International Journal of Pharmaceutics (v.371, #1-2)

Overcome side identification in PPOP by making orifices on both layers by Zhi-hong Zhang; Wei Li; Shu-fang Nie; Xin Tang; Bo Peng; Lei Tian; Wei-san Pan (1-7).
The original purpose of this research was to build a database for an expert system. Unexpectedly, it was found that the color-identifying device in push–pull osmotic pump (PPOP) manufacturing process could be unnecessary. Water-insoluble drug indapamide, gliclazide and dipyridamole were employed as model drugs. Bunches of conventional formulations were designed; and traditional preparation procedures were used. In vitro drug release was studied; and the similarity between the conditions of orifice only on the side of the drug layer and orifices of the same diameter on both sides was evaluated. It was found that the drug release from PPOP could be influenced by formulation and core hardness while it could hardly be influenced by orifice size. No significant difference was observed between the dissolution profiles of orifice only on the side of the drug layer and orifices of the same diameter on both sides. Mechanism of drug release was discussed. The conclusion was that the disadvantage of side identification in PPOP manufacturing process could be overcome by drilling orifices on both sides.
Keywords: Push–pull osmotic pump (PPOP); Indapamide; Gliclazide; Dipyridamole; Orifice; Color Identification;

High amylose cross-linked to different degrees with sodium trimetaphosphate by varying base strength (2% or 4%) and contact time (0.5–4 h) was evaluated as non-compacted systems for sodium diclophenac controlled release. The physical properties and the performance of these products for sodium diclophenac controlled release from non-compacted systems were related to the structures generated at each cross-linking degree. For samples at 2% until 2 h the swelling ability, G′ and η* values increased with the cross-linking degree, because the longer polymer chains became progressively more entangled and linked. This increases water uptake and holding, favoring the swelling and resulting in systems with higher viscosities. Additionally, the increase of cross-linking degree should contribute for a more elastic structure. The shorter chains with more inter-linkages formed at higher cross-linking degrees (2%4 h and 4%) make water caption and holding difficult, decreasing the swelling, viscosity and elasticity. For 2% samples, the longer drug release time exhibited for 2%4 h sample indicates that the increase of swelling and viscosity contribute for a more sustained drug release, but the mesh size of the polymeric network seems to be determinant for the attachment of drug molecules. For the 4% samples, smaller meshes size should determine less sustained release of drug.
Keywords: High amylose; Cross-linking; Swelling; Controlled release; Rheology;

Graft copolymerization of amino group-terminated poly((2-dimethylamino) ethyl methacrylate) (PDMAEMA-NH2) onto oxidized sodium alginate (OSA) was reacted without using a catalyst. The structure of the graft was investigated by Fourier transform infrared (FT-IR) spectroscopy. The OSA-g-PDMAEMA gel beads were prepared by dropping the aqueous solution of the graft copolymer into CaCl2 aqueous solution. The effects of pH and ionic strength on the swelling behaviors of the gel beads were studied. The results indicate that the gel beads have pH and ionic strength sensitivity. Bovine serum albumin (BSA) was entrapped in the beads and the in vitro drug release profiles were established in buffer solution with pH 1.8 (HCl), pH 7.4 (KH2PO4–NaOH), and 0.9% (w/v) NaCl at 37 °C. The results showed that the oral delivery of proteins can be controlled by adjusting the graft percentage (G, %), pH and ionic strength. According to this study, the OSA-g-PDMAEMA gel beads could be suitable for the oral delivery of proteins.
Keywords: Sodium alginate; Poly((2-dimethylamino) ethyl methacrylate); Oxidation; Protein delivery;

The maximum fluxes of a series of alkyloxycarbonyloxymethyl (AOCOM) ethers of acetaminophen (APAP) through hairless mouse skin from isopropyl myristate, IPM (J MMIPM) were measured. The J MMIPM, solubilities in IPM (S IPM), water (S AQ) and pH 4.0 buffer (S 4.0) and molecular weights MW were then fitted to the Roberts–Sloan (RS) equation: log  J M  =  x  +  y  log  S LIPID  + (1 −  y) log  S AQ  −  zMW. Only one of the prodrugs gave an improvement in the flux obtained by APAP itself. The general lack of improvement in flux seems to be due to the fact that there was no improvement in the S AQ values of the AOCOM derivatives compared to APAP. When the n  = 5 members of the AOCOM series were added to the n  = 66 database of J MMIPM to give n  = 71 and fitted to the RS equation where S LIPID was S IPM, the following coefficients were obtained: x  = −0.562, y  = 0.501, z  = 0.00248, r 2  = 0.923. These results demonstrate the importance of improving S AQ for prodrugs to improve their solubilities in the skin and hence the flux of the parent drug. The RS equation, which is derived directly from Fick's law, explains this dependence of flux on S AQ.
Keywords: Prodrugs; Diffusion cell experiments; Roberts–Sloan equation; Lipid solubility; Water solubility; Topical delivery;

Injectable in situ forming depot systems: PEG-DAE as novel solvent for improved PLGA storage stability by K. Schoenhammer; H. Petersen; F. Guethlein; A. Goepferich (33-39).
Injectable in situ forming depots (ISFD) that contain a peptide or a protein within a polymeric solution comprise an attractive, but challenging application system. Beyond chemical compatibility, local tolerability and acute toxicity, an important factor for an ISFD is its storage stability as a liquid. In this study, poly(d,l-lactide-co-glycolide) (PLGA) degradation in the presence of poly(ethyleneglycol) (PEG) as biocompatible solvent was investigated as a function of storage temperature and water content. The PLGA molecular weight (M w) was determined by gel permeation chromatography (GPC), and monitored by NMR during degradation.Rapid PLGA degradation of 75% at 25 °C storage temperature was shown to be the result of a transesterification using conventional PEG as solvent. A significant improvement with only 3% M w loss was obtained by capping the PEG hydroxy- with an alkyl- endgroup to have poly(ethyleneglycol) dialkylether (PEG-DAE). The formation of PEG-PLGA block co-polymers was confirmed by NMR, only for PEG300. Reaction rate constants were used to compare PLGA degradation dissolved in conventional and alkylated PEGs. The degradation kinetics in PEG-DAE were almost completely insensitive to 1% additional water in the solution. The transesterification of the hydroxy endgroups of PEG with PLGA was the major degradation mechanism, even under hydrous conditions. The use of PEG-DAE for injectable polymeric solutions, showed PLGA stability under the chosen conditions for at least 2 months. Based on the results obtained here, PEG-DAE appears to be a promising excipient for PLGA-based, parenteral ISFD.
Keywords: Poly(d,l-lactide-co-glycolide); Degradation; Stability; In situ forming depot;

Decaarginine-PEG-liposome enhanced transfection efficiency and function of arginine length and PEG by Masahiko Furuhata; Tomohiro Izumisawa; Hiroko Kawakami; Kazunori Toma; Yoshiyuki Hattori; Yoshie Maitani (40-46).
Oligoarginine-conjugated lipids ((Arg)n-PEG-lipid) (n  = 4, 6, 8, and 10: number of arginine residues) are novel gene delivery vectors. We prepared two oligoarginine-modified liposomes using (Arg)n-lipid without and with poly(ethylene glycol) (PEG) spacer ((Arg)n-L and (Arg)n-PEG-L), and investigated the effect of PEG spacer and oligoarginine length of liposomes on cellular uptake, gene transfection, and its mechanism in HeLa cells, using complexes with plasmid DNA (DNA) or oligodeoxynucleotide. Transfection efficiency increased as the number of arginine residues increased and Arg10-PEG-L/DNA complexes (lipoplexes) showed the highest gene transfection efficiency among (Arg)n- and (Arg)n-PEG-lipoplexes. Arg4- and Arg4-PEG-lipoplexes were taken up greatly into cells, but showed lower transfection efficiency than Arg10- and Arg10-PEG-lipoplexes, respectively. The different gene expression by Arg4-L to Arg10-L with or without PEG spacer may be explained by the different intracellular uptake mechanism. The main cellular uptake mechanism of Arg10-L and Arg10-PEG-L was the macropinocytosis pathway, whereas that of Arg4-L and Arg4-PEG-L was not. PEG spacer was more effective for intracellular trafficking than Arg length and surface charge of lipoplex which depends on Arg length at the almost same size of lipoplexes. The findings suggested that Arg10-PEG-L was a superior vector since Arg10 induced the macropinocytosis uptake pathway.
Keywords: Cell-penetrating peptide; Oligoarginine; Liposome; Nonviral gene delivery vector; Macropinocytosis;

A voltammetric sensor based on a molecularly imprinted polymer (MIP) brush grafted to sol–gel film on graphite electrode is reported for the selective and sensitive analysis of barbituric acid (BA) in aqueous, blood plasma, and urine samples. The modified electrode was preanodised at +1.6 V (vs. saturated calomel electrode), where encapsulated BA involved hydrophobically induced hydrogen bondings, in MIP cavities exposed at the film/solution interface, at pH 7.0. Scanning electron microscopy (SEM) was employed to characterise the surface morphology of the resultant imprinted film of MIP brush. The differential pulse, cathodic stripping voltammetry (DPCSV) technique was employed to investigate the binding performance of the sol–gel-modified imprinted polymer brush, which yielded a linear response in the range of 4.95–100.00 μg mL−1 of BA with a detection limit of 1.6 μg mL−1 (S/N  = 3).
Keywords: Barbituric acid sensor; Molecularly imprinted polymer; Sol–gel; Graphite electrode; Differential pulse; Cathodic stripping voltammetry;

Topical delivery of lipophilic drugs from o/w Pickering emulsions by Justyna Frelichowska; Marie-Alexandrine Bolzinger; Jocelyne Pelletier; Jean-Pierre Valour; Yves Chevalier (56-63).
Surfactant-free emulsions stabilized by solid particles (Pickering emulsions) have been evaluated in the terms of skin absorption of lipophilic drugs. The behavior of three formulations: a surfactant-based emulsion, a Pickering emulsion stabilized by silica particles and a solution in triglyceride oil, were compared in order to assess the effect of the surface coating of Pickering emulsions as new dosage forms for topical application. Such comparative investigation was performed in vitro on excised pig skin in Franz diffusion cells with all-trans retinol as model lipophilic drug. Surfactant-based (classical, CE) and Pickering (PE) oil-in-water emulsions containing retinol were prepared with the same chemical composition (except the stabilizing agent: surfactant or silica particles), the same droplet size and the same viscosity. No permeation through the skin sample was observed after 24 h exposure because of the high lipophilic character of retinol. Penetration of retinol was 5-fold larger for both CE and PE than for the solution in triglyceride. The distribution of retinol inside the skin layers depended significantly on the emulsions type: the classical emulsion allowed easy diffusion through the stratum corneum, so that large amounts reached the viable epidermis and dermis. Conversely, high storage of retinol inside the stratum corneum was favored by the Pickering emulsion. The retinol content in stratum corneum evaluated by skin stripping, demonstrated the increased retinol accumulation from PE. Therefore Pickering emulsions are new drug penetration vehicles with specific behavior; they are well-suited either for targeting the stratum corneum or aimed at slow release of drug from stratum corneum used as a reservoir to the deeper layers of skin.
Keywords: Skin penetration; Skin delivery; Emulsion; Solid particles;

Novel polymeric film coatings for colon targeting: How to adjust desired membrane properties by Youness Karrout; Christel Neut; Daniel Wils; Florence Siepmann; Laetitia Deremaux; Pierre Desreumaux; Jürgen Siepmann (64-70).
The major aim of this work was to optimize the properties of novel polymeric films based on blends of ethylcellulose and Nutriose (a water-soluble, branched dextrin). Such blends were recently shown to be highly promising for the site-specific delivery of drugs to the colon in patients suffering from inflammatory bowel diseases, in particular Crohn’s disease and ulcerative colitis. Importantly, and in contrast to various other colon targeting approaches, the system is adapted to the pathophysiological conditions in the disease state. However, it is yet unknown how desired membrane properties, especially water uptake and dry mass loss kinetics as well as mechanical stability can be adjusted to the specific needs of particular drug treatments. Different highly efficient and easy to apply tools were identified altering the membrane’s properties, in particular their mechanical resistance required to withstand the shear forces resulting from the motility of the upper GIT and the hydrostatic pressure built up within the devices upon contact with aqueous media. This includes the variation of the Nutriose:ethylcellulose blend ratio and initial plasticizer content. Importantly, Nutriose also exhibits significant pre-biotic activity, normalizing the microflora in the patients’ colon, which is of major clinical benefit in the case of inflammatory bowel diseases.
Keywords: Colon targeting; Coating; Mechanical properties; Controlled release; Starch derivative;

Despite the extensive research into the freeze-drying of aqueous solutions of proteins, it remains unknown whether proteins can survive the lyophilization process in a water-organic co-solvent system and how the process and additives affect the structural stability and activity of the proteins. In the present study, a conformational analysis of insulin in the absence/presence of bile salt and trehalose was carried out, before and after freeze-drying of a tert-butyl alcohol (TBA)/water co-solvent system at volume ratios of TBA to water ranging from 50/50 to 0/100. The study involved the use of ultraviolet derivative and fluorescence spectroscopy, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Also the bioactivity of insulin was evaluated in vivo using the streptozotocin (STZ)-induced diabetic mice as an animal model. Initial investigations indicate that the extent of the structural change of insulin depends significantly both on the TBA content and on the concentration of additives, such as sodium deoxycholate, prior to lyophilization. This could be accounted for by the phase behavior properties of the TBA/water co-solvent system, surface denaturation together with the selective and/or forced dispersion of insulin during phase separation. Lyophilized insulin in the presence of bile salt and trehalose retained more of its bioactivity and native-like structure in the solid state compared with that in the absence of additives at various TBA/water ratios, although in all cases there was a major and reversible rearrangement of secondary structure after rehydration, except for insulin at 50% TBA (v/v). Furthermore, both lyophilization in non-eutectic systems and less structural changes in the formulation process lead to more bioactivity.
Keywords: Conformation; Bioactivity; Insulin; Surfactant; Sugar; Lyophilization; Co-solvent; Alcohol;

The diffusion mechanism of vitamin B12 in two types of crosslinked hydrogels, poly(acrylic acid) (cPAA) and copolymers of acrylic acid and N-vinyl pyrrolidinone (cP(AA-NVP)) was studied. The PAA and P(AA-NVP) synthesized by three different degrees of crosslinking have limited water absorption capabilities ranging from 3% to 18%. In the copolymers permeability of B12 is controlled by both intramolecular and intermolecular hydrogen-bonding between the pyrrolidinone and carboxylic acid side chains. The diffusion kinetic data in two types of polymers were best described by Peppas models instead of Higuchi models. Permeation from both crosslinked PAA and P(AA-co-NVP) copolymers followed a Super Case II transport mechanism, most likely driven by macromolecular chain relaxation and swelling of hydrophilic polymers. A special FTIR spectroscopic method for drug binding study, FTIR difference spectroscopy, is used to probe the strong interactions between vitamin B12 and the side chains of the hydrogels. The FTIR differential spectra of B12 in PAA hydrogels revealed dramatic changes of the spectral marker bands of B12 after binding in the crosslinked gels, indicating significant interactions occurring in the amide and phosphate moieties of B12. Such interactions retard the diffusion of vitamin B12.
Keywords: Hydrogel; Swelling; Permeation; Controlled release; Vitamin B12;

Novel biodegradable hydrogels based on pachyman and its derivatives for drug delivery by Yan Hu; Xiaoju Zhou; Yong Lu; Chengyang Hu; Xianming Hu (89-98).
Two kinds of hydrogels were synthesized based on pachyman and its hydroxyethyl derivatives (hydroxyethyl pachyman, HEP) by the crosslinking reactions with confunctional crosslinker agent epichlorohydrin (ECH). Hydrogels with different crosslinking ratio were obtained by varying the content of the crosslinker and the polymer. The structure and morphology of hydrogels were characterized and the pH-dependent swelling of hydrogels was confirmed to be strongly influenced by the polymer properties, structure and the crosslinker contents. In the swelling assays, the hydrogels based on pachyman exhibited significant pH sensitivity, while the hydrogels based on hydroxyethyl pachyman tended to have notable swelling capability. In the drug release study, two drugs salicylic acid and bovine serum albumin (BSA) were chosen as model drugs. The results indicated that both two kinds of hydrogels showed better drug sustained release behavior for protein drug BSA than salicylic acid. In addition, evaluated by two model equations, the drug transport mechanism showed anomalous in both two kinds of hydrogels. Importantly, this study offers an entirely new window of developing hydrogels based on this natural polysaccharide, which has great potential for using as a novel sustained release carrier for protein drugs.
Keywords: Hydrogels; Pachyman; Protein drug; Sustained release;

Influence of realistic airflow rate on aerosol generation by nebulizers by Laurent Vecellio; Paul Kippax; Stephane Rouquette; Patrice Diot (99-105).
Mathematical models are available which predict aerosol deposition in the respiratory system assuming that the aerosol concentration and size are constant during inhalation. In this study, we constructed a sinusoidal breathing model to calculate the aerosol concentration produced by a nebulizer as a function of inhalation time. The laser diffraction technique (Spraytec™, Malvern Instruments Ltd., Malvern, UK) was used to validate this model as it allows the aerosol concentration and particle size to be measured in real time. Each nebulizer was attached to a special glass measurement cell and a sine-wave pump. Two standard jet nebulizers (Mistyneb® and Microneb®), two breath-enhanced jet nebulizers (Pari® LC+ and Atomisor® NL9 M) and three mesh nebulizers (Eflow®, Aeroneb® Go and Aeroneb® Pro with Idehaler®) were characterized. Results obtained were consistent in terms of curve profile between the proposed model and the laser diffraction measurements. The standard jet and mesh nebulizers produced significant variations in aerosol concentration during inhalation, whereas the breath-enhanced jet nebulizers produced a constant aerosol concentration. All of the nebulizers produced a relatively constant particle size distribution. Our findings confirm that the concentration observed during inhalation is often not constant over time. The laser diffraction method allows the concentration and size of particles for each unit volume of air inhaled to be measured and could therefore be used to predict the aerosol deposition pattern more precisely.
Keywords: Laser; Size; Concentration; Nebulizer; Aerosol;

Solubility behavior and biopharmaceutical classification of novel high-solubility ciprofloxacin and norfloxacin pharmaceutical derivatives by Susana A. Breda; Alvaro F. Jimenez-Kairuz; Ruben H. Manzo; María E. Olivera (106-113).
The hydrochlorides of the 1:3 aluminum:norfloxacin and aluminum:ciprofloxacin complexes were characterized according to the Biopharmaceutics Classification System (BCS) premises in comparison with their parent compounds. The pH–solubility profiles of the complexes were experimentally determined at 25 and 37 °C in the range of pH 1–8 and compared to that of uncomplexed norfloxacin and ciprofloxacin. Both complexes are clearly more soluble than the antibiotics themselves, even at the lowest solubility pHs. The increase in solubility was ascribed to the species controlling solubility, which were analyzed in the solid phases at equilibrium at selected pHs. Additionally, permeability was set as low, based on data reported in the scientific literature regarding oral bioavailability, intestinal and cell cultures permeabilities and also considering the influence of stoichiometric amounts of aluminum. The complexes fulfill the BCS criterion to be classified as class 3 compounds (high solubility/low permeability). Instead, the active pharmaceutical ingredients (APIs) currently used in solid dosage forms, norfloxacin and ciprofloxacin hydrochloride, proved to be BCS class 4 (low solubility/low permeability). The solubility improvement turns the complexes as potential biowaiver candidates from the scientific point of view and may be a good way for developing more dose-efficient formulations. An immediate release tablet showing very rapid dissolution was obtained. Its dissolution profile was compared to that of the commercial ciprofloxacin hydrochloride tablets allowing to dissolution of the complete dose at a critical pH such as 6.8.
Keywords: Biopharmaceutics Classification System (BCS); Dissolution rate; Solubility; Formulation; Permeability; Tableting; Complexation;

Transient drug supersaturation kinetics of beclomethasone dipropionate in rapidly drying films by Monica L. Reid; Stuart A. Jones; Marc B. Brown (114-119).
Supersaturation is an effective method to enhance the delivery of active compounds into the skin, however the long-term instability of the drug in these formulations that exceed thermodynamic unity prevents clinical use. The creation of supersaturation in situ by volatile solvent evaporation after application may overcome this. The aim of this study was to determine how altering the kinetics of transient supersaturation and recrystallisation would effect the rate of beclomethasone dipropionate (BDP) release from metered dose aerosols (MDA) that also consisted of hydrofluoroalkane 134a, ethanol (EtOH), and poly(vinyl pyrrolidone) (PVP) K90. An MDA containing 10% EtOH generated a sub-saturated concentration of BDP immediately after dose actuation and did not become supersaturated until 30 min post-actuation. Increasing the EtOH to 20% (w/w) and thus the BDP to 1.76% created supersaturation upon dose actuation but the drug recyrstallised within minutes of application. It was shown that the formulations with higher DS had accelerated rates of release despite rapid recrystallisation (444.9 ± 79.3 μg/(cm2  h) for the fastest compared to 206.5 ± 23.0 μg/(cm2  h) for the slowest). In highly volatile sprays maintaining BDP supersaturation for extended periods of time was less important than generating instantaneous, high levels of supersaturation to enhance drug release.
Keywords: Topical drug delivery; Corticosteroid; Supersaturation; Diffusion;

A novel powder sample holder for the determination of glass transition temperatures by DMA by Denny Mahlin; John Wood; Nicholas Hawkins; Jas Mahey; Paul G. Royall (120-125).
The use of a new sample holder for dynamic mechanical analysis (DMA) as a means to characterise the T g of powdered hydroxypropyl methyl cellulose (HPMC) has been investigated. A sample holder was constructed consisting of a rectangular stainless steel container and a lid engineered to fit exactly within the walls of the container when clamped within a TA instruments Q800 DMA in dual cantilever configuration. Physical mixtures of HPMC (E4M) and aluminium oxide powders were placed in the holder and subjected to oscillating strains (1 Hz, 10 Hz and 100 Hz) whilst heated at 3 °C/min. The storage and loss modulus signals showed a large reduction in the mechanical strength above 150 °C which was attributed to a glass transition. Optimal experimental parameters were determined using a design of experiment procedure and by analysing the frequency dependence of T g in Arrhenius plots. The parameters were a clamping pressure of 62 kPa, a mass ratio of 0.2 HPMC in aluminium oxide, and a loading mass of either 120 mg or 180 mg. At 1 Hz, a T g of 177 ± 1.2 °C (n  = 6) for powdered HPMC was obtained. In conclusion, the new powder holder was capable of measuring the T g of pharmaceutical powders and a simple optimization protocol was established, useful in further applications of the DMA powder holder.
Keywords: Amorphous powders; Dynamic mechanical analysis (DMA); Amorphous content; Glass transition; HPMC; Powder holder;

The apparent diffusion coefficients, D app , and the release mechanisms of salicylic acid from salicylic acid-loaded polyacrylamide hydrogels, SA-loaded PAAM, and salicylic acid-doped poly(phenylene vinylene)/polyacrylamide hydrogels, SA-doped PPV/PAAM, were investigated. In the absence of an electric field, the diffusion of SA from the SA-doped PPV/PAAM is delayed in the first 3 h due to the ionic interaction between the anionic drug (SA anion) and the PPV. Beyond this period, SA is dissolved in and can diffuse into the buffer solution through the PAAM matrix. The D app of the SA-doped PPV/PAAM is higher than that of the SA-loaded PAAM, and the former increases with increasing electric field strength due to combined mechanisms: the expansion of PPV chains inside the hydrogel; the reduction reaction under a negative potential driving the anionic SA through the PAAM matrix; and the expansion of the matrix pore. The D app of SA from the SA-loaded PAAM and the SA-doped PPV/PAAM apparently obey the scaling behavior: D app /D 0=  (drug size/pore size) m with the scaling exponent m equal to 0.50 at 0.1 V for both SA-loaded PAAM and SA-doped PPV/PAAM. Thus, the presence of the conductive polymer and the applied electric field can be combined to control the drug release rate at an optimal desired level.
Keywords: Polyacrylamide hydrogels; Salicylic acid-doped poly(phenylene vinylene); Diffusion coefficients; Electrically controlled drug release; Electro-responsive hydrogel;

Chitosan-dibasic orthophosphate hydrogel: A potential drug delivery system by Hang T. Ta; Han Han; Ian Larson; Crispin R. Dass; Dave E. Dunstan (134-141).
Injectable thermo-activated hydrogels have shown great potential in biomedical applications including use in therapeutic delivery vehicles. In addition to their biocompatibility, the feasibility of these delivery systems is significantly contributed by their ability to gel at physiological conditions and to release entrapped molecules in a sustained manner. In this study, parameters affecting the gelling behavior and the release characteristics of a neutral hydrogel system based on chitosan and an inorganic orthophosphate salt have been investigated. Monobasic and tribasic phosphate salts were not effective in inducing gelation of chitosan solution. However, in the presence of dibasic phosphate salt such as dipotassium hydrogen orthophosphate (DHO), the acidic chitosan solution was neutralized and gelling at temperature and time regulated by varying chitosan and salt concentrations in the formulation. The release rate of the entrapped macromolecules depended on chitosan concentration, DHO concentration, structural conformation and molecular weight of entrapped agents. The relationship between the morphology of the hydrogel and the release profiles are discussed. Chitosan/DHO (Chi/DHO) hydrogels were found to be cytocompatible as evaluated in an in vitro study using a human cell line. These results indicate the potential of Chi/DHO hydrogels as delivery systems for different therapeutic agents with controlled release kinetics.
Keywords: Chitosan; Orthophosphate; Hydrogel; Gelation; Delivery; Cytocompatible;

Enhanced electrostatic interaction between chitosan-modified PLGA nanoparticle and tumor by Rui Yang; Won-Sik Shim; Fu-De Cui; Gang Cheng; Xu Han; Qing-Ri Jin; Dae-Duk Kim; Suk-Jae Chung; Chang-Koo Shim (142-147).
In our previous study, lung tumor-specific targeting of paclitaxel was achieved in mice by intravenous administration of chitosan-modified paclitaxel-loaded PLGA nanoparticles (C-NPs-paclitaxel). Transient formation of aggregates in the blood stream followed by enhanced trapping in the capillaries was proposed as a mechanism of the lung-specific accumulation of paclitaxel. In the present study, the mechanism of tumor lung preferential accumulation of paclitaxel from C-NPs-paclitaxel was investigated. Zeta potential and in vitro cellular cytotoxicity (A549 cells and CT-26 cells) of C-NPs-paclitaxel, and in vitro uptake of coumarin 6 to these cells from chitosan-modified coumarin 6 containing PLGA nanoparticles (C-NPs-coumarin 6) were examined as a function of pH (6.8, 7.4 and 8.0). The zeta potential of C-NPs-paclitaxel increased as the medium pH became more acidic. In vitro uptake of coumarin 6 by A549 cells and CT-26 cells was enhanced at lower pH for C-NPs-coumarin 6. In vitro cytotoxicity experiment with C-NPs-paclitaxel demonstrated enhanced cytotoxicity as the pH became more acidic. Therefore, enhanced electrostatic interaction between chitosan-modified PLGA nanoparticles and acidic microenvironment of tumor cells appears to be an underlying mechanism of lung tumor-specific accumulation of paclitaxel from C-NPs-paclitaxel.
Keywords: PLGA nanoparticles; Chitosan; Paclitaxel; Coumarin 6; Electrostatic interaction; Extracellular tumor pH; Zeta potential; Tumor; Targeting;

Enhancement of oral absorption of curcumin by self-microemulsifying drug delivery systems by Jing Cui; Bo Yu; Yu Zhao; Weiwei Zhu; Houli Li; Hongxiang Lou; Guangxi Zhai (148-155).
Curcumin is a poorly water-soluble drug and its oral bioavailability is very low. A new self-microemulsifying drug delivery system (SMEDDS) has been successfully developed to improve the solubility and oral absorption of curcumin. Suitable compositions of SMEDDS formulation were screened via solubility studies of curcumin and compatibility tests. The formulation of curcumin-loaded SMEDDS was optimized by a simplex lattice experiment design. The optimal formulation of SMEDDS was comprised of 57.5% surfactant (emulsifier OP:Cremorphor EL = 1:1), 30.0% co-surfactant (PEG 400) and 12.5% oil (ethyl oleate). The solubility of curcumin (21 mg/g) significantly increased in SMEDDS. The average particle size of SMEDDS-containing curcumin was about 21 nm when diluted in water. No significant variations in particle size and curcumin content in SMEDDS were observed over a period of 3 months at 4 °C. The spherical shape of microemulsion droplet was observed under TEM. The dissolution study in vitro showed that more than 95% of curcumin in SMEDDS could be dissolved in pH 1.2 or pH 6.8 buffer solutions in 20 min, however, less than 2% for crude curcumin in 60 min.The in situ absorption property of curcumin-loaded SMEDDS was evaluated in intestines of rats. The results showed the absorption of curcumin in SMEDDS was via passive transfer by diffusion across the lipid membranes. The results of oral absorption experiment in mice showed that SMEDDS could significantly increase the oral absorption of curcumin compared with its suspension. Our study illustrated that the developed SMEDDS formulation held great potential as a possible alternative to traditional oral formulations of curcumin.
Keywords: Curcumin; Self-microemulsifying drug delivery system (SMEDDS); Simplex lattice experiment design; Microemulsion;

The aim of this work is to investigate the effect of betaine substitution degree of chitosan N-betainates (CsB) on cellular uptake, cytotoxicity and transfection efficiency of CsB/DNA complex nanoparticles (CsBNs) against COS-7 and MDA-MB-468 cells. The polymers with three substitution degrees (CsB12, CsB47 and CsB85) complexed with pDNA formed CsBN12s, CsBN47s and CsBN85s. The CsBNs showed less pH dependency with smaller particle size and higher zeta potential than that of chitosan/pDNA complex nanoparticles (CsNs) at neutral pH. CsBN85s showed stronger cellular uptake than that of CsBN47s or CsBN12s. CsBNs showed higher cytotoxicity than CsNs, and a trend increasing toxicity with substitution degree increasing. In COS-7 cells, the transfection efficiency increased with the substitution degree increasing, while the opposite result was observed in MDA-MB-468 cells. Chitosan modified with betaine could increase its ability to facilitate DNA uptake and its cytotoxicity, both of which showed the influence on transfection efficiency. It was able to increase cellular uptake and transfection efficiency of complex nanoparticles in COS-7 cells to increase betaine substitution of CsB, however, the higher sensitivity of MDA-MB-468 cells to CsBs led to decreased transfection efficiency due to the increased cytotoxicity with betaine substitution increasing. The predominant role of cellular uptake or toxicity in affecting transfection efficiency was different in two cell lines. These results provided an important guidepost for further development of chitosan derivatives/pDNA complexes as non-viral gene vectors.
Keywords: Chitosan N-betainates; Gene vector; DNA; Chitosan; Nanoparticles;

SPION-loaded chitosan–linoleic acid nanoparticles to target hepatocytes by Chang-Moon Lee; Hwan-Jeong Jeong; Se-Lim Kim; Eun-Mi Kim; Dong Wook Kim; Seok Tae Lim; Kyu Yoon Jang; Yong Yeon Jeong; Jae-Woon Nah; Myung-Hee Sohn (163-169).
The aim of this study was to develop a novel polymeric magnetic nanoprobe as an MRI contrast agent to target hepatocytes, as well as to evaluate the targeting ability of the nanoprobe with MRI in vivo. Superparamagnetic iron oxide nanocrystals (SPIONs) were synthesized by a thermal decomposition and seed growth method. An 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide (EDC)-mediated reaction coupled water-soluble chitosan (WSC) to linoleic acid (LA). Twelve-nanometer-sized SPIONs were incorporated into the core of self-assembled WSC–LA nanoparticles. The morphology and size distribution of the SPION-loaded WSC–LA nanoparticles (SCLNs) were determined by transmittance electron microscopy (TEM) and dynamic light scattering (DLS), respectively. The encapsulation of SPIONs in the WSC–LA nanoparticles reduced the cytotoxicity of bare iron particles and enhanced their dispersion ability in water. The clustering of SPIONs into WSC–LA nanoparticles showed ultrasensitive magnetic behavior. After in vivo intravascular SCLN injection, MRI revealed relative signal enhancement in the liver. The localization of SCLN in hepatocytes was confirmed by Prussian blue staining and TEM analysis. We have successfully developed an ultrasensitive SCLN that effectively targets hepatocytes. The SCLN can be used as a contrast agent to aid in the diagnosis of hepatic diseases.
Keywords: Hepatocytes; Iron oxide; Superparamagnetic nanoparticle; Chitosan; Linoleic acid; Self-assembled nanoparticle; MRI; Molecular imaging;

Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors by Erem Bilensoy; Can Sarisozen; Güneş Esendağlı; A. Lale Doğan; Yeşim Aktaş; Murat Şen; N. Aydın Mungan (170-176).
Cationic nanoparticles of chitosan (CS), poly-ɛ-caprolactone coated with chitosan (CS-PCL) and poly-ɛ-caprolactone coated with poly-l-lysine (PLL-PCL) were developed to encapsulate intravesical chemotherapeutic agent Mitomycin C (MMC) for longer residence time, higher local drug concentration and prevention of drug loss during bladder discharge. Nanoparticle diameters varied between 180 and 340 nm depending on polymer used for preparation and coating. Zeta potential values demonstrated positive charge expected from cationic nanoparticles. MMC encapsulation efficiency depended on hydrophilicity of polymers since MMC is water-soluble. Encapsulation was increased by 2-fold for CS-PCL and 3-fold for PLL-PCL as a consequence of hydrophilic coating. Complete drug release was obtained with only CS-PCL nanoparticles. On the other hand, CS and PLL-PCL nanoparticles did not completely liberate MMC due to strong polymer–drug interactions which were elucidated with DSC studies. As far as cellular interaction was concerned, CS-PCL was the most efficient formulation for uptake of fluorescent markers Nile Red and Rhodamine123 incorporated into nanoparticles. Especially, CS-PCL nanoparticles loaded with Rhodamine123 sharing hydrophilic properties with MMC were selectively incorporated by bladder cancer cell line, but not by normal bladder epithelial cells. CS-PCL nanoparticles seem to be promising for MMC delivery with respect to anticancer efficacy tested against MB49 bladder carcinoma cell line.
Keywords: Mitomycin C; Chitosan; Poly-ɛ-caprolactone; Poly-l-lysine; Nanoparticle; Intravesical delivery;

Nanoparticle infiltration to prepare solvent-free controlled drug delivery systems by Isabel M. Rodríguez-Cruz; Clara L. Domínguez-Delgado; Jose J. Escobar-Chávez; Gerardo Leyva-Gómez; Adriana Ganem-Quintanar; David Quintanar-Guerrero (177-181).
The purpose of this work was to propose a drug delivery system based on a biodegradable porous membrane, whose surface is covered by a nanoparticle film, thus achieving a controlled drug release rate. Furthermore, due to the fact that the assembly of the system is performed in aqueous medium, contact with organic solvents is avoided. The method is performed in two steps: (i) preparation of biodegradable porous membranes (by a solvent casting and particulate leaching technique) and biodegradable nanoparticles (by the emulsification–diffusion method), extensively eliminating the solvent in both of them; (ii) infiltration into membranes of an aqueous solution of a model drug (carbamazepine) and a nanoparticle dispersion. In both cases, poly(dl-lactic-co-glycolic acid) (PLGA 50:50) was used as a biodegradable polymer. Carbamazepine adsorbed onto biodegradable porous membranes shows an immediate release behavior (95% released in <15 min). Infiltration of different amounts of nanoparticles (50, 100, 400 and 600 mg of nanoparticles/0.625 g of membrane) into biodegradable porous membranes shows a Fickian diffusion according to Peppas model, and fits Higuchi's model. This behavior was attributed to the diffusional barrier constituted by the nanoparticle film. As expected, the carbamazepine release rate was dependent on the amount of infiltrated/adsorbed nanoparticles into biodegradable porous membrane. DSC studies show molecular dispersion of the drug throughout the membrane.
Keywords: Infiltration; Nanoparticles; Biodegradable porous membranes; Emulsification–diffusion technique; Drug delivery system;

Production and characterization of Hesperetin nanosuspensions for dermal delivery by Prabhat R. Mishra; Loaye Al Shaal; Rainer H. Müller; Cornelia M. Keck (182-189).
Nanosuspensions of Hesperetin were produced using four different stabilizers, Poloxamer 188, Inutec SP1, Tween 80 and Plantacare 2000, possessing different mechanisms of stabilisation. The nanosuspensions were characterized with regard to size (photon correlation spectroscopy (PCS), laser diffractometry (LD)) and charge (zeta potential measurements). A nanocrystal PCS size of about 300 nm was obtained after 30 homogenization cycles at 1500 bar with the stabilizers Poloxamer, Inutec and Plantacare. Tween was slightly less efficient to preserve the nanocrystal size directly after production (347 nm). The short-term stability was assessed by storage of the nanosuspensions at 4 °C, room temperature and 40 °C. As predicted from the zeta potential measurements, Inutec and Plantacare stabilized nanosuspensions were stable with no change in PCS diameter and LD diameter 99%. Slight increases in size were found for the Poloxamer and the Tween stabilized nanosuspensions, which is not considered to impair their use in dermal formulations.
Keywords: Nanosuspension; Nanocrystals; Hesperetin; Drug delivery; Poorly soluble; High-pressure homogenization;

Well-defined amphiphilic diblock copolymers with different poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) content were successfully synthesized via RAFT polymerization technology. The structure of the copolymers was confirmed using GPC, 1H NMR and FTIR. The polymers have very low critical micelles concentration (6.32 × 10−7  mol/L to 1.01 × 10−6  mol/L), which indicates their high thermodynamic stability needed for intravenous injection. Blank and paclitaxel-loaded polymeric micelles were prepared from the PMPC–b–PBMA copolymers using self-emulsion/evaporation method. TEM analysis revealed a regular spherical shape, small diameter (less than 30 nm) and narrow size distribution of the micelles. The paclitaxel-loaded polymeric micelles had high loading content (above 13%). In vitro release kinetics of paclitaxel from the micelles was also investigated. Less than 30% of the paclitaxel was released within 320 h and the increase of the length of PMPC leads to slower release rate.
Keywords: Amphiphilic diblock copolymer; Micelle; PMPC; Paclitaxel; Controlled drug release;

Development of nobiliside A loaded liposomal formulation using response surface methodology by Yang Xiong; Dan Guo; Lili Wang; Xiaoli Zheng; Yue Zhang; Jianming Chen (197-203).
To reduce the hemolysis and toxicity of nobiliside A (Nob), liposomes were used as a carrier in this study. Response surface methodology (RSM) based on central composite rotatable design (CCRD) was applied for formulation optimization. Phosphatidyl choline (PC) proportion, cholesterol (CH) proportion, and lipids/drug ratio were selected as the independent variables while the encapsulation efficiency (EE) and hemolytic rate (HR) of the liposomes as the dependent variables. The results indicated CH proportion and lipids/drug ratio were the major contributing variables for EE and PC/CH ratio was the major contributing variables for HR. The optimum formulation of Nob liposomes, in which PC proportion of 2% (w/v), CH proportion of 0.9% (w/v), and lipids/drug ratio (w/w) of 40, had higher EE (>95%) and lower HR (<1% at the concentration of 80 μg mL−1) with spherical shape and uniform sizes. The intravenous LD50 increased to 9.5 mg kg−1 compared to 4.1 mg kg−1 of Nob solution. In conclusion, the liposome was a safety and effective carrier for intravenous Nob.
Keywords: Nobiliside; Liposome; Response surface methodology; Hemolysis; Encapsulation efficiency; Acute toxicity;

Noticeboard (204).