International Journal of Pharmaceutics (v.312, #1-2)

This work examines the influence of various process parameters on α-amylase entrapped in crosslinked κ-carrageenan beads for stability improvement. A three level full factorial design was employed to investigate the effect of three process variables namely κ-carrageenan concentration, potassium chloride concentration and hardening time on % entrapment, time required for 50% (T 50) and 90% (T 90) of enzyme release and particle size.The beads were prepared by dropping the κ-carrageenan-containing α-amylase to magnetically stirred potassium chloride solution. The composite index was applied to optimize the process under study. ‘In vitro’ enzyme release profile of the beads was fitted to various release kinetics models to understand the release mechanism. Topographical characterization was carried out by SEM and entrapment was confirmed by FTIR and DSC. Stability testing according to the ICH guidelines for zone III and IV was carried out.With the use of ionotropic gelation method, a polymeric matrix prepared by 3.5% (w/v) κ-carrageenan, 0.7 M potassium chloride and hardening time of 30 min resulted in the production of beads characterized by disc shaped with collapsed center, absence of aggregates, % entrapment of 73.79, T 90 of 74.4 min, and composite index of 83.01. Moreover, shelf-life of the enzyme loaded beads was found to increase up to 3.53 years compared to 0.99 year of the conventional formulation.It can be inferred that the proposed method can be used to prepare α-amylase loaded κ-carrageenan beads for stability improvement. Also the proper selection of rate-controlling carrageenan concentration and its interactive potential for crosslinking is important and will determine the overall size and shape of beads, the duration and pattern of dissolution profiles and enzyme loading capacity.
Keywords: α-Amylase; κ-Carrageenan; Ionotropic gelation; Release kinetics; Composite index; Stability study;

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation by James Birchall; Sion Coulman; Alexander Anstey; Chris Gateley; Helen Sweetland; Amikam Gershonowitz; Lewis Neville; Galit Levin (15-23).
The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a β-galactosidase reporter plasmid DNA applied to ViaDerm™ treated skin. Skin treated with ViaDerm™ using 50 μm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm™ was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm™) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.
Keywords: Radiofrequency-microchannels; Radiofrequency ablation; Plasmid DNA; Skin; Gene therapy;

The purpose of this study was to develop a new monolithic matrix system to completely deliver glipizide, a Biopharmaceutics Classification System (BCS) Class II drug in a zero order manner over an extended time period. Two approaches were examined using drug in formulations that contain swellable hydroxypropylmethylcellulose (HPMC) or erodible polyethylene oxide (PEO). The matrices were prepared by dry blending selected ratios of polymers and ingredients using direct compression technique. Dissolution was assessed using modified USP apparatus II. Glucotrol XL push-pull osmotic pump (PPOP) was used as the reference. The interrelationship between matrix hydration, erosion and textural properties were determined and analyzed under the dissolution test conditions. Linear and reproducible release similar to that of Glucotrol XL was achieved for optimized matrices (f 2  > 50) independent of hydrodynamic conditions. The kinetics of drug delivery was directly related to the synchronization of swelling, erosion and fractional release. HPMC matrices showed a significantly greater degree of hydration and swelling and stronger texture property relative to PEO matrices. Results indicate that in the case of low dose/low soluble drug, total drug release in a zero order manner heavily depends on the synchronization of erosion and swelling fronts during the entire dissolution study.
Keywords: Glipizide; Controlled release; Textural behavior; Swelling; Matrix;

In order to develop and optimize a pulsed electric field (PEF) extraction method and evaluate it against conventional extraction methods for the extraction of polysaccharide from Rana temporaria chensinensis David, we have investigated various experimental conditions, respectively, such as electric field intensity (10–30 kV/cm), pulse duration (2–6 μs) and concentration of distilling solvent (0–1% KOH, v/v), and then optimized them by an orthogonal experiment. The result showed that the largest extraction ratio is 55.59% by PEF on the conditions of 0.5% KOH, 20 kV/cm electric field intensity and 6 μs pulse duration. Comparing it with the conventional extraction methods, such as alkali extraction method, enzyme extraction method and compound extraction method, the extraction ratio and polysaccharide content of PEF method are higher than the other three methods. The PEF extraction ratio for 6 μs is 1.77 times the compound extraction method for 6 h, the total sugar contents are more than 26.34% of that for the compound extraction method and the impurity of extraction material is less. So the PEF method is a novel and promising method to extract polysaccharide of R. temporaria chensinensis David.
Keywords: Pulsed electric field (PEF); Polysaccharide; Rana temporaria chensinensis David; Extraction;

The influence of variation of gastric pH on the gelation and release characteristics of in situ gelling pectin formulations by Kunihiko Itoh; Wataru Kubo; Mariko Fujiwara; Tomohiro Hirayama; Shozo Miyazaki; Masatake Dairaku; Mitsuo Togashi; Ryozo Mikami; David Attwood (37-42).
The aim of this study was to examine the influence of variation of gastric pH over the range 1–3 on the gelation of liquid formulations of pectin and on the in vitro and in vivo release of paracetamol and ambroxol from the resultant gels. The formulations were dilute solutions of pectin containing complexed calcium ions that form gels when these ions are released in the acidic environment of the stomach. Gels suitable as vehicles for sustained delivery of these drugs were formed in vitro at pH < 3 from pectin solutions of concentrations 1.0–2.0% (w/v). Very weak gels were formed at pH 3.0 resulting in poor sustained release characteristics compared with those at pH 1.2; no significant in vitro gelation was observed at pH 3.5. The bioavailabilities of paracetamol and ambroxol from gels formed in the stomach following oral administration of the liquid formulations were investigated using gastric-acidity controlled rabbits. Visual observations showed in situ gelation of 1.5% (w/v) pectin formulations under conditions of both high (pH 1.0–1.6) and low gastric acidity (pH 3.3–3.6). The bioavailabilities of these drugs were not significantly different when released from gels formed at the two pH limits suggesting that normal variations of gastric acidity in the fasting state will have no effect on the bioavailability of these drugs when delivered using this vehicle.
Keywords: In situ gelation; Oral drug delivery; Sustained release; Pectin gels; Gastric acidity; Paracetamol; Ambroxol;

Biotin uptake and cellular translocation in human derived retinoblastoma cell line (Y-79): A role of hSMVT system by Viral Kansara; Shuanghui Luo; Budda Balasubrahmanyam; Dhananjay Pal; Ashim K. Mitra (43-52).
The objective of this research was to investigate the presence of a specialized carrier-mediated system for biotin and delineate uptake mechanism and intracellular trafficking of biotin in the human derived retinoblastoma cell line (Y-79). Human derived retinoblastoma cell line, Y-79, was used for uptake studies. Uptake of [3H]Biotin was determined at various concentrations, pH, temperatures, in the absence of sodium and in the presence of other vitamins and metabolic inhibitors to delineate the mechanism of uptake. Uptake was determined in the presence of various intracellular regulatory pathways (protein kinase A & C, protein tyrosine kinase and calcium-calmodulin) modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of human sodium-dependent multivitamin transporter (hSMVT). Uptake of [3H]Biotin in Y-79 cells were found to be saturable at micromolar concentration range, with apparent K m of 8.53 μM and V max of 14.12 pmol/min/mg protein, but linear at nanomolar concentration range. Uptake was sodium, pH, temperature and energy-dependent, but chloride independent; inhibited by the structural analogue desthiobiotin, pantothenic acid and lipoic acid at milimolar concentrations and not at nanomolar concentrations. Uptake of [3H]Biotin was trans-stimulated by the intracellular biotin. Ca2+/calmodulin pathways appeared to play important roles in the regulation of riboflavin uptake in Y-79 cells via significant reduction in V max (66%) and K m (28%) of the uptake process. A human sodium-dependant multivitamin transporter, hSMVT, was identified by RT-PCR in Y-79. These studies demonstrated for the first time the existence of a human sodium dependant multivitamin transporter (hSMVT), a specialized carrier-mediated system for biotin uptake, in human derived retinoblastoma cells.
Keywords: Biotin; Human derived retinoblastoma cell line (Y-79); Uptake mechanism; Cellular translocation; Human sodium-dependant multivitamin transporter (hSMVT);

Effect of preparation technique on the properties of liposomes encapsulating ketoprofen–cyclodextrin complexes aimed for transdermal delivery by Francesca Maestrelli; Maria Luisa González-Rodríguez; Antonio Maria Rabasco; Paola Mura (53-60).
The combined approach of cyclodextrin complexation and entrapment in liposomes was investigated in order to develop an effective topical formulation of ketoprofen. Equimolar complex of drug and hydroxypropyl-β-cyclodextrin (HPβCyd) was added at different concentrations to the aqueous phase of liposomes consisting of phosphatidylcholine and cholesterol (60%/40%, w/w). Liposomes were prepared with different techniques, such as thin layer evaporation, freezing and thawing, extrusion through microporous membrane, and reverse phase evaporation method, obtaining, respectively, multi-lamellar vesicles (MLV), frozen and thawed MLV (FATMLV), small uni-lamellar vesicles (SUV) and large uni-lamellar vesicles (LUV). Size and morphology of the different types of liposomes were investigated by light scattering analysis, transmission electron microscopy, and confocal laser scanning microscopy, whereas drug entrapment efficiency was determined by dialysis experiments. Cyclodextrin complexation improved drug solubilization and allowed a strong improvement of its entrapment into the aqueous liposomal phase. Liposome preparation method and operating conditions clearly affected both liposome size and drug loading capacity. Encapsulation efficiency increased with increasing the complex concentration up to 10 mM, and was in the order MLV > LUV > SUV. An opposite behaviour was observed for FATMLV, probably due to the freezing phase required by such a preparation method, which reduced the complex solubility. Moreover, it was not possible to use higher complex concentrations, due to the destabilizing effect of cyclodextrins toward the liposomal membrane. Permeability studies of drug–HPβCyd complexes, directly in solution or incorporated in liposomes, performed across artificial membranes simulating the skin behaviour, highlighted, as expected, a prolonged release effect of liposomal formulations. Furthermore, the drug permeation rate depended on the vesicle characteristics and varied in the order: SUV > MLV = FATMLV > LUV. Therefore, the most suitable liposome preparation method can be suitably selected on the basis of drug encapsulation efficiency and/or desired drug release rate.
Keywords: Ketoprofen; Cyclodextrin; Liposome; Permeation studies;

HyperDSC studies of amorphous polyvinylpyrrolidone in a model wet granulation system by Graham Buckton; Akintayo A. Adeniyi; Mark Saunders; Ameet Ambarkhane (61-65).
Measurements of the properties of amorphous materials are very important to help in the understanding of how materials behave during manufacture, storage and use of medicines. However, there are few methods that are suited to the study of amorphous materials, especially if in multi-component systems or model formulations. The goal here was to explore the potential for the use of HyperDSC to study a model granulation system. It was found that the sensitivity of HyperDSC was such that the glass transition (T g) of polyvinylpyrrolidone (PVP) could be detected in granules made with realistic levels of this binder. The measured T g in the granules, even after drying, was very different to that of PVP alone and to PVP in physical mixtures with lactose. It is argued that the granulation process has resulted in the dissolution of some lactose and that the amorphous binder holding the granules together is in fact a solid dispersion of PVP and lactose. Based on the standard Gordon–Taylor equation it was estimated that the solid dispersion contained 50% of PVP and lactose. Given that solid dispersions have a tendency to crystallise on storage, it could be expected that changes in the binder properties will occur with time after granulation. We believe that this is the first measurement of in situ properties of a binder in this way and opens the possibility of studies on formulated systems.
Keywords: Amorphous; Granulation; Binder; PVP; Lactose; HyperDSC (or fast scan DSC);

Acrylic/cyclodextrin hydrogels with enhanced drug loading and sustained release capability by Ulf Siemoneit; Christoph Schmitt; Carmen Alvarez-Lorenzo; Asteria Luzardo; Francisco Otero-Espinar; Angel Concheiro; José Blanco-Méndez (66-74).
The influence of the proportion of acrylamidomethyl-γ-cyclodextrin (γ-CD-NMA) on loading and release of the hydrophobic triamcinolone acetonide (TA) and the hydrophilic propranolol (PR) by acrylic acid hydrogels was evaluated. γ-CD-NMA was synthesized by condensation of γ-cyclodextrin (γ-CD) with N-(hydroxymethyl) acrylamide. Hydrogels were prepared with γ-CD-NMA and sodium acrylate (3 M or 4 M), using N,N′-methylen(bisacrylamide) (BIS) as cross-linker, by free radical polymerization into glass moulds of 2 mm wide and were cut as discs (10 mm diameter). γ-CD-NMA did not modify the pH-dependent swelling of the hydrogels, but significantly increased the swelling degree in the 40:60 ethanol:water, medium in which TA can be dissolved. Hydrogels prepared with γ-CD-NMA above 5% (w/w of total monomers) showed a remarkably higher capacity to load TA, e.g., 33 mg/g dry hydrogel versus 0.6 mg/g dry hydrogel without γ-CD-NMA. This is explained by the formation of 1:1 inclusion complexes of TA with γ-CD mers that overcomes the lack of interactions with the acrylic groups of the network. The release of TA in water, 0.1 N HCl, or pH 6.8 phosphate buffer was sustained for at least 24 h, whatever the pH and the composition of the medium used. In contrast, loading of PR from the water solutions was greater for hydrogels prepared with 3 M acrylate than with 4 M acrylate, irrespective to their content in γ-CD-NMA, and in less than 2 h ca. 80% PR was released. The lower affinity of PR for the γ-CD cavities, compared to the strong intensity of the electrostatic interactions with the acrylic acid groups, explains why the incorporation of γ-CD-NMA did not increased the loading and control release capacity of the hydrogels of this hydrophilic drug. In summary, the copolymerisation of CD with acrylic monomers can provide highly hydrophilic pH-sensitive networks which load large amounts of hydrophobic drugs and release them in a sustained way.
Keywords: Acrylic hydrogels; γ-Cyclodextrin; Inclusion complexes; Triamcinolone acetonide; Propranolol; Controlled release;

Betamethasone-in-cyclodextrin-in-liposome: The effect of cyclodextrins on encapsulation efficiency and release kinetics by Géraldine Piel; Marie Piette; Valery Barillaro; Delphine Castagne; Brigitte Evrard; Luc Delattre (75-82).
Lipophilic drugs have limited solubility in phospholipid systems, hence maximum entrapment levels in liposomes are known to be low. “Drugs-in-cyclodextrin-in-liposome” systems were previously proposed to overcome this drawback but studies were limited to βCD and HPβCD. In some cases, other cyclodextrins may be more interesting than βCD or HPβCD, such as methylated cyclodextrins. However, these cyclodextrins are known to extract lipid components from the lipid membrane, which may destabilize liposomes.We tested the influence of several cyclodextrins (βCD, γCD, Dimeb, Trimeb, Crysmeb, Rameb, HPβCD and HPγCD) on the aqueous solubility of betamethasone by phase solubility diagrams and on the encapsulation efficiency in liposomes. The release kinetics of betamethasone was studied using Franz diffusion cells. We showed that release kinetics are directly correlated with encapsulation efficiency, which is closely related to betamethasone concentration in cyclodextrin complex solution. No liposome destruction was observed, even with the testing of methylated cyclodextrins at the highest concentration (40 mM). This can be explained by the fact that these cyclodextrins have a higher affinity for betamethasone than for cholesterol. This was proved by the comparison of phase solubility diagrams of both betamethasone and cholesterol.
Keywords: Liposome; Cyclodextrin; Cholesterol; Betamethasone; Release kinetics;

Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin by Yasuyuki Sadzuka; Ikumi Sugiyama; Tomoko Tsuruda; Takashi Sonobe (83-89).
Liposomes are recognized as one of the useful drug carriers, but have many problems to overcome before their clinical application. Liposomes, bonding peculiarly with serum protein (opsonization), are taken up by reticuloendothelial system (RES) cells in the liver and spleen. It is known that polyethyleneglycol (PEG) modification of the liposome surface induces the formation of a fixed aqueous layer around the liposomes due to the interaction between the PEG-polymer and water molecule, and thus prevents the attraction of opsonins. Namely, PEG-modified liposomes are able to escape trapping by the RES cells, and have a prolonged circulation time. In this study, the effects of different anchors with the same PEG molecular weight on the cell uptake and cytotoxicity of mixed PEG-modified liposomal doxorubicin (DOX) were examined.The fixed aqueous layer thickness (FALT) of liposomes covered with mixtures of PEG-molecules which differ in their chain length were increased, compared to that of the single PEG2000-modified liposome. Mixed PEG-modification of liposomes with different anchors (PEG2000-(1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (DSG): cholesterol (CHO) = 1:1)-modified liposome) led to an increase in the FALT, compared to that of each single PEG-modification. The uptake of DOX into Ehrlich ascites carcinoma cells by the liposomes covered with PEG-CHO was higher than the other liposomes. Thus, liposomes covered with PEG-DSG and PEG-CHO have an enhanced cytotoxicity.In conclusion, it was confirmed that mix-modified liposomes using PEG-lipid with different anchors were superior.
Keywords: Liposome; Polyethyleneglycol; Doxorubicin; Fixed aqueous layer thickness (FALT);

The objective was to use normal flora to deliver protein/peptide drugs orally. A probiotic bacterium, Lactococcus lactis subsp. lactis (L. lactis) transformed with Plasmid ss80, which made it able to synthesize and secrete β-lactamase, a 29 kDa protein, was used as the delivery system for β-lactamase. Oral absorption of β-lactamase in rats when delivered by this L. lactis system was investigated. The oral bioavailability of β-lactamase delivered by 3 × 107 of the L. lactis was equivalent to 209 mU of i.v. dose, and the estimated relative bioavailability was 16.7%. When delivered by β-lactamase free solution form, the relative oral bioavailability was 4.7%, which increased to 6.0% when co-administered with 3 × 107 of the untransformed L. lactis. The results demonstrated that the L. lactis significantly increased the β-lactamase oral bioavailability by 2–3-folds (p  < 0.01), the mean residence time (MRT) by 3–4 times (p  < 0.01), and the mean absorption time (MAT) by 6–14 times (p  < 0.01), as compared to the free solution form with/without the untransformed L. lactis. In conclusion, the L. lactis is more efficient in delivering β-lactamase orally compared with the free solution form. It also provides a sustained delivery mechanism for β-lactamase. Gene-transformed normal flora may be used as an efficient and sustained delivery system for protein drugs through oral route.
Keywords: Lactococcus lactis; β-Lactamase; Normal flora; Protein oral administration; Pharmacokinetics; Bioavailability; Sustained delivery;

Interactions of α-tocopherol with biomembrane models: Binding to dry lecithin reversed micelles by D. Bongiorno; L. Ceraulo; M. Ferrugia; F. Filizzola; A. Longo; A. Mele; A. Ruggirello; V. Turco Liveri (96-104).
The state of α-tocopherol (Vitamin E) in solutions of dry lecithin reversed micelles dispersed in an apolar medium has been investigated as a function of the Vitamin E to surfactant molar ratio (R VE) at fixed surfactant concentration by FT-IR, 1H NMR and SAXS with the aim to emphasize the role played by anisotropic intermolecular interactions and confinement effects as driving forces of its partitioning between apolar bulk solvent and polar nanodomains and of mutual Vitamin E/reversed micelle effects. It has been found that its binding strength to reversed micelles, triggered by steric and orientational constrains, is mainly regulated by specific interactions between the hydrophilic groups both of Vitamin E and surfactant. Moreover, the R VE dependence of the Vitamin E distribution constant and of the micellar size suggest that the inclusion of increasing amounts of Vitamin E in reversed micelles involves substantial changes in the structural and dynamical properties of the micellar aggregates.The occurrence of mutual effects and the partitioning of Vitamin E between hydrophilic/hydrophobic interfaces and apolar domains allow to infer some important biological implications concerning the capacity of Vitamin E to scavenge free radicals arising from hydrophilic and/or hydrophobic domains, possible variations of its local reactivity respect to that observed in bulk as well as its significant influence on the stability of biomembranes.
Keywords: α-Tocopherol; Vitamin E; Lecithin; Reversed micelles; Intermolecular interactions; Solubilization;

Gadolinium neutron capture therapy (Gd-NCT) is a promising cancer therapy modality. One of the key factors for a successful Gd-NCT is to deliver and maintain a sufficient amount of Gd in tumor tissues during neutron irradiation. We proposed to prepare a Gd delivery system by complexing a Gd-containing compound, diethylenetriaminepentaacetic acid (Gd-DTPA), with a polycationic peptide, poly-l-lysine (pLL), and then encapsulate the complexed Gd-DTPA into PEGylated liposomes. Complexation of Gd-DTPA with pLL not only enhanced the encapsulation efficiency of Gd-DTPA in liposomes, but also significantly limited the release of Gd-DTPA from the liposomes. A Gd-DTPA-encapsulated liposome formulation that contained 6.8 ± 0.3 mg/mL of pure encapsulated Gd was prepared. The blood half-life of the Gd encapsulated into the liposome formulation was estimated to be about 24 h in healthy tumor-free mice. About 12 h after the Gd-encapsulated liposomes were intravenously injected into mice with pre-established model tumors, the Gd content in the tumors reached an average of 159 μg/g of wet tumor tissue. This Gd-DTPA encapsulated liposome may be used to deliver Gd into solid tumors for NCT and tumor imaging.
Keywords: Complexation; Release; Biodistribution; Tumor uptake;

This study describes the preparation of mucoadhesive alginate/chitosan microparticles containing prednisolone intended for colon-specific delivery. Two methods have been used for the preparation of the particles: the one-step method is the method in which prednisolone was dispersed within sodium alginate solution and this dispersion was then dropped in a solution containing both calcium chloride and chitosan. The two-step method consisted also of the dispersion of prednisolone in alginate solution and then dropping this dispersion into a solution containing calcium chloride, the particles were then transferred to a chitosan solution. The concentration of sodium alginate solution at 2% (w/v), various concentrations of calcium chloride solution (0.5–1.0%, w/v), chitosan solutions (0.5, 1.0 and 1.5%, w/v) and prednisolone drug load (2, 5, 10 and 15%, w/v) have been used. The results for both preparation methods show that the particle size and drug content were mainly depending on the amount of the drug concentration and not the amount of chitosan and calcium chloride. The in vitro mucoadhesive tests for particles prepared from both methods were carried out using the freshly excised gut of pigs. The particles prepared by the one-step method exhibited excellent mucoadhesive properties after 1 h test. Increased chitosan concentrations from 0, 0.5, 1.0 to 1.5% (w/v) resulted in 43, 55, 82 and 88% of the particle remaining attached on the gut surface after 1 h, respectively. However, the particles prepared by the two-step method showed significant less mucoadhesion under the same experimental conditions. At chitosan concentrations of 0, 0.5, 1.0 and 1.5% (w/v) the amount of particles remaining attached to the mucosal surface of the pig gut after 1 h was 43, 3, 11 and 11%, respectively. The prednisolone release at a pH of 6.8 after 4 h was between 63 and 79% for the particles prepared by the one-step method and between 57 and 88% for the particles prepared by the two-step method with a prednisolone drug load of 5 and 10% (w/v), respectively. The results show that depending on the preparation method these chitosan coated alginate particles show different mucoadhesiveness whereas their other properties are not statistically significant different.
Keywords: Alginate/chitosan; Prednisolone; Microparticles; Mucoadhesive;

Incorporation of a model protein into chitosan–bile salt microparticles by Maria Helena Lameiro; António Lopes; Lígia O. Martins; Paula M. Alves; Eurico Melo (119-130).
In order to develop a mucosal delivery system based on biocompatible polymers, a new methodology for production of protein-loaded microparticles is developed. Chitosan anionic precipitation/coacervation is accomplished by the addition of sodium deoxycholate (DCA). These microparticles were prepared under mild conditions, where bovine serum albumin (BSA) and DCA were simply dipped into a chitosan solution under stirring. Platelet-like and/or spherical microparticles, having high protein loading efficiency and relatively low protein external exposure, are obtained. To achieve a better compaction of the microparticle matrix, block copolymers and other non-ionic surfactants are added to the formulation. BCA analysis and fluorescence quenching were used to assess the degree of protein exposure. BSA release profiles for chitosan–DCA formulations in PBS pH 7.4 and HCl 0.1N revealed, in most cases, an initial burst release, but more than 55% of the BSA remains protected inside the microparticles. It is also observed that in acidic environment (HCl 0.1N) the protein is better shielded from the environment. Some of the formulations show good properties for mucosal protein delivery, and one of those here developed is now being tested in vivo, for mucosal administration of an adenovirus vaccine.
Keywords: Microparticles; Chitosan; Protein delivery; Mucosal drug delivery; Bile salts;

Freeze-drying of liposomes using tertiary butyl alcohol/water cosolvent systems by JingXia Cui; ChunLei Li; YingJie Deng; YongLi Wang; Wei Wang (131-136).
The objective of this study was to obtain dehydrated liposomes using a novel procedure that involves freeze-drying (FD) of liposomes with TBA/water cosolvent systems. The effects of TBA on the integrity/stability of vesicles of HSPC (or SPC):Cholesterol (4:1) were investigated. TBA used as a cosolvent was detrimental to SPC liposomes, leading to increased particle size and leakage of trapped calcein. However, this was not the case for HSPC liposomes. The vesicle size and the retention of trapped calcein after lyophilization from cosolvents were similar to those after FD from water alone. Moreover, the addition of TBA can significantly enhance the sublimation of ice resulting in short FD cycles. The resulting lyophilized cake can form a loose powder upon agitation, which flowed well enough to be easily poured from the vial. Thus FD of HSPC liposomes using TBA/water cosolvent systems can provide sterile powder for specialized applications. In addition, in conjunction with a modified injection method, this FD technology might be used to produce dehydrated HSPC liposomes on a large scale.
Keywords: Liposomes; Preparation; Freeze-drying; Cosolvents; Tertiary butyl alcohol;

The interaction of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and a poorly water-soluble flavonoid, baicalein (Ba), chemically 5,6,7-trihydroxy flavone in solution and solid-state was studied. Ba/HP-β-CD solid systems were prepared by freeze-drying method. The formation of Ba/HP-β-CD complex in aqueous solution was demonstrated by UV spectroscopy, while Ba/HP-β-CD co-lyophilized product was characterized by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). Through complexation with HP-β-CD, the solubility of Ba in neutral aqueous solution was improved significantly. The phase-solubility profile was AP-type, indicating the formation of higher-order complexes or complex aggregates. Ba/HP-β-CD solid powders were amorphous and show a significantly improved dissolution rate in comparison with free Ba. Comparison of the pharmacokinetics between Ba/HP-β-CD co-lyophilized product and free Ba was also performed in rats. The concentration of Ba and its mainly conjugated metabolite, 7-O-glucuronide of baicalein (BG) in rat plasma was determined by HPLC method. The in vivo results show that Ba/HP-β-CD co-lyophilized product exhibits the similar pharmacokinetics as that of free Ba after intravenous administration. Ba/HP-β-CD co-lyophilized product displays earlier t max and higher C max of BG than free Ba after oral dosing. By comparing the AUC0–∞ of BG between oral dosing, the relative bioavailability of Ba/HP-β-CD co-lyophilized product to free Ba was 165.0%, which highlighted the evidence of significantly improved bioavailability of formulation of Ba with HP-β-CD.
Keywords: Baicalein; 2-Hydroxypropyl-β-cyclodextrin; Solubilization; Co-lyophilized product; Pharmacokinetics;

Effects of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles by Antoine Richard; Julie Delvaux; Line Bourel-Bonnet (144-150).
Spherulites™ are new promising multilamellar vesicles that we study in a drug delivery context. The sterilization of spherulites™ suspensions is a necessary step before biological tests and later, before pharmaceutical applications (for example, parenteral or local injections). Among all sterilizing operations, the filtration through 0.22 μm sterilizing-grade filters (of the type Millex® (Ø 4 mm) by Millipore) is easy and rapid, and we decided to study it as a mean to obtain sterile suspensions. The spherulites™ diameter is usually comprised between 0.2 and 0.5 μm but bigger vesicles occur and reach Ø 1 μm. The effects of such filters on vesicles’ size and lipids’ concentration were then compromised. After examination of this challenging operation, results proved that the sterilizing filtration had no effect on these two parameters whatever the formulation chosen. Then, the possible release of amaranth, an encapsulated hydrophilic dye was followed. With the formulations and in spite of a filter diameter inferior to that of the vesicles, the encapsulation yields were not significantly different before and after the filtration and no leakage could be detected. Finally, the spherulites™’ functionality after sterilizing filtration was studied under the chemical angle: vesicles containing an amphiphilic reactive anchor (CholE3ONH2) were still able to bind covalently a peptidic molecular recognition pattern. The ligation was quantified by fluorimetry as high as for non-filtrated suspensions. Thus, though spherulites™ can present a diameter superior to that of the sterilizing filters, their passage through them do not alter the physico-chemical properties of these vesicles.
Keywords: Vesicles; Encapsulation; Sterilizing filtration; Cell targeting; Drug delivery;

pH-Sensitive hydrogels based on bovine serum albumin for oral drug delivery by Francesca Iemma; U. Gianfranco Spizzirri; Francesco Puoci; Rita Muzzalupo; Sonia Trombino; Roberta Cassano; Sonia Leta; Nevio Picci (151-157).
pH-Sensitive bovine serum albumin (BSA) hydrophilic microspheres were prepared by free radical polymerization of methacrylate derivatized BSA and methacrylic acid sodium salt. Incorporation of both monomers in hydrogels was confirmed by Fourier transform infrared spectroscopy. Morphological analysis by scanning electron microscopy showed spherical shape and porous surface of all prepared samples. The microspheres showed high water affinity at neutral pH values and a narrow dimensional distribution. Network density of hydrogels depends on derivatization degree (DD%) of BSA and/or concentration of modified BSA in the polymerization feed. In order to employ the prepared samples such as pH-sensitive hydrogels, in vitro release studies, in media simulating biological fluids, were performed using diflunisal (DF) and β-propranolol (PR) as model drugs. Experimental data showed that the release profiles depend on drug–matrix interactions and diffusional limitation awardable to crosslinking degree of microparticles. β-Propranolol is quickly released at pH 1.0 and a complete release after 1 h at pH 6.8 was observed. In the case of diflunisal pH-sensitive release was observed. At pH 1.0 low amounts of drug were released (w/w < 10% after 2 h). When the pH is 6.8, the diflunisal release increased in the amount (w/w > 75% after 24 h).
Keywords: BSA; Radical polymerization; Hydrogel; Microspheres; pH-Controlled release;

The objective of the study was to determine whether lubrication of pharmaceutical powders with magnesium stearate (MgSt) results in a change in the surface energy of the powder, and to assess whether surface energy changes, if any, are correlated to lubricant concentration and blend time. The surface energies of microcrystalline cellulose (MCC), lactose, and blends of each material with MgSt, prepared at a range of concentrations and blending times were measured using inverse gas chromatography. The physical distribution of MgSt in the blend was mapped by energy dispersive spectrometry. Overall, there was a reduction in the dispersive surface energy of MCC–MgSt blends with increase in MgSt concentration, that was attributed to increasing coverage of the high-energy sites on microcrystalline cellulose by magnesium stearate. MgSt concentration had a larger effect on dispersive energy than the blending time of the powder with lubricant. X-ray maps of blend samples indicated a heterogeneous distribution of the lubricant in the blend and on the excipient particles. Measurement of the specific component of surface energy indicated that MgSt interacts with excipient powders through non-specific forces rather than acid–base interactions. No distinction among lactose–MgSt blends could be made on the basis of dispersive energy because of similar surface energies of the native materials.
Keywords: Inverse gas chromatography (IGC); Surface energy; Lubrication; Magnesium stearate;

Ionotropic cross-linked chitosan microspheres for controlled release of ampicillin by Anil K. Anal; Willem F. Stevens; Carmen Remuñán-López (166-173).
The solubility of non cross-linked chitosan in weak acid solutions restricts its utility in microspheres for drug delivery. The primary aim of this study was to produce pentasodium tripolyphosphate cross-linked chitosan microspheres with higher acid resistance for controlled release of ampicillin. The microspheres were prepared by two different microencapsulation procedures (by emulsification and by spray-drying) and characterized by their particle size, surface morphology, stability, drug entrapment efficiency and drug release. The size of the microspheres was <10 μm with a narrow size distribution. The entrapment of ampicillin in the microspheres was more than 80%. Stability of uncross-linked and cross-linked microspheres was affected by the pH of simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5). The inclusion of the enzymes pepsin and pancreatin did not affect the stability of the microspheres. The inclusion of lysozyme in phosphate buffer saline resulted in increased solubilization. The release of the drug was affected by cross-linking of microspheres with tripolyphosphate (TPP). The cross-linked microspheres were more stable in simulated gastric fluid and showed slower but sustained release of ampicillin. The antimicrobial activity of the released ampicillin was confirmed by Staphylococcus aureus bioassay.
Keywords: Ampicillin; Chitosan microspheres; Controlled release; Emulsification-solvent evaporation; Spray-drying; Tripolyphosphate;

Partitioning of parabens between phases of submicron emulsions stabilized with egg lecithin by Dorota Watrobska-Swietlikowska; Malgorzata Sznitowska (174-178).
Partitioning of methyl and propyl parabens (methyl and propyl hydroxybenzoate, paraben M and P) between the major phases in the parenteral submicron emulsions was studied. The investigated emulsions contained 10% or 20% soya-bean oil, 1.2% or 2.4% egg lecithin, 0.18% or 0.36% paraben M and 0.02% or 0.04% paraben P. The aqueous phase was obtained by ultracentrifugation, and subsequently, it was subjected to ultrafiltration, which procedure allowed to distinguish between the fractions of free preservatives (F w) and incorporated in the liposomal or micellar region (F lm). The fractions present in the oily phase and in the interface were calculated. Depending on the formulation, F w was 17–31% and 2.3–6.0% for paraben M and P, respectively. The F lm values were in a very narrow range, i.e. 3.0–6.0% for both preservatives. Substantial accumulation, i.e. 38–58% was found in the interface and the partitioning into this region was related to the oil/lecithin ratio rather than to lipophilicity of the preservative.
Keywords: Emulsion; Preservatives; Parabens; Lecithin;

Poorly water-soluble compounds are difficult to develop as drug products using conventional formulation techniques and are frequently abandoned early in discovery. In the present study, the melt emulsification method traditionally used to prepare solid lipid nanoparticles was adapted to produce drug nanosuspensions. The method was evaluated in comparison with the well known solvent diffusion process for ibuprofen as a model drug. Control of the preparation variables (stabilizers, drug content, homogenization procedure and cooling conditions) allowed formation of nanosuspensions with diameters less than 100 nm. The major advantage of the melt emulsification method over the solvent diffusion method is the avoidance of organic solvents during production, although the mean particle size is slightly greater. The combination of Tween 80 and PVP K25 as stabilizers yields nanosuspensions with the smallest average particle size. The formulation of ibuprofen as a nanosuspension, either in the form of lyophilized powder or granules, was very successful in enhancing dissolution rate, more than 65% of the drug being dissolved in the first 10 min compared to less than 15% of the micronized drug. The increase in in vitro dissolution rate may favourably affect bioavailability and improve safety for the patient by decreasing gastric irritancy.
Keywords: Nanoparticles; Ibuprofen; Melt emulsification method; Solvent diffusion method; Granules; Dissolution rate;

Light scattering measurements on microemulsions: Estimation of droplet sizes by C. Goddeeris; F. Cuppo; H. Reynaers; W.G. Bouwman; G. Van den Mooter (187-195).
Different scattering methods were used as tools to assess the size of droplets in highly diluted microemulsions. These were obtained after dilution of a self-emulsifying system made up of an oil, a surfactant and ethanol. Typical methods, often used in size and shape determination of particles, such as SAXS and USAXS suffer in the present case from a lack of electrondensity contrast. It becomes clear from our extensive use of dynamic light scattering that one should be careful in interpreting the latter data as well. Sample preparation and the subsequent handling of the samples during the experiments strongly affect reproducibility of the results. There is a need for well-defined protocols at the level of sample preparation and data handling. In the present research one uses extensively dynamic light scattering (DLS) in the back scattering mode and strengths and pitfalls, inherent to the backscattering technique, are discussed. It is crucial to be aware of droplet size distributions (monomodal/bimodal/multimodal) while reporting mean radii (R h) as this radius is only relevant in the case of well-defined monomodal distributions. Moreover, one should asses the shape of the droplets prior to data interpretation, as usual in scattering methods, by an independent method. Anyway the shape of the time correlation functions of the scattered intensity should be reported or at least inspected as they provide information on the reproducibility of the experiments hence safegarding the value of the physical meaning of the final value of droplet size (R h). Preferentially static light scattering (SLS) measurements should always support DLS experiments as the angular dependence is very sensitive to the presence of large particles.
Keywords: Self-emulsifying systems; Microemulsion; Dynamic light scattering; Droplet radius determination;

Comparative assessment of two indices of drug induced permeability changes in the perfused rat intestine by Majella E. Lane; Karl Levis; George S.A. McDonald; Owen I. Corrigan (196-199).
In the present study, two indices of acute intestinal permeability changes were investigated as measurements of drug induced intestinal damage. The first method was based on 14C-polyethylene glycol (PEG) 4000 permeability assessment and the second was based on histological evaluation of the intestine. The test compounds were ibuprofen, ketoprofen and naproxen and the alanine, glycine and phenylalanine amide derivatives of ibuprofen. Perfusion studies were carried out using a rat model. Post-perfusion, the gut was fixed and tissue changes were assessed and scored. Ibuprofen, ketoprofen and naproxen altered the barrier properties of the intestine to PEG 4000 with significantly higher scores (p  < 0.05) for gastrointestinal toxicity relative to blank buffer. For ketoprofen, PEG 4000 permeability and intestinal damage scores increased with increasing ketoprofen concentration. Ibuprofen amide derivatives did not induce significant histological damage or PEG 4000 permeability when compared with ibuprofen. A correlation coefficient of 0.91 is obtained when intestinal damage scores are plotted against PEG 4000 permeability for all compounds. Both indices are proposed as rapid and useful measures of drug induced acute intestinal damage.
Keywords: Intestine; Permeability; Histology; Non-steroidal anti-inflammatory drugs;

Noticeboard (200-202).