a zinc phosphate coating in vitro and in vivo inducing the retention of theophylline release. Zn-pectinate beads exhibit interesting properties due to its potential as pulsatile delivery system induced by the in situ formation of Zn phosphate, while Ca-pectinate was found to be of limited suitability for controlled release of theophylline.
Keywords: Zinc-pectinate; Pulsatile delivery; Ionotropic gelation; In vivo self-assembling particles;

The use of polyethylene glycol 400 (PEG 400) as an excipient in oral formulations can have profound and differing effects on drug bioavailability in men and women; therefore an understanding of the pharmacokinetics of this excipient is required. A direct injection electrospray selected ion monitoring mass spectrometry methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration. The most abundant ions corresponding to PEG 400 oligomers at m/z 365, 409, 453, 497, 541, and 585 were used for selected ion monitoring (SIM). Pre-dose urine of volunteers was spiked with various amounts of PEG 400 to generate calibration curves over the concentration range 2.5–90 μg/mL for all SIM channels. The relative standard deviations of intra- and inter-day analysis of PEG 400 in human urine were lower than 11.8% and bias percentage was less than 9.7%. This specific method for relative quantitation of PEG 400 was then used to analyse urine samples with minimal sample preparation. Urine samples of twelve healthy volunteers (six men and six women) who received 0.75 g and 1.5 g PEG 400 on two separate occasions were collected over 24 h. On average 36.5% of the orally administered dose of PEG 400 was recovered in the urine of the volunteers, with no significant difference observed between men and women.
Keywords: Polyoxyethylene polymers; Permeability probes; Ranitidine; Paracellular pathway; Gastrointestinal transit; Motility; ES-MS; Selected ion monitoring;

Prednisolone-α-cyclodextrin-star poly(ethylene glycol) polypseudorotaxane with delayed pH-sensitivity as a targeted drug delivery system by Eliška Bílková; Miloš Sedlák; Aleš Imramovský; Petra Chárová; Petr Knotek; Ludvík Beneš (42-47).
The acylation of prednisolone 20-hydrazone with star poly(ethylene glycol) tetracarboxylic acid (M  = 20,000) has been used to prepare the corresponding pH-sensitive conjugate. With α-cyclodextrin, this conjugate forms a polypseudorotaxane, which was characterised by means of 1H NMR spectra, powder X-ray diffraction patterns and STM microscopy. The rate of acid-catalysed hydrolysis of the conjugate was studied under in vitro conditions in model media of hydrochloric acid solutions, phosphate and acetate buffers (pH 2–5.8). The acid-catalysed hydrolysis (at pH 2) of the polypseudorotaxane was ca 3.5 times slower than that of the original conjugate. After 1 h in this medium, 86% of the covalently attached prednisolone remained unchanged. The prepared polypseudorotaxane represents a promising peroral transport system of prednisolone with a pH-sensitive linker with delayed acid-catalysed hydrolysis thanks to protection at the molecular level using α-cyclodextrin.
Keywords: pH-sensitivity; Pro-drugs; PEG; Polypseudorotaxane; Prednisolone;

Investigation of physicochemical factors affecting the stability of a pH-modulated solid dispersion and a tablet during storage by Phuong Ha-Lien Tran; Thao Truong-Dinh Tran; Jun-Bom Park; Dong Hun Min; Han-Gon Choi; Hyo-Kyung Han; Yun-Seok Rhee; Beom-Jin Lee (48-55).
Microenvironmental pH at surface and center of a compressed tablet containing pH modulated solid dispersion of a poorly water-soluble drug during storage for 3 months at pH 6.8 intestinal fluid: refrigerator (5–8 °C), 25 °C/32% RH, 25 °C/55% RH, 25 °C/75% RH, 40 °C/32% RH, 40 °C/55% RH, and 40 °C/75% RH.The stability of solid dispersions (SD) during storage is of concern. We prepared the pH-modulated SD (pSD) and compressed tablets consisting of polyethylene glycol (PEG) 6000 as a carrier, drug and MgO (alkalizer). Telmisartan (TEL), an ionizable poorly water-soluble drug, was chosen as a model drug. The changes in physicochemical factors such as the dissolution rate, drug crystallinity, microenvironmental pH (pHM) and intermolecular interactions of the pSD and the tablets were investigated over 3 months under different temperature and relative humidity (RH) conditions: refrigerator (5–8 °C), 25 °C/32% RH, 25 °C/55% RH, 25 °C/75% RH, 40 °C/32% RH, 40 °C/55% RH, and 40 °C/75% RH. Differential scanning calorimetry (DSC) analysis of all samples revealed no distinct changes in the drug melting point. In contrast, powder X-ray diffraction (PXRD) diffractograms revealed that samples stored at 40 °C/75% RH for 1 month, 25 °C/75% RH for 3 months and 40 °C at all humidity conditions for 3 months showed gradual recrystallization of the drug. Fourier transform infrared (FTIR) spectra indicated a reduced intensity of intermolecular interactions between TEL and MgO in the pSD and tablet. The pHM also gradually decreased. These altered physicochemical factors under the stressed conditions resulted in decreased dissolution profiles in intestinal fluid (pH 6.8). In contrast, the dissolution rate in gastric fluid (pH 1.2) was almost unchanged because of the high intrinsic solubility of TEL at this pH.
Keywords: Telmisartan; Alkalizer; pH-modulated solid dispersion; Temperature; Humidity; Stability test; Microenvironmental pH;

Development and characteristics of temperature-sensitive liposomes for vinorelbine bitartrate by Hui Zhang; Zhi-yuan Wang; Wei Gong; Zhi-ping Li; Xing-guo Mei; Wan-liang Lv (56-62).
A novel liposome with temperature-sensitivity for vinorelbine bitartrate (VB) was designed to enhance VB targeted delivery and antitumor effect. Liposomes without drugs were prepared by thin film hydration, and then VB was entrapped into liposomes by pH gradient loading method. The mean particle size of the liposomes was about 100 nm, and the drug entrapment efficiency was more than 90%. Stability data indicated that the liposome was physically and chemically stable for at least 6 months at 4 °C. In vitro drug release study showed that drugs hardly released at 37 °C; while at 42 °C, drugs released quickly. For in vivo experiments, the lung tumor model was established by subcutaneous inoculation of cell suspension on mice, liposomes and free VB were injected i.v. in mice, followed by exposure the tumors to hyperthermia (HT) for 30 min after administration. The ratio of inhibition tumor of temperature-sensitive liposomes group was significantly higher than the normal injection group. Combining temperature-sensitive liposomes with HT enhanced the delivery of VB and, consequently, its antitumor effects. This liposome could potentially produce viable clinical strategies for improved targeting and delivery of VB for treatment of cancer.
Keywords: Temperature-sensitive; Liposomes; Vinorelbine; pH gradient; Thin film hydration; Hyperthermia;

A theoretical approach to evaluate the release rate of acetaminophen from erosive wax matrix dosage forms by Yasuyoshi Agata; Yasunori Iwao; Kai Shiino; Atsuo Miyagishima; Shigeru Itai (63-68).
To predict drug dissolution and understand the mechanisms of drug release from wax matrix dosage forms containing glyceryl monostearate (GM; a wax base), aminoalkyl methacrylate copolymer E (AMCE; a pH-dependent functional polymer), and acetaminophen (APAP; a model drug), we tried to derive a novel mathematical model with respect to erosion and diffusion theory. Our model exhibited good agreement with the whole set of experimentally obtained values pertaining to APAP release at pH 4.0 and pH 6.5. In addition, this model revealed that the eroding speed of wax matrices was strongly influenced by the loading content of AMCE, but not that of APAP, and that the diffusion coefficient increased as APAP loading decreased and AMCE loading increased, thus directly defining the physicochemical properties of erosion and diffusion. Therefore, this model might prove a useful equation for the precise prediction of dissolution and for understanding the mechanisms of drug release from wax matrix dosage forms.
Keywords: Wax matrix; Mathematical model; Diffusion; Erosion;

Scale-up of in vitro permeation assay data to human intestinal permeability using pore theory by Makoto Kataoka; Katsuaki Iwai; Yoshie Masaoka; Toshiyasu Sakane; Shinji Sakuma; Shinji Yamashita (69-76).
The aim of this study is to establish a theoretical method for the prediction of human intestinal permeability from in vitro permeation assay. Pore radius and porosity/length and ion selectivity of the paracellular pathway were calculated using the Renkin function using permeabilities of mannitol and urea and potential difference study to evaluate paracellular permeability in Caco-2 cell monolayer; they were calculated to be 5.91 Å, 7.51 cm−1 and 2.75, respectively. These values in the human epithelium were calculated from the reported intestinal permeability. The area factor, which can correct the difference in the transcellular permeability between Caco-2 cell monolayer and human epithelium, was obtained using the ratio of permeability of high lipophilicity compounds (human/Caco-2) and was calculated to be 13.3. Paracellular and transcellular permeabilities of 9 compounds in human epithelium were estimated on the basis of the characteristics of the paracellular pathway using physicochemical properties of compounds and the area factor, respectively. Human intestinal permeabilities were predicted by the sum of estimated transcellular and paracellular permeabilities. A linear correlation whose slope and intercept were nearly 1 and 0, respectively, was observed between predicted and reported human intestinal permeabilities. We successfully predicted human intestinal permeability from in vitro data.
Keywords: Absorption; Caco-2 cells; Intestine; Permeability; Renkin function;

Targeting SVCT for enhanced drug absorption: Synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir by Shuanghui Luo; Zhiying Wang; Mitesh Patel; Varun Khurana; Xiaodong Zhu; Dhananjay Pal; Ashim. K. Mitra (77-85).
In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [3H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 μM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [14C]AA diminished by about 50–70% relative to control in the presence of 200 μM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5 mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5 mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors.
Keywords: Saquinavir (Saq); Vitamin C conjugated prodrug of saquinavir (AA-Su-Saq); Synthesis; Uptake; Transport; Metabolism; SVCT;

Investigation of the riddle of sulfathiazole polymorphism by Mohd R. Abu Bakar; Zoltan K. Nagy; Chris D. Rielly; Sandy E. Dann (86-103).
Since the discovery of sulfathiazole as an antimicrobial agent in 1939, numerous works in the screening for its different polymorphic forms, which is an essential part of drug development, have been conducted and published. These works consequently result in the availability of various methods for generating a particular polymorph. By following these methods, however, one cannot be guaranteed to obtain the intended pure polymorph because most of the methods do not clearly and adequately describe the crystallisation conditions, such as cooling rates and initial solute concentrations. In this paper, the available methods for generating all the known polymorphs of sulfathiazole are reviewed and selected methods for generating certain polymorphs, performed with their processes monitored using process analytical technology tools, i.e. focussed beam reflectance measurement and attenuated total reflectance ultraviolet spectroscopy, are presented. The properties of the obtained crystals, examined using various characterisation methods, are also presented and whenever possible, are compared with those of other workers.
Keywords: Sulfathiazole; Process analytical technology; Solid state analysis;

Biocompatible hydrogels based on hyaluronic acid cross-linked with a polyaspartamide derivative as delivery systems for epithelial limbal cells by Calogero Fiorica; Richard A. Senior; Giovanna Pitarresi; Fabio Salvatore Palumbo; Gaetano Giammona; Pallavi Deshpande; Sheila MacNeil (104-111).
The aim of this work was to evaluate the potential use of hydrogels based on hyaluronic acid (HA) chemically cross-linked with α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-d,l-aspartamide (PHEA-EDA) as substitutes for the amniotic membrane able to release limbal cells for corneal regeneration. Hydrogels, shaped as films, with three different molar ratios (X) between PHEA-EDA and HA (X  = 0.5, 1.0 and 1.5) have been investigated. First, it has been evaluated their swelling ability, hydrolytic resistance in simulated physiological fluid and cell compatibility by using human dermal fibroblasts chosen as a model cell line. Then adhesion studies in comparison with collagen gel, have been performed by using immortalized cells, such as human corneal epithelial cells (HCEC) or primary cells, such as rabbit limbal epithelial cells (RLEC) and/or rabbit limbal fibroblasts (RLF). HA/PHEA-EDA hydrogels allow a moderate/poor adhesion of all investigated cells thus suggesting their potential ability to act as cell delivery systems. Finally, commercial contact lenses have been coated, in their inner surface, with each HA/PHEA-EDA film and it has been found that in these conditions, a greater cell adhesion occurs, particularly when RLEC are in co-culture with RLF. However, this adhesion is only transitory, in fact after three days, viable cells are released in the culture medium thus suggesting a potential application of HA/PHEA-EDA hydrogels, for delivering limbal cells in the treatment of corneal damage.
Keywords: Polyaspartamide; Hyaluronic acid; Hydrogels; Cell release; Corneal epithelium regeneration;

Scanning electron micrograph of rifampicin-loaded PLGA microspheres prepared from O/W (a and b) or W/O/W (c and d) emulsions homogenized by a premix membrane homogenization process (3 cycles).The solvent evaporation method with premix membrane homogenization was applied, with class-3 ethyl acetate as organic solvent, to produce narrowly size-distributed rifampicin (RIF)-loaded poly(lactide-co-glycolide) (PLGA) microspheres for sustained lung delivery as aerosol. Microsphere formulations (simple or multiple emulsions, different PLGA and RIF concentrations) and process parameters (transmembrane pressure, SPG membrane pore diameter) were investigated as their effects on RIF content, microsphere size, aerodynamic properties of the freeze-dried powder and in vitro release profiles. Narrowly size distributed microspheres with diameters from 2 to 8 μm, satisfactory RIF contents (from 4.9 to 16.5%), 80% RIF release from 12 h to 4 days, and adequate aerodynamic properties were prepared from a multiple emulsion and using SPG membrane pore diameter of 19.9 μm. The premix membrane homogenization appeared to be a rapid and efficient method to prepare monodisperse drug-loaded microspheres suitable for lung delivery as sustained-release microsphere aerosol.
Keywords: PLGA microspheres; Rifampicin; Lung delivery; Premix membrane emulsification; Aerosol; Sustained release;

The conflict between in vitro release studies in human biorelevant media and the in vivo exposure in rats of the lipophilic compound fenofibrate by Thao Thi Do; Michiel Van Speybroeck; Raf Mols; Pieter Annaert; Johan Martens; Jan Van Humbeeck; Jan Vermant; Patrick Augustijns; Guy Van den Mooter (118-124).
The performance of four different lipid-based systems for delivery of fenofibrate was studied and compared to a commercial formulation (Lipanthyl Micronized®) in vitro in various biorelevant media and in vivo in rats. The result of this study clearly points to the conflicting situation that might arise during the preclinical phase of the development of lipid based formulations of lipophilic drugs as the performance of such systems is very often evaluated by both in vitro release studies in human biorelevant media as well as in vivo studies in rats. Care must be taken to select a relevant animal model.The performance of four different lipid-based (Tween 80–Captex 200P, Tween 80–Capmul MCM, Tween 80–Caprol 3GO and Tween 80–soybean oil) and one commercially available micronized formulation (Lipanthyl Micronized®) of the lipophilic compound fenofibrate was compared in vitro in various biorelevant media and in vivo in rats. In simulated gastric fluid without pepsin (SGFsp) and fasted state simulated intestinal fluid (FaSSIF), only Tween 80–Captex 200P system resulted in a stable fenofibrate concentration, but no supersaturation was obtained. The other three lipid based systems created fenofibrate supersaturation; however they did not maintain it. In fed state simulated intestinal fluid (FeSSIF), all lipid-based formulations resulted in complete dissolution of fenofibrate during the experiment, which represented a supersaturated state for Tween 80–Capmul MCM and Tween 80–Caprol 3GO systems. In both FaSSIF and FeSSIF, all lipid-based formulations yielded a higher fenofibrate concentration than the micronized formulation. Contrary to the in vitro results, no significant difference in the in vivo performance was observed among the four tested lipid-based formulations both in the fasted and the fed states. The in vivo performance of all lipid-based formulations was better than that of Lipanthyl Micronized®, in the fasted as well as in the fed state. The fact that for the lipid based systems the in vitro differences in pharmaceutical performance were not translated into in vivo differences can be attributed to the continuous excretion of bile in the gastrointestinal tract of rats, causing enhanced solubilizing capacity for lipophilic drugs. This study clearly points to the conflicting situation that might arise during the preclinical phase of the development of lipid based formulations of lipophilic drugs as the performance of such systems is very often evaluated by both in vitro release studies in human biorelevant media as well as in vivo studies in rats. Care must be taken to select a relevant animal model.
Keywords: Fenofibrate; Lipid-based formulation; In vitro study; In vivo study;

Hildebrand solubility parameter to predict drug release from hydroxypropyl methylcellulose gels by P. Bustamante; J. Navarro-Lupión; M.A. Peña; B. Escalera (125-130).
An equation including the Hildebrand solubility parameter δ of the drugs is used for the first time to model drug release from hydroxypropyl methylcellulose (HPMC) gels: ln  M  = −21.578 + 2.102δ  − 0.037δ 2  + 0.48 ln  t  + 1.028 ln  C i (r 2  = 0.94 for a total of 286 cases). The experimentally determined release data of six drugs having different polarity (caffeine, theophylline, paracetamol, salicylic acid, naproxen and diclofenac) at several initial concentrations C i were included in the equation. In general, the amount of drug delivered is linear at the first 5–6 h of the release profiles and the zero order constants K o increase as the solubility parameter of the drugs become larger. The Peppas exponential law M/M  =  Kt n is applicable to larger fractional release, until 67–87% (48–51 h) for the less polar drugs (diclofenac and naproxen, lower δ values) and more than 80% (26–28 h) for the more polar drugs (higher δ values, theophylline, salicylic acid, caffeine and paracetamol). The Peppas release rate (ln  K) shows a parabolic relationship with the drug solubility parameter. The diffusional exponent n varies between 0.40 and 0.58 indicating that drug release is mainly controlled by diffusion. An extended form of the Peppas equation is also tested for each drug including all the initial concentrations: ln  M  =  a  +  b  ln  t  +  c  ln  C i (r 2  = 0.88–0.94). The logarithm of the octanol–water partition coefficients can also be used in combination with the drug concentrations.
Keywords: Solubility parameters; Drug release; Hydroxypropyl methylcellulose; Caffeine; Theophylline; Paracetamol; Salicylic acid; Naproxen; Diclofenac;

Compressibility of wet processed granules of same model placbo mixture was superior to dry processed mixtures measured via Heckel and Walker models and net energy using force–displacement data.The purpose of this study was to investigate the influence of various powder agglomeration processes on tableting mixture flow and compaction properties. Four different granulation methods of the same model placebo formulation were tested at a semi-industrial scale and their properties were compared to those of the directly compressed mixture. The wet granulated mixtures had superior flow properties compared to other mixtures and showed better compressibility, measured by the Heckel and Walker models. This was attributed to work hardening due to the double particle processing and also to shorter contact times due to higher initial densities of dry granulated mixtures, allowing a shorter time for deformation. A strong linear correlation was established between the Heckel and Walker coefficients, which was further confirmed by the net energy results of force–displacement measurements. It was shown that the Walker model had slightly better discriminative power to differentiate tableting mixtures according to compressibility. The compactibility was considerably lower for the slugged mixture; however, the roller-compacted mixture produced tablets with unexpectedly high tensile strength. In conclusion, it is important to emphasize that general assumptions like higher porosity ⇒ better compressibility or better compressibility ⇒ better compactibility cannot be established for complex tableting mixtures.
Keywords: Wet granulation; Dry granulation; Compressibility; Compactibility; Heckel; Waker;

Exposure to subzero temperature leads to loss of vaccine potency. This can happen due to degradation of adjuvant surface and/or inactivation of the antigen. When adsorbed on aluminium hydroxide and subjected to freeze-thawing, tetanus toxoid was desorbed from the gel matrix and the preparation was found to lose its antigenicity. Analyses showed that the gel particles were denatured after freezing. When freeze-thawing was carried out in the presence of glucose, sorbitol and arginine, the degradation of gel particles was inhibited. A higher fraction of the protein could be retained on the gel. However, the antigenicity of these preparations was quite low. In the presence of trehalose, the protein could be partially retained on aluminium hydroxide. Being a cryoprotectant, trehalose was also able to inhibit the freezing-induced denaturation of tetanus toxoid, which resulted in retention of antigenicity of the adjuvanted toxoid.
Keywords: Adjuvant; Aluminium hydroxide; Cryoprotectant; Tetanus toxoid;

Comparison of mesoporous silicon and non-ordered mesoporous silica materials as drug carriers for itraconazole by Päivi Kinnari; Ermei Mäkilä; Teemu Heikkilä; Jarno Salonen; Jouni Hirvonen; Hélder A. Santos (148-156).
Mesoporous materials have an ability to enhance dissolution properties of poorly soluble drugs. In this study, different mesoporous silicon (thermally oxidized and thermally carbonized) and non-ordered mesoporous silica (Syloid AL-1 and 244) microparticles were compared as drug carriers for a hydrophobic drug, itraconazole (ITZ). Different surface chemistries pore volumes, surface areas, and particle sizes were selected to evaluate the structural effect of the particles on the drug loading degree and on the dissolution behavior of the drug at pH 1.2. The results showed that the loaded ITZ was apparently in amorphous form, and that the loading process did not change the chemical structure/morphology of the particles’ surface. Incorporation of ITZ in both microparticles enhanced the solubility and dissolution rate of the drug, compared to the pure crystalline drug. Importantly, the physicochemical properties of the particles and the loading procedure were shown to have an effect on the drug loading efficiency and drug release kinetics. After storage under stressed conditions (3 months at 40 °C and 70% RH), the loaded silica gel particles showed practically similar dissolution profiles as before the storage. This was not the case with the loaded mesoporous silicon particles due to the almost complete chemical degradation of ITZ after storage.
Keywords: Drug loading; Drug release; Itraconazole; Silica gel; Mesoporous silicon; Dissolution;

In this work, near infrared (NIR) hyperspectral imaging was used to quantify the spatial distribution of drug in tablets containing tolmetin sodium dihydrate. Hyperspectral data cubes were generated by imaging the same spatial region of a sample while illuminated by a laser at a different wavelength for each image. Images were generated for wavelengths ranging from 1100 to 2200 nm. Ten tablets with concentrations ranging from 0.0 to 10.0% w/w tolmetin were imaged, and the scales of segregation were calculated for the tablets. Lactose anhydrous was used as the diluent, and all mixtures contained 0.5% magnesium stearate as a lubricant. This research has shown hyperspectral imaging to be viable tool for quantifying segregation of low dose drugs in tablets.
Keywords: NIR; Chemical imaging; Hyperspectroscopy; Scale of segregation; Low dose;

Doxorubicin and paclitaxel loaded microbubbles for ultrasound triggered drug delivery by Michael C. Cochran; John Eisenbrey; Richard O. Ouma; Michael Soulen; Margaret A. Wheatley (161-170).
Ultrasound contrast agents are destroyed with focused ultrasound, resulting in drug loaded polymer fragments less than 400 nm in diameter capable of escaping the leaky vasculature of a tumor and providing a sustained release of drug.A polymer ultrasound contrast agent (UCA) developed in our lab has been shown to greatly reduce in size when exposed to ultrasound, resulting in nanoparticles less than 400 nm in diameter capable of escaping the leaky vasculature of a tumor to provide a sustained release of drug. Previous studies with the hydrophilic drug doxorubicin (DOX) demonstrated enhanced drug delivery to tumors when triggered with ultrasound. However the therapeutic potential has been limited due to the relatively low payload of DOX. This study compares the effects of loading the hydrophobic drug paclitaxel (PTX) on the agent's acoustic properties, drug payload, tumoricidal activity, and the ability to deliver drugs through 400 nm pores. A maximum payload of 129.46 ± 1.80 μg PTX/mg UCA (encapsulation efficiency 71.92 ± 0.99%) was achieved, 20 times greater than the maximum payload of DOX (6.2 μg/mg), while maintaining the acoustic properties. In vitro, the tumoricidal activity of paclitaxel loaded UCA exposed to ultrasound was significantly greater than controls not exposed to ultrasound (p  < 0.0016). This study has shown that PTX loaded UCA triggered with focused ultrasound have the potential to provide a targeted and sustained delivery of drug to tumors.
Keywords: Ultrasound contrast agent; Microbubble; Paclitaxel; Nanoparticle; Targeted drug delivery;

Real-time in-die compaction monitoring of dry-coated tablets by Jingfei Liu; James D. Stephens; Brian R. Kowalczyk; Cetin Cetinkaya (171-178).
The practicability of a pulse–echo ultrasonic approach developed for the real-time quality monitoring of dry-coated tablets in the tablet press during compaction is evaluated. The punch–tablet interface (i.e., steel–tablet) is the boundary condition that dictates the viability of acoustic in-die compaction monitoring. The current study utilizes compacted tablets with a simulated punch–tablet interface to achieve the required waveform detectability levels needed for in-die compaction monitoring. The geometric and mechanical properties of a dry-coated tablet are crucial to its structural functions and therapeutic effectiveness, therefore they are monitored especially when the control of dissolution rates of their active ingredients are critically important. Acquired pulse–echo ultrasonic waveforms in the tablet could provide the time-of-flight information needed to determine the thickness, elasticity and/or integrity of the relevant layer, and bonding quality between layers depending on the given parameters. Since the amplitudes of the reflected waves are extremely low due to the high acoustic impedance mismatches of tablet materials and die/punch materials, signal processing techniques are required to extract the wave arrival times. In current study, it is demonstrated that the reflection of an ultrasonic pulse generated by a transducer embedded in a die or a punch from the coat–core interface can be acquired by the same transducer.
Keywords: Dry-coated tablets; Real-time quality monitoring systems; In-die online monitoring; Core eccentricity; Process Analytical Technology (PAT);

Drug targeting to inflammation: Studies on antioxidant surface loaded diclofenac liposomes by Raju Jukanti; Gopinath Devaraj; Rambhau Devaraj; Shashank Apte (179-185).
The drug targeting potential of prepared antioxidant surface loaded diclofenac liposomes was evaluated in air pouch induced rats. The results envisage better targeting potential for antioxidant surface loaded liposomes to the inflammatory site assessed by measuring the diclofenac content in air pouch fluid.Inflammation is associated with enhanced vascular permeability, production of inflammatory markers and over production of reactive oxygen species (ROS) with depletion of endogenous antioxidants. Several drug targeting approaches to inflammation taking clues from these events have been evolved. Surprisingly, a drug targeting approach utilizing abundant oxidative stress at inflammatory site has not been followed. Antioxidant surface loaded liposomes might preferentially localize at inflammatory sites via redox interaction where at high level of ROS exist. The present study was focused to investigate the role of antioxidant as a targeting ligand on the surface of liposome employing rat granuloma air pouch model of inflammation. We developed conventional and antioxidant loaded diclofenac (DFS) liposomes (co-enzyme Q10 and ascorbyl palmitate) for i.v. administration and characterized for vesicle size, zeta potential and percent entrapment. In vivo drug targeting studies showed an increase in AUC, therapeutic availability of DFS in air pouch fluid (APF) and APF/serum DFS concentration ratios from antioxidant loaded liposomes compared to conventional liposomes and drug solution. The promising results suggest the role of antioxidant as a possible ligand in drug targeting to a site where at abundant ROS exist.
Keywords: Antioxidant; Liposomes; Diclofenac; Inflammation; Targeting;

Physicochemical and pharmacokinetic characterization of a spray-dried malotilate emulsion by Jianjun Zhang; Yuan Gao; Shuai Qian; Xin Liu; Hui Zu (186-192).
Spray-dried malotilate emulsion exhibited a 2.9-fold higher peak plasma concentration (C max) and 2.3-fold higher area under the curve (AUC) than MT suspension, a 2.11-fold higher C max and 1.83-fold higher AUC than CE.Malotilate (MT) is a hepatoprotective drug administered orally. However, MT was found to be a poorly water-soluble drug with low oral bioavailability. In the present investigation, a novel spray-dried emulsion (SDE) loaded with MT was prepared, and its physicochemical properties were characterized by rheological evaluation, particle size measurement, in vitro release, and surface morphology. The pharmacokinetic study of SDE, in comparison to MT suspension with the pure MT powder homogeneously dispersed in 0.5% CMC–Na solution, was also performed in rats after a single oral dose. It was found that SDE exhibited a 2.9-fold higher peak plasma concentration (C max) and 2.3-fold higher area under the curve (AUC) than MT suspension.
Keywords: Malotilate; Spray-dried emulsion; Rheological; Pharmacokinetics;

The characterization of protein release from sericin film in the presence of an enzyme: Towards fibroblast growth factor-2 delivery by Ayumu Nishida; Tsuyoshi Naganuma; Takanori Kanazawa; Yuuki Takashima; Masaki Yamada; Hiroaki Okada (193-202).
The drug-release and the biodegradation of sericn film were evaluated in enzymatic conditions. And FGF-2 was incorporated in the films and delivered at a bone defection.Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form β-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate–albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds.
Keywords: Sericin; Sustained release; Film; Biodegradation; FGF-2; Bone defection;

The effect of poly(l-lactide-co-TMC) chain microstructure (and its changes during degradation) on immunosuppressive drugs’ release process was analyzed. Three kinds of poly(l-lactide-co-TMC) (PLATMC) – two semiblock and one random were used to prepare matrices containing cyclosporine A or rapamycine and drug free matrices. All of them degraded slowly enough to provide long term delivery of immunosuppressive agents. Moreover, copolymer chain microstructure determined the effect of drug loading on the degradation process. It was observed that matrices without drug obtained from semiblock copolymer degraded differently than matrices containing cyclosporine A or rapamycine, whereas all kinds of matrices obtained from random PLATMC degraded in similar way. This is the evidence that the only in case of semiblock copolymer factors concerning the presence of drug and the kind of drug influenced degradation process. Based on the obtained results, correlations between copolymer degradation and drug release process are proposed. According to our outcomes, regular drug release process may be obtained from highly randomized copolymers (R  ≈ 1) that remain amorphous during degradation process. Determination of this factor may help in development of biodegradable systems, in which drug release rate and profile can be tailored by synthesis of polymer with appropriate chain microstructure.
Keywords: Cyclosporine A; Rapamycine; Biodegradable polymers; Microstructure; Controlled release; Poly(l-lactide-co-trimethylene carbonate);

The stability of solid dispersions of felodipine in polyvinylpyrrolidone characterized by nanothermal analysis by Jianxin Zhang; Matthew Bunker; Andrew Parker; Claire E. Madden-Smith; Nikin Patel; Clive J. Roberts (210-217).
Nanothermal analysis (NTA) supported by atomic force microscopy imaging has been used to study the changes that occur at the surfaces of solid dispersions of the drug felodipine and the water soluble polymer, polyvinylpyrrolidone (PVP) on exposure to standard pharmaceutical environmental stress conditions. Exposure to relative humidities above 75% (at 40 °C) was sufficient to achieve phase separation of the drug and polymer into areas which displayed a glass transition temperature consistent with pure drug and polymer over a period of a few days. Higher values of humidity at 25 °C (e.g. 95%RH) were also sufficient to cause such phase separation within a day. Extended studies of up to two months showed an eventual crystallization of the drug. NTA is shown to be effective at the early detection of instabilities in solid dispersions and the quantifiable identification of the relative composition of phase separated domains based upon their glass transition temperatures. The combined nanoscale analytical approach employed here is able to systematically study the influence of storage conditions and different drug loadings and to evaluate physical stability as a function of environmental conditions.
Keywords: Solid solution; Solid dispersion; Felodipine; PVP; Stability; Nanothermal analysis;

An investigation into silk fibroin conformation in composite materials intended for drug delivery by Francesco Cilurzo; Chiara G.M. Gennari; Francesca Selmin; Laura A. Marotta; Paola Minghetti; Luisa Montanari (218-224).
Regenerated silk fibroin/hydrophilic polymers composite materials prepared using mild evaporation conditions can be used to design drug delivery systems since they lead to water stable constructs.Regenerated silk fibroin (SF) is a promising biomaterial to design drug delivery systems. To guarantee satisfactory prolonged release of loaded drugs, the native β-sheet conformation of SF is generally induced by a final curing which can determine instability of the loaded drug. This work aimed to investigate the influence on SF conformation of the addition of hydrophilic polymers, namely poloxamer 188 (PEO), a range of poly(ethylenglycol) (PEG)and poly(vinyl pyrrolidone) (PVP) and drying conditions, namely spray-drying or evaporation at 60 °C. DSC data on spray-dried products indicated that SF in composite materials was in the random coil conformation. ATR-FTIR spectroscopy with Fourier self-deconvolution of the amide I band revealed that SF in spray dried products was partially organized in the β-sheet structure only in presence of PEG4000.Both DSC and ATR-FTIR spectra registered on composite materials obtained by the slowest evaporation method indicated that all hydrophilic polymers favoured the β-sheet conformation. This feature was attributed to the formation of H-bonds with the tyrosine residues of the semicrystalline region in SF. In conclusion, this approach to prepare of SF/hydrophilic polymer composites at slow evaporation rate leads to water insoluble materials which could be used in the development of drug delivery systems.
Keywords: Silk fibroin; Spray-drying; Casting; ATR-FTIR spectroscopy;

A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation by Matthias Wacker; Anja Zensi; Jürgen Kufleitner; Aaron Ruff; Jessica Schütz; Tobias Stockburger; Thomas Marstaller; Vitali Vogel (225-232).
Nanoparticles consisting of human serum albumin (HSA) play an emerging role in the development of new drug delivery systems. Many of these protein-based colloidal carriers are prepared by the well-known desolvation technique, which has shown to be a robust and reproducible method for the laboratory-scale production of HSA nanoparticles.The aim of the present study was to upscale the ethanolic desolvation process utilizing the paddle stirring systems Nanopaddle I and II in combination with a HPLC pump in order to find the optimal conditions for the controlled desolvation of up to 2000 mg of the protein.For characterization of the HSA nanoparticles particle size, zeta potential as a function of the pH, polydispersity index and particle content were investigated. The particle content was determined by microgravimetry and by a turbidimetry to allow optimized in-process control for the novel desolvation technique. Furthermore the sedimentation coefficient was measured by analytical ultracentrifugation (AUC) to gain deeper insight into the size distribution of the nanoparticles.The formed nanocarriers were freeze dryed to achieve a solid preparation for long-term storage and further processing. Particles ranging in size between 251.2 ± 27.0 and 234.1 ± 1.5 nm and with a polydispersity index below 0.2 were achieved.
Keywords: HSA; Albumin; Preparation; Analytical ultracentrifugation; Coacervation; Industrial production;

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using hydrophobized hyaluronic acid–spermine conjugates (HHSCs), which were previously synthesized and their properties were also characterized in our published papers. Fluorescein isothiocyanate (FITC) labeled specific Silencer Select siRNAs were used as a model system suppressing the cyclooxygenase-2 (COX-2) gene expression. The polymers were able to effectively bind siRNA, self-assemble into micelles, protect siRNA from degradation by nuclease and release complexed siRNA efficiently in the presence of low concentrations of polyanionic heparin. The cytotoxicity of siRNA/HHSCs complex to SGC-7901 cells was lower than that of siRNA/PEI 25k and Lipofectamine 2000 complex according to the MTT assay. When SGC-7901 and GES-1 cells were treated with FITC labeled siRNA/HHSCs complexes, SGC-7901 cells, with a cluster determinant 44 receptor (CD44), showed higher green fluorescent intensity than GES-1 cells because of the HA receptor mediated endocytosis of the complex. In addition, the inhibitory effect on the uptake in the presence of free HA in the transfection medium revealed that siRNA/HHSC-1 complex was selectively taken up to SGC-7901 cells via HA-receptor mediated endocytosis. Based on flow cytometry and microscopy, observation revealed that siRNA/HHSC complex was taken up preferentially through caveolae-mediated endocytosis, which may be a desirable pathway for avoiding the lysosomal degradation of delivered genes. All these results demonstrated that the intracellular delivery of siRNA/HHSC complex could be facilitated by the HA-receptor mediated endocytosis.
Keywords: Hyaluronic acid; Small interfering RNA; HA-receptor; Tumor-targeted; HA-receptor mediated endocytosis;

Preparation of polymeric nanoparticles of cyclosporin A using infrared pulsed laser by Gen Takebe; Tokio Takagi; Masumi Suzuki; Mitsuo Hiramatsu (244-250).
We showed that laser irradiation of drugs can be a novel tool for dispersing drug nanoparticles in water.Nanoparticle formation of poorly water-soluble drugs is a means of providing much benefit for improving solubility and bioavailability. We showed that laser irradiation of drugs can be a novel tool for dispersing drug nanoparticles in water. Using our method, we were able to produce nanoparticles containing immunosuppressant drug, cyclosporin A, which shows poor solubility toward water, with high levels of the drug using polyvinyl pyrrolidone and sodium dodecyl sulfate as stabilizing agents. The absence of degradation products was confirmed and the loss of pharmaceutical activity with an inhibitory effect on the interleukin-2 production of Jurkat T cells did not occur. Cyclosporin A nanoparticles showed a spherical shape and their particle size was distributed uniformly around 200 nm. Powder X-ray diffraction analysis suggested that cyclosporin A in the nanoparticles was in an amorphous state. In the measurement of solubility rate, the nanoparticle formulation showed a higher rate than that which had not been processed. At present, although this laser irradiation technology has low productivity, it is expected as a new technology for drug nanoparticle manufacturing together with the development of a new laser device.
Keywords: Polymeric nanoparticles; Cyclosporin A; Laser technology; Polyvinyl pyrrolidone;

Characterization, pharmacokinetics and disposition of novel nanoscale preparations of paclitaxel by Cheng Wang; YingJing Wang; YuJun Wang; Min Fan; Feng Luo; ZhiYong Qian (251-259).
Polymeric nanoparticles (NPs) have great potential application in achieving targeted delivery of anticancer drugs. Paclitaxel (PTX) loaded NPs were developed using biodegradable methoxy poly (ethylene glycol)–poly (ɛ-caprolactone) (MPEG–PCL) diblock copolymer by solid dispersion technique without toxic organic solvent. The lyophilized powder has been stored at room temperature for more than six months and still unchanged. PTX-loaded MPEG–PCL nanoparticles (PTX-NPs) displayed that the highest drug loading of PTX was about 25.6% and entrapment efficiency was over 98%, and the optimized average diameter and polydispersity index (PDI) were about 27.6 ± 0.1 nm and 0.05, respectively. Moreover, experimental results shown PTX-NPs had sustained-release effects and its curve fitting followed the Higuchi model. The maximum tolerated dose (MTD) of PTX-NPs after single dose in Balb/c mice was above 80 mg PTX/kg body weight (b.w), which was 2.6-fold higher than that of Taxol® (30 mg PTX/kg b.w). The levels of PTX administrated PTX-NPs had obvious distinction to Taxol® in plasma, liver, spleen, kidneys, lungs, heart and tumor. Especially, the concentration of PTX in tumor administrated PTX-NPs was higher than administration of Taxol®. All results suggested that we had contrived a simple, biodegradable, effective and controllable drug delivery system for paclitaxel.
Keywords: Polymeric nanoparticle; Paclitaxel; Amphiphilic diblock copolymer; MPEG–PCL; Solid dispersion technique;

Absorption of the novel artemisinin derivatives artemisone and artemiside: Potential application of Pheroid™ technology by J. Dewald Steyn; Lubbe Wiesner; Lissinda H. du Plessis; Anne F. Grobler; Peter J. Smith; Wing-Chi Chan; Richard K. Haynes; Awie F. Kotzé (260-266).
Artemisinins have low aqueous solubility that results in poor and erratic absorption upon oral administration. The poor solubility and erratic absorption usually translate to low bioavailability. Artemisinin-based monotherapy and combination therapies are essential for the management and treatment of uncomplicated as well as cerebral malaria. Artemisone and artemiside are novel artemisinin derivatives that have very good antimalarial activities. Pheroid™ technology is a patented drug delivery system which has the ability to entrap, transport and deliver pharmacologically active compounds. Pharmacokinetic models were constructed for artemisone and artemiside in Pheroid™ vesicle formulations. The compounds were administered at a dose of 50.0 mg/kg bodyweight to C57 BL/6 mice via an oral gavage tube and blood samples were collected by means of tail-bleeding. Drug concentrations in the samples were determined using an LC/MS/MS method. There was 4.57 times more artemisone in the blood when the drug was entrapped in Pheroid™ vesicles in comparison to the drug only formulation (p  < 0.0001). The absorption of artemiside was not dramatically enhanced by the Pheroid™ delivery system.
Keywords: Malaria; Artemisone; Artemiside; Pheroid™ technology; Pharmacokinetic analysis;

Lipid nanocarriers for dermal delivery of lutein: Preparation, characterization, stability and performance by Khalil Mitri; Ranjita Shegokar; Sven Gohla; Cecilia Anselmi; Rainer H. Müller (267-275).
Topical application of lutein as an innovative antioxidant, anti-stress and blue light filter, which is able to protect skin from photo damage, has got a special cosmetic and pharmaceutical interest in the last decade. Lutein is poorly soluble, and was therefore incorporated into nanocarriers for dermal delivery: solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and a nanoemulsion (NE). Nanocarriers were produced by high pressure homogenization. The mean particle size was in the range of about 150 nm to maximum 350 nm, it decreased with increasing oil content of the carriers. The zeta potential in water was in the range −40 to −63 mV, being in agreement with the good short term stability at room temperature monitored for one month. In vitro release was studied using a membrane free model. Highest release in 24 h was observed for the nanoemulsion (19.5%), lowest release (0.4%) for the SLN. Release profiles were biphasic (lipid nanoparticles) or triphasic (NE). In vitro penetration study with a cellulose membrane showed in agreement highest values for the NE (60% in 24 h), distinctly lower values for the solid nanocarriers SLN and NLC (8–19%), lowest values for lutein powder (5%). Permeation studies with fresh pig ear skin showed that no (SLN, NLC) or very little lutein (0.4% after 24 h) permeated, that means the active remains in the skin and is not systemically absorbed. The nanocarriers were able to protect lutein against UV degradation. In SLN, only 0.06% degradation was observed after irradiation with 10 MED (Minimal Erythema Dose), in NLC 6–8%, compared to 14% in the NE, and to 50% as lutein powder suspended in corn oil. Based on size, stability and release/permeation data, and considering the chemical protection of the lutein prior to its absorption into the skin, the lipid nanoparticles are potential dermal nanocarriers for lutein.
Keywords: Lutein; Solid lipid nanoparticles; Nanostructured lipid carriers; Dermal delivery; Antioxidant; Photo stability;

A photoprotective formulation was developed with an increased sunprotection factor (SPF), compared to a conventional nanoemulsion, but having the same concentration of three molecular sunscreens, namely ethylhexyl triazone, bis-ethylhexyloxyphenol methoxyphenyl triazine, and ethylhexyl methoxycinnamate. The sunscreen mixture was incorporated into nanostructured lipid carriers (NLCs). The ability of nine different solid lipids to yield stable aqueous NLC suspensions was assessed. After the production by hot high pressure homogenization, the NLC were analyzed in terms of particle size, physical state, particle shape, ultraviolet absorbance and stability. The particle size for all NLC was around 200 nm after production. The NLC suspension with carnauba wax had superior UV absorbance, NLC from bees wax showed similar efficiency as the reference emulsion. The NLC formulations were incorporated into hydrogel formulations and the in vitro SPF was measured. This study demonstrated that approximately 45% higher SPF values could be obtained when the organic UV filters were incorporated into carnauba wax NLC, in comparison to the reference nanoemulsion and bees wax NLC. The data showed that the synergistic effect of NLC and incorporated sunscreens depends not only on the solid state of the lipid but also on its type.
Keywords: Lipid nanoparticles; Skin sunprotection; Hot high pressure homogenization; SPF;

A new nanomedicine based on didanosine glycerolipidic prodrug enhances the long term accumulation of drug in a HIV sanctuary by Rym Skanji; Karine Andrieux; Muriel Lalanne; Joachim Caron; Claudie Bourgaux; Jéril Degrouard; François Brisset; Claire Gueutin; Hélène Chacun; Nathalie Dereuddre-Bosquet; Angelo Paci; Gilles Vassal; Laurent Bauduin; Sébastien Garcia-Argote; Bernard Rousseau; Pascal Clayette; Didier Desmaële; Patrick Couvreur (285-297).
New nanomedicines could improve drug accumulation in HIV sanctuaries and ameliorate their antiretroviral efficiency. In this view, we propose herein a combined strategy based on a biomimetic prodrug of ddI and its formulation in well-characterized lipid nanoobjects. The glycerolipidic prodrug of ddI (ProddINP) has been synthesized and its bulk structure was characterized. An appropriate formulation of this prodrug has been designed using a rational approach combining different physicochemical techniques. The high incorporation ratio of the prodrug into dipalmitoylphosphatidylcholine (DPPC) bilayers was determined by DSC. Then two liposome preparation methods were compared, with respect to size, incorporation yield and molecular/supramolecular organization of vesicles. The best liposomal formulation of ProddINP has been checked to keep intact the anti-HIV activity of ddI. This formulation was finally compared to ddI after oral route in rat. The animal experiments evidenced the increase of ddI blood half life (3-fold) and its enhanced accumulation as prodrug form at 24 h in numerous organs and especially intestine after administration of ProddINP in comparison with free drug. Finally, the tested liposomal formulation of ProddINP seems to be a promising approach to eradicate HIV infection from intestinal sanctuaries where the virus can concentrate.
Keywords: Antiviral activity; Didanosine; In vivo experiments; Liposomes; Prodrug;

Three amphiphilic random copolymers poly(2-(dimethylaminoethyl)methacrylate-co-alkylacrylate) (where, alkyl = hexyl, octyl, dodecyl) with 16 mol% hydrophobic substitution were synthesized. Surface tension, viscosity, fluorescence probe, dynamic light scattering (DLS), as well as transmission electron microscopic (TEM) techniques were utilized to investigate self-assembly formation by the hydrophobically modified polymers (HMPs) in pH 5. Formation of hydrophobic domains through inter-polymer chain interaction of the copolymer in dilute solution was confirmed by fluorescence probe studies. Average hydrodynamic diameter of the copolymer aggregates at different polymer concentration was measured by DLS studies. The copolymer with shorter hydrophobic chain exhibits larger hydrodynamic diameter in dilute solution, which decreased with either increase of concentration or increase of hydrophobic chain length. TEM images of the dilute solutions of the copolymers with shorter as well as with longer hydrophobic chain exhibit spherical aggregates of different sizes. The antimicrobial activity of the copolymers was evaluated by measuring the minimum inhibitory concentration value against one Gram-positive bacterium Bacillus subtilis and one Gram-negative bacterium Escherichia coli. The copolymer with the octyl group as pendent hydrophobic chain was found to be more effective in killing these microorganisms. The interaction of the cationic copolymers with calf-thymus DNA was studied by fluorescence quenching method. The polymer-DNA binding was found to be purely electrostatic in nature. The hydrophobes on the polymer backbone were found to have a significant influence on the binding process. Biocompatibility studies of the copolymers in terms of cytotoxicity measurements were finally performed at different concentrations of the HMPs to evaluate their potential application in biomedical fields.
Keywords: Hydrophobically modified polymer; Transmission electron microscopy; Antimicrobial activity; DNA binding; Biocompatibility;

Posintro™-HBsAg, a modified ISCOM including HBsAg, induces strong cellular and humoral responses by Åsa Schiött; Kristina Larsson; Søren Manniche; Suzanne Kalliomäki; Annette Vinther Heydenreich; Kristian Dalsgaard; Nikolai Kirkby (312-320).
This paper shows the immune responses induced by the novel hepatitis B vaccine formulation Posintro™-HBsAg.To improve the hepatitis B vaccines on the market new adjuvant systems have to substitute aluminium. In this study the hepatitis B surface antigen (HBsAg) was incorporated into a novel adjuvant system, the Posintro™, a modification of the traditional immune stimulatory complexes (ISCOMs). This new HBsAg vaccine formulation, Posintro™-HBsAg, was compared to two commercial hepatitis B vaccines including aluminium or monophosphoryl lipid A (MPL) and the two adjuvant systems MF59 and QS21 in their efficiency to prime both cellular and humoral immune responses. The Posintro™-HBsAg induced the strongest humoral response with high titers of HBsAg specific antibody, high number of antigen specific B-cells and a strong T helper 1 (Th1) antibody profile when compared to the other adjuvant formulations. The Posintro™-HBsAg was also a strong inducer of cellular immune responses with induction of delayed type hypersensitivity (DTH) reaction and CD4+ T-cell proliferation. In addition, Posintro™-HBsAg was the only vaccine tested that also induced a strong cytotoxic T lymphocyte (CTL) response, with high levels of antigen specific CD8 T-cells secreting IFN-gamma mediating cytolytic activity. The results demonstrate that this novel experimental vaccine formulation, the Posintro™-HBsAg, is strongly immunogenic and can induce both class I and class II responses in experimental animals. This shows promise both for the protection against hepatitis B virus infection and as a potential therapeutic vaccine.
Keywords: Adjuvant; HBsAg; Hepatitis B; ISCOM; Posintro™; Vaccine;

This study establishes and compares structure–property–processing relationships on three drug delivery systems containing an anionic active pharmaceutical ingredient (API) in the following excipient carriers: (a) an inorganic anionic nanoclay, (b) pH responsive acrylic polymers, and (c) combinations thereof. The effects of the excipients on the APIs dissolution rate were studied from their release profile in simulated body fluids (SBFs) with different pH.In the API-nanoclay system, calcination of the clay followed by its reconstitution in an API solution was successfully used to intercalate the API in its amorphous state in the clay. As a result, the API showed increased apparent solubility vs. its crystalline form with its release mechanism from the clay being predominantly diffusion controlled and depending on the pH of the SBFs. In melt mixed ternary polymer systems containing the above hybrids, as a result of an additional diffusional step due to presence of nanoplatelets, the API showed a more controlled release vs. polymer systems that contained only API. By comparison to the low pH SBF, the ternary system in the pH 7.4 SBF showed a reduced diffusion contribution due to the presence of clay platelets, the latter unaffected by the high pH value. Reasonable agreement was found with predictions from literature diffusion/erosion models.It is confirmed that hot melt mixing offers opportunities to produce systems with enhanced API apparent solubility. The presence of nanoclays can also increase the API's apparent solubility and affect its release in a controlled manner.
Keywords: Controlled release; Melt mixing; Nanoclays; Aqueous solubility; Diffusion; Ion exchange;