International Journal of Pharmaceutics (v.453, #2)

A comparison between two powder compaction parameters of plasticity: The effective medium A parameter and the Heckel 1/K parameter by Foad Mahmoodi; Ingvild Klevan; Josefina Nordström; Göran Alderborn; Göran Frenning (295-299).
A correlation between the effective medium equation's A parameter and the Heckel equation's 1/K parameter. Error bars are standard deviations.The purpose of the research was to introduce a procedure to derive a powder compression parameter (EM A) representing particle yield stress using an effective medium equation and to compare the EM A parameter with the Heckel compression parameter (1/K).16 pharmaceutical powders, including drugs and excipients, were compressed in a materials testing instrument and powder compression profiles were derived using the EM and Heckel equations. The compression profiles thus obtained could be sub-divided into regions among which one region was approximately linear and from this region, the compression parameters EM A and 1/K were calculated.A linear relationship between the EM A parameter and the 1/K parameter was obtained with a strong correlation. The slope of the plot was close to 1 (0.84) and the intercept of the plot was small in comparison to the range of parameter values obtained. The relationship between the theoretical EM A parameter and the 1/K parameter supports the interpretation of the empirical Heckel parameter as being a measure of yield stress. It is concluded that the combination of Heckel and EM equations represents a suitable procedure to derive a value of particle plasticity from powder compression data.
Keywords: Powder compression; Compression parameter; Yield stress; Effective medium equation; Heckel equation;

Profiling biopharmaceutical deciding properties of absorption of lansoprazole enteric-coated tablets using gastrointestinal simulation technology by Chunnuan Wu; Le Sun; Jin Sun; Yajun Yang; Congcong Ren; Xiaoyu Ai; He Lian; Zhonggui He (300-306).
The aim of the present study was to correlate in vitro properties of drug formulation to its in vivo performance, and to elucidate the deciding properties of oral absorption. Gastrointestinal simulation technology (GST) was used to simulate the in vivo plasma concentration–time curve and was implemented by GastroPlus™ software. Lansoprazole, a typical BCS class II drug, was chosen as a model drug. Firstly, physicochemical and pharmacokinetic parameters of lansoprazole were determined or collected from literature to construct the model. Validation of the developed model was performed by comparison of the predicted and the experimental plasma concentration data. We found that the predicted curve was in a good agreement with the experimental data. Then, parameter sensitivity analysis (PSA) was performed to find the key parameters of oral absorption. The absorption was particularly sensitive to dose, solubility and particle size for lansoprazole enteric-coated tablets. With a single dose of 30 mg and the solubility of 0.04 mg/ml, the absorption was complete. A good absorption could be achieved with lansoprazole particle radius down to about 25 μm. In summary, GST is a useful tool for profiling biopharmaceutical deciding properties of absorption of lansoprazole enteric-coated tablets and guiding the formulation optimization.
Keywords: Gastrointestinal simulation technology; GastroPlus™; Lansoprazole; Parameter sensitivity analysis; Formulation optimization;

A novel lipid-based solid dispersion for enhancing oral bioavailability of Lycopene – In vivo evaluation using a pig model by Waleed Faisal; Therese Ruane-O’Hora; Caitriona M. O’Driscoll; Brendan T. Griffin (307-314).
Lycopene is a potent anti-oxidant, which has been widely reported for its potential benefits at reducing the risks of certain types of cancer e.g. prostate cancer. The oral bioavailability of this highly lipophilic carotenoid is low and highly influenced by the extent of intestinal lymphatic uptake. The aim of this study was to develop an optimised formulation, which allows for efficient absorption following oral administration. A self-emulsifying drug delivery system (SEDDS) and solid dispersion of Lycopene were developed initially. Subsequently, a novel lipid based solid dispersion (LBSD) was designed. Processing via a solid dispersion approach was found to alter the solid state characteristics of Lycopene, as determined by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The bioavailability of Lycopene was significantly increased after oral administration of LBSD to fasted pigs, relative to the commercial product (Lycovit®). A clear distinction in terms of C max and AUC was observed between Lycovit® and LBSD. In conclusion, a novel LBSD formulation was developed to enhance the oral bioavailability of the model lipophilic compound, Lycopene, by enhancing dissolution in the gastrointestinal tract and promoting intestinal lymphatic uptake utilising digestible lipid excipients.
Keywords: Lycopene bioavailability; Lipid-based formulation; SEDDS; Solid dispersions; Gelucire 44/14; Pig model;

Crystallization of a new polymorph of acetohexamide from 2-hydroxybutyl-β-cyclodextrin solution: Form VI with a high aqueous solubility by Hibah Aldawsari; Abdulmalik Altaf; Zainy M. Banjar; Daisuke Iohara; Mami Nakabayashi; Makoto Anraku; Kaneto Uekama; Fumitoshi Hirayama (315-321).
A new polymorph of acetohexamide (Form VI) was prepared via the formation of a complex with 2-hydoxybutyl-β-cyclodextrin (HB-β-CD) in aqueous solution. An alkaline solution of acetohexamide and HB-β-CD was adjusted to pH 4.0 by titration with hydrochloric acid. The resulting opaque solution was filtered through paper and allowed to stand at 4 °C for 24 h. The resulting precipitate was isolated on a filter and analyzed for polymorph content by powder X-ray diffractometry and thermal analysis. The diffraction pattern and thermal behavior of the precipitate was different from those of previously reported acetohexamide polymorphs (Forms I, III, IV and V), indicating that a new polymorph of the drug, i.e. Form VI was produced. This new polymorph was fairly stable against conversion to a stable form even at accelerated storage conditions. Crystalline Form VI was highly soluble in water and dissolved more rapidly than the other known polymorphs. This property was reflected in the blood concentrations of the drug after oral administration to rats.
Keywords: 2-Hydroxybutyl-β-cyclodextrin; Acetohexamide; New polymorph; Dissolution rate; In vivo-absorption;

Work hardening of the drug layer (DL) blend can be avoided by low roll force compression during roller compaction; however, this can result in flow issues of the DL blend and carry over into the sweller layer during compression. The addition of a compression aid can help improve granule properties without degrading tablet performance.Swellable core technology (SCT) represents a broadly applicable oral osmotic drug delivery platform for the controlled release of drugs. SCT tablets control drug delivery by using osmosis to regulate the influx of water into the tablet's core. The tablet consists of two layers; drug layer and sweller layer, with a semi-permeable membrane coating and delivery port located in the drug layer side of the tablet.The key component of SCT formulations is polyethylene oxide (PEO), which is typically wet granulated with organic solvents to prevent rapid gel hydration observed during contact with aqueous environments. However, the use of organic solvents has their own environmental and cost considerations which make this form of processing undesirable.To overcome this issue, dry granulation can be employed. However, PEO is a very plastic material and problems may be encountered during the tableting process, when work hardening occurs upon double compression. The addition of compression aids to the drug layer will help to increase the roll force when generating ribbons – reducing fines and segregation potential – while also reducing work hardening effects which impact tablet friability. The five compression aids used in this study were microcrystalline cellulose (MCC), xylitol, di-calcium phosphate (anhydrous), lactose monohydrate and starch.The work undertaken here studies the compression properties of the drug layer blends with different levels of the five compression aids as part of the formulation. Roller compaction properties are also varied to provide granules with differing solid fractions. The results of this study indicate that addition of microcrystalline cellulose in the formulation in levels between 10% and 30% significantly improve the tablet hardness at lower tablet compression forces. Further work is required to investigate the impact on dissolution.
Keywords: Swellable core technology; Osmotic; Controlled release; Roller compaction;

Submicron-sized lipid emulsions have significant potential to deliver drugs to the posterior segment via eye drops, and their efficacy could be significantly influenced by their surface modification using chitosan (CS) and poloxamer 407 (P407).This work explored submicron-sized lipid emulsion as potential carriers for intraocular drug delivery to the posterior segment via eye drops. The effects of physicochemical properties of lipid emulsion on drug delivery were evaluated in vivo using mice. Different formulations of submicron-sized lipid emulsions were prepared using a high pressure homogenization system. Using coumairn-6 as a model drug and fluorescent marker, fluorescence could be observed in the retina after administration of the lipid emulsion. The fluorescence intensity observed after administration of medium chain triglycerides containing the same amount of coumarin-6 was much lower than that observed after administration of lipid emulsions. The inner oil property and phospholipid emulsifier did not affect the drug delivery efficiency to the retina. However, compared with unmodified emulsions, the fluorescence intensity in the retina increased by surface modification using a positive charge inducer and the functional polymers chitosan (CS) and poloxamer 407 (P407). CS-modified lipid emulsions could be electrostatically interacted with the eye surface. By its adhesive property, poloxamer 407, a surface modifier, possibly increased the lipid emulsion retention time on the eye surface. In conclusion, we suggested that surface-modified lipid emulsions could be promising vehicles of hydrophobic drug delivery to the ocular posterior segment.
Keywords: Lipid emulsion; Topical administration; Posterior segment; Retina; Surface modification;

The evaluation of encapsulated insulin intestinal absorption enhancement was investigated by in vitro methods. Insulin-loaded microparticles (INS-MP) made of whey protein (WP) and alginate (ALG) were prepared by a cold gelation technique. Effect of INS encapsulation toward trypsin and chymotrypsin degradation was performed. Permeability studies using in vitro (Caco-2 cells) experiments were conducted. INS was partially protected by encapsulation toward enzymatic degradation. Moreover INS transport experiments showed that WP and, in lesser extent, ALG were able to enhance INS absorption both as MP and as polymeric solutions by opening the tight junctions. These experiments reinforced the interest of encapsulation in WP/ALG hydrogel combination.
Keywords: Hydrogel microparticle; Alginate; Whey protein; Insulin; Intestinal absorption;

This work investigates the flocculation effect of polyethylene glycol (PEG) on typical aqueous dispersions, such as O/W emulsions and solid/liquid suspensions. Hereby, sunflower oil and flubendazole were selected as model ingredients, whereas microfluidization at variable driving air pressure was used to enable particle size distribution variations for both systems. The molecular weight of PEG varied from 2000 to 12,000 g/mol while its concentration ranged from 50 to 100 mg/ml. Statistical analysis revealed that both PEG concentration and molecular weight showed a flocculation enhancing effect. Hereby the inhibiting effect of particle size toward the formation of voluminous and easily resuspendable sediment could at least partially be overcome by selecting appropriate PEG characteristics.
Keywords: Flocculation; Polyethylene glycol; Emulsion; Flubendazole suspension; Sedimentation; Resuspendability;

Detailed batch to batch variability and process induced physical changes in BSA samples were observed using Taylor dispersion analysis which were not detectable using DLS. Taylor dispersion analysis profiles for BSA (A2058, 10 mg/ml) after heat shock treatment in PBS showing first (black) and second (grey) passes through the Actipix detector highlighting pre-peaks.Biosimilar pharmaceuticals are complex biological molecules that have similar physicochemical properties to the originator therapeutic protein. They are produced by complex multi-stage processes and are not truly equivalent. Therefore, for a biosimilar to be approved for market it is important to demonstrate that the biological product is highly similar to a reference product. This includes its primary and higher order structures and its aggregation behaviour. Representative lots of both the proposed biosimilar and the reference product are analysed to understand the lot-to-lot variability of both drug substances in the manufacturing processes. Whilst it is not easy to characterise every variation of a protein structure at present additional analytical technologies need to be utilised to ensure the safety and efficacy of any potential biosimilar product. We have explored the use of Taylor dispersion analysis (TDA) to analyse such batch to batch variations in the model protein, bovine serum albumin (BSA) and compared the results to that obtained by conventional dynamic light scattering analysis (DLS). Inter and intra batch differences were evident in all grades of BSA analysed. However, the reproducibility of the TDA measurements, enabled the stability and reversibility of BSA aggregates to be more readily monitored. This demonstrates that Taylor dispersion analysis is a very sensitive technique to study higher order protein states and aggregation. The results, here, also indicate a correlation between protein purity and the physical behaviour of the samples after heat shocking. Here, the protein with the highest quoted purity resulted in a reduced increase in the measured hydrodynamic radius after heat stressing, indicating that less unfolding/aggregation had occurred. Whilst DLS was also able to observe the presence of aggregates, its bias towards larger aggregates indicated a much larger increase in hydrodynamic radii and is less sensitive to small changes in hydrodynamic radii. TDA was also able to identify low levels of larger aggregates that were not observed by DLS. Therefore, given the potential for immunogenicity effects that may result from such aggregates it is suggested that TDA may be suitable in the evaluating detailed batch to batch variability and process induced physical changes of biopharmaceuticals and biosimilars.
Keywords: Taylor dispersion analysis; Hydrodynamic radius; Dynamic light scattering; Aggregation; Biosimilar;

Enhanced transdermal drug delivery of zaltoprofen using a novel formulation by Jong-Suep Baek; Ji-Ho Lim; Jong-Seong Kang; Sang-Chul Shin; Sang-Hun Jung; Cheong-Weon Cho (358-362).
Mean plasma concentration–time profiles of zaltoprofen after transdermal administration of zaltoprofen gel.Zaltoprofen is a non-steroidal anti-inflammatory drug (NSAID) belonging to the propionic acid class. It has strong inhibitory effects on acute and chronic inflammation. Although zaltoprofen is well tolerated orally compared to other NSAIDs, it has to be administered in three to four doses per day and was associated with ulcerogenicity, bellyache and indigestion. This makes administration of zaltoprofen unsuitable for patients with gastric ulcer and is also associated with drug interactions. Therefore, it is important to develop an alternative dosage form which is easier to administer and avoids first-pass metabolism. The transdermal route meets all the above advantages. In this study, zaltoprofen gels were prepared using carbomer with mixture solution of polyethylene glycol (PEG) 400, Tween 80 and (2-hydroxypropyl)-β-cyclodextrin (HPCD) (called as T2), subsequently oleic acid as a penetration enhancer was added. Zaltoprofen gel containing T2 and oleic acid could promote the percutaneous absorption of zaltoprofen and increase AUC by 183% compared to zaltoprofen gel without T2 and oleic acid. Also, there was a finding zaltoprofen gel containing T2 and oleic acid did not cause dermal irritations in an experimental animal.
Keywords: Zaltoprofen; Transdermal drug delivery; Cytotoxicity; Skin irritation;

Effects of pharmaceutical excipients on membrane permeability in rat small intestine by Yusuke Takizawa; Hisanao Kishimoto; Minami Nakagawa; Nasa Sakamoto; Yoshifusa Tobe; Takahito Furuya; Mikio Tomita; Masahiro Hayashi (363-370).
Pharmaceutical excipients should not disturb the effects of drug therapy. In recent years, however, it has been reported that excipients induce some changes to the tight junction (TJ) and P-glycoprotein (P-gp), which can affect drug disposition. In this study, we examined the effects of 20 common pharmaceutical excipients from different classes on mucosal membrane and the differences of such effects among regions of the small intestine. We used the in vitro sac method in rat jejunum and ileum to study the effects of excipients on the membrane permeation of 5(6)-carboxyfluorescein (5-CF). 5-CF was used as a model of water-soluble compounds. In some dosage conditions of methyl-β-cyclodextrin, the membrane permeability of 5-CF was significantly increased in the jejunum, but such change was not observed in the ileum. Similarly, in the cases of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and croscarmellose sodium, the membrane permeability of 5-CF was significantly increased in the jejunum, but no change was observed in the ileum. On the other hand, in both the jejunum and the ileum, the membrane permeation of 5-CF was decreased with 0.02% (w/v) hydroxypropyl cellulose, but significantly increased with it at 0.20% (w/v). It was shown that excipients affected the membrane permeability of water-soluble compounds via the paracellular route, and these effects on absorption differed among regions of the small intestine. Moreover, in the case of 20 excipients, not only an increase in membrane permeability but also a decrease was observed. Therefore, it was suggested that a more effective formulation could be designed by changing the combination of excipients.
Keywords: Pharmaceutical excipient; Membrane permeability; Paracellular route; Small intestine;

The interaction between human breast cancer resistance protein (BCRP) and five bisbenzylisoquinoline alkaloids by Ye Tian; Sainan Qian; Yan Jiang; Qi Shen; Jiang Zheng; Hui Zhou; Su Zeng (371-379).
BCRP is one of the key factors to drug absorption, distribution and elimination. Bisbenzylisoquinoline alkaloids are a large family of natural phytochemicals with great potential for clinical use. In this study, the interaction between BCRP and five bisbenzylisoquinoline alkaloids (neferine, isoliensinine, liensinine, dauricine and tetrandrine) were evaluated using LLC-PK1/BCRP cell model. The intracellular accumulation and bi-directional transport studies were conducted, and then molecular docking analysis was carried out employing a homology model of BCRP. Our study revealed that the permeability of these five alkaloids was not high, the P app values were all less than 6.5 × 10−6  cm/s. Liensinine and dauricine were substrates of BCRP: at lower concentration (10 μM), the net efflux ratios were 2.87 and 1.64 respectively. And their cellular accumulation was lower in LLC-PK1/BCRP cells than in LLC-PK1 cells. On the other hand, tetrandrine, isoliensinine and neferine were not substrates of BCRP. On the basis of docking studies, a direct hydrogen bond was formed between liensinine and arginine 482 which is a hot spot of BCRP for substrate specificity; and dauricine had hydrophobic interaction with BCRP. In conclusion, our study indicated that BCRP could mediate the excretion of liensinine and dauricine, thus influence their pharmacological activity and disposition.
Keywords: LLC-PK1/BCRP; Bisbenzylisoquinoline alkaloid; Cellular accumulation; Bi-directional transport; Docking analysis; Substrate;

Pharmaceutical characterisation and evaluation of cocrystals: Importance of in vitro dissolution conditions and type of coformer by Irena Tomaszewska; Shyam Karki; Jag Shur; Robert Price; Nikoletta Fotaki (380-388).
The objectives of this study were to demonstrate the importance of experimental set-up and type of coformer for the enhanced dissolution properties of cocrystals. Carbamazepine–saccharin and carbamazepine–nicotinamide cocrystals were prepared by the sonic slurry method and characterised with SEM, DSC, XRPD and particle size analysis. Solubility and dissolution testing (closed and open system) were performed in compendial media and media with a physiologically relevant amount of surfactant. Carbamazepine cocrystals (1:1 molar ratio) did not show a difference in the equilibrium solubility compared to the carbamazepine in compendial media but a substantial difference was observed in modified media. In compendial media, a faster dissolution rate was obtained only from the carbamazepine–saccharin cocrystal, whereas in modified media both cocrystals had a substantial higher dissolution compared to carbamazepine. With the selected method a clear difference in the dissolution profiles of each cocrystal is shown, driven by the characteristics of the coformer used. This study demonstrated that improved dissolution of carbamazepine from the cocrystal forms can be revealed only by appropriate selection of in vitro conditions. The characteristics of the coformer define a critical variable for dissolution of pharmaceutical cocrystals with important implications for their in vivo performance.
Keywords: Carbamazepine; Cocrystal; Coformer; Solubility; Dissolution;

Pharmaceutical powders exhibit more or less plastic and viscos-elastic behavior when they are compressed. The comparison between numerical (with and without viscous effect) and experimental results emphasizes the improvement of numerical model by using creep behavior in the numerical modeling of die compaction process.This work studies the influence of visco-elastic behavior in the finite element method (FEM) modeling of die compaction of pharmaceutical products and how such a visco-elastic behavior may improve the agreement between experimental and simulated compression curves.The modeling of the process was conducted on a pharmaceutical excipient, microcrystalline cellulose (MCC), by using Drucker–Prager cap model coupled with creep behavior in Abaqus® software. The experimental data were obtained on a compaction simulator (STYLCAM 200R). The elastic deformation of the press was determined by performing experimental tests on a calibration disk and was introduced in the simulation. Numerical optimization was performed to characterize creep parameters.The use of creep behavior in the simulations clearly improved the agreement between the numerical and experimental compression curves (stresses, thickness), mainly during the unloading part of the compaction cycle. For the first time, it was possible to reproduce numerically the fact that the minimum tablet thickness is not obtained at the maximum compression stress.This study proves that creep behavior must be taken into account when modeling the compaction of pharmaceutical products using FEM methods
Keywords: Die compression; FEM simulation; Creep behavior; Pharmaceutical products;

A novel electronic skin patch for delivery and pharmacokinetic evaluation of donepezil following transdermal iontophoresis by Sonal Saluja; Purna C. Kasha; Jyotsna Paturi; Carter Anderson; Russell Morris; Ajay K. Banga (395-399).
The nature of Alzheimer's disease limits the effectiveness of available oral treatments. The aim of this study was to assess the feasibility of transdermal iontophoretic delivery of donepezil in a hairless rat model as a potential treatment modality in Alzheimer's and to evaluate the effect of current densities on its pharmacokinetics. Donepezil loaded integrated Wearable Electronic Drug Delivery (WEDD®) patches supplied current levels of 0, 0.13, 0.26 and 0.39 mA. Plasma extracted donepezil was analyzed by HPLC. Noncompartmental analysis was used to characterize disposition of the drug. The amount delivered across hairless rat skin and areas under the curve (AUC) were found to rise in proportion to the current levels. Peak plasma levels of 0.094, 0.237 and 0.336 μg/ml were achieved at 0.13, 0.26 and 0.39 mA respectively. Time to peak plasma concentrations was after termination of current and same for all current levels. Transdermal elimination half-life was significantly increased from the true value of 3.2 h due to depot formation, prolonging complete absorption of the drug. Donepezil was successfully delivered iontophoretically at levels sufficient to produce pharmacodymanic effect. Pharmacokinetic analysis demonstrated linear kinetics at the current levels used and flip flop kinetics following iontophoretic administration.
Keywords: Transdermal; Iontophoresis; Electronic patches; Alzheimers; Donepezil;

The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity.
Keywords: Anti-tumor immunity; W/O/W multiple emulsions; gp100 encoding plasmid DNA; Melanoma; Vaccine;

Optimizing the taste-masked formulation of acetaminophen using sodium caseinate and lecithin by experimental design by Thanh Huong Hoang Thi; Mohamed Lemdani; Marie-Pierre Flament (408-415).
In a previous study of ours, the association of sodium caseinate and lecithin was demonstrated to be promising for masking the bitterness of acetaminophen via drug encapsulation. The encapsulating mechanisms were suggested to be based on the segregation of multicomponent droplets occurring during spray-drying. The spray-dried particles delayed the drug release within the mouth during the early time upon administration and hence masked the bitterness. Indeed, taste-masking is achieved if, within the frame of 1–2 min, drug substance is either not released or the released amount is below the human threshold for identifying its bad taste. The aim of this work was (i) to evaluate the effect of various processing and formulation parameters on the taste-masking efficiency and (ii) to determine the optimal formulation for optimal taste-masking effect. Four investigated input variables included inlet temperature (X 1), spray flow (X 2), sodium caseinate amount (X 3) and lecithin amount (X 4). The percentage of drug release amount during the first 2 min was considered as the response variable (Y). A 24-full factorial design was applied and allowed screening for the most influential variables i.e. sodium caseinate amount and lecithin amount. Optimizing these two variables was therefore conducted by a simplex approach. The SEM and DSC results of spray-dried powder prepared under optimal conditions showed that drug seemed to be well encapsulated. The drug release during the first 2 min significantly decreased, 7-fold less than the unmasked drug particles. Therefore, the optimal formulation that performed the best taste-masking effect was successfully achieved.
Keywords: Pediatric formulation; Taste-masking; Spray-drying; Component segregation; Factorial design; Simplex optimization;

Roll compaction of mannitol: Compactability study of crystalline and spray-dried grades by Carl Moritz Wagner; Miriam Pein; Jörg Breitkreutz (416-422).
Purpose of this project was to investigate the roll compaction behavior of various mannitol grades. Therefore, five spray-dried grades as well as unprocessed β-d-mannitol were roll compacted with different compaction forces. The resulting granules were characterized with regard to their particle size distribution, flow properties, and BET surface area and compressed to tablets. Granules of unprocessed mannitol, even when applying high compaction forces during dry granulation, were characterized by a high amount of fines (about 21%), a small surface area (0.83 m2/g), and solely fair flowability (ff c  = 7.2). Tablets revealed either high friability or insufficient disintegration behavior. However, the use of spray-dried mannitol led to better results. Granules showed improved flow properties and a reduced amount of fines. Robust tablets with low friability were produced. Within the various spray-dried grades huge differences concerning the compactability were observed. Large BET surface areas of the granules resulted in advanced tensile strengths of the tablets, but acceptable disintegration behavior was maintained. These findings are relevant for the development of mannitol based drug formulations, in particular (oro)dispersible tablets containing a low dose or poor flowing active pharmaceutical ingredient, where direct compression is inappropriate and a granulation process prior to tableting is mandatory.
Keywords: Roll compaction; Dry granulation; Mannitol; Compactability; Work-hardening;

Despite of advancements in dosage form design and use of multifunctional excipients, improvement in dissolution characteristics of molecules like Telmisartan (TEL) having exceedingly pH dependent and poor solubility profile is still challenging. The present research work explores an innovative particle engineering approach which synergistically coalesce two principally different solubility enhancement strategies namely ternary β-cyclodextrin complexation and top-down nanonization in a unit process. The research was aimed to improve solubility and reduce in vivo variability in pharmacokinetic parameters of TEL irrespective to physiological pH conditions. Ternary β-cyclodextrin nanocomposites of TEL were prepared with high pressure homogenization using meglumine as ternary component. TEL nanocomposites were thoroughly characterized for particle size, surface topology, surface charge, inclusion complexation, crystalinity, dissolution and in vivo pharmacokinetic performance in male wistar rats at fed and fasted state. TEL nanocomposites exhibited average particle size of 698 ± 23 nm. Remarkable improvement in in vitro dissolution characteristics in multimedia and biorelevant media was observed in comparison with plain drug and marketed formulation. Results of in vivo pharmacokinetic studies revealed that, nanocomposites effectively bypass variation in pharmacokinetic parameters at fed and fasted states with 346%, 315%, 301% and 321% increase in relative bioavailability compared to marketed formulation and pure TEL in fed and fasted conditions respectively.
Keywords: Poorly soluble drugs; Cyclodextrins; Complexation; Amorphous; Nanocomposites; Nanotechnology; Pharmacokinetics;

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens by César Burgos-Díaz; Raquel Martín-Venegas; Verónica Martínez; Carolina Emilia Storniolo; José Antonio Teruel; Francisco José Aranda; Antonio Ortiz; Ángeles Manresa; Ruth Ferrer; Ana M. Marqués (433-440).
The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.
Keywords: Biosurfactants; Haemolysis; Apoptosis; Caco-2 cells; 3T3 fibroblasts; HaCaT keratinocytes;

Design and evaluation of biodegradable enteric microcapsules of amifostine for oral delivery by Gula A; Lei Ren; Zhe Zhou; Dandan Lu; Shengqi Wang (441-447).
Amifostine is the first FDA approved cytoprotective and chemoprotective agent in the treatment of cancer. However, it is not used widely because of its ineffectiveness when administered orally. The objective of this study was to prepare and evaluate the radioprotective efficacy of orally active amifostine enteric microcapsules (amifostine mc). The microcapsules were prepared by spray drying technique using Eudragit L100-55, and the yield was more than 80%. The particle size and surface morphology were determined by particle analyzer and scanning electron microscopy. Thermal characterization and infrared spectroscopy were evaluated as well. In vitro release assay found that more than 60% amifostine was released during the first 4 h and the cumulative release ratio was up to approximately 90% in 24 h at 37 °C. The radioprotective efficacy was determined by 30-day survival study in mice acutely exposed to 6 Gy γ-ray irradiation. The results showed that all dose groups of amifostine microcapsules could significantly improve survival animal numbers and time. Furthermore, tissue distribution studies indicated the concentrations of the active metabolite WR-1065 in mice tissues of microcapsule group were higher than that of oral amifostine group at 180 min (p  < 0.01). These results demonstrated that oral administration of amifostine microcapsules provided effective radioprotection compared to the bulk drug.
Keywords: Amifostine; Enteric microcapsule; Radioprotection; Oral delivery; Eudragit;

Comparative static curing versus dynamic curing on tablet coating structures by Claire Gendre; Muriel Genty; Barbara Fayard; Ali Tfayli; Mathieu Boiret; Olivier Lecoq; Michel Baron; Pierre Chaminade; Jean Manuel Péan (448-453).
Curing is generally required to stabilize film coating from aqueous polymer dispersion. This post-coating drying step is traditionally carried out in static conditions, requiring the transfer of solid dosage forms to an oven. But, curing operation performed directly inside the coating equipment stands for an attractive industrial application. Recently, the use of various advanced physico-chemical characterization techniques i.e., X-ray micro-computed tomography, vibrational spectroscopies (near infrared and Raman) and X-ray microdiffraction, allowed new insights into the film-coating structures of dynamically cured tablets. Dynamic curing end-point was efficiently determined after 4 h. The aim of the present work was to elucidate the influence of curing conditions on film-coating structures. Results demonstrated that 24 h of static curing and 4 h of dynamic curing, both performed at 60 °C and ambient relative humidity, led to similar coating layers in terms of drug release properties, porosity, water content, structural rearrangement of polymer chains and crystalline distribution. Furthermore, X-ray microdiffraction measurements pointed out different crystalline coating compositions depending on sample storage time. An aging mechanism might have occur during storage, resulting in the crystallization and the upward migration of cetyl alcohol, coupled to the downward migration of crystalline sodium lauryl sulfate within the coating layer. Interestingly, this new study clearly provided further knowledge into film-coating structures after a curing step and confirmed that curing operation could be performed in dynamic conditions.
Keywords: Curing; Film-coating structures; Aqueous ethylcellulose dispersion; Near infrared spectroscopy; X-ray micro-computed tomography; X-ray microdiffraction;

Evaluation of matrix type mucoadhesive tablets containing indomethacin for buccal application by Yuri Ikeuchi-Takahashi; Masanaho Sasatsu; Hiraku Onishi (454-461).
Nonsteroidal anti-inflammatory drugs (NSAIDs) are administered for pain relief from oral mucositis. However, the systemic administration of NSAIDs is limited due to systemic side effects. To avoid these side effects and treat local lesions effectively, a matrix type mucoadhesive tablet was developed. A mixture of hard fat, ethylcellulose (EC) and polyethylene glycol (PEG) was used as a matrix base, and indomethacin (IMC) was used as the principal agent. In tablets consisting of hard fat, EC and IMC, the drug release was sustained. In tablets consisting of hard fat, EC, considerable amounts of PEG and IMC, the drug release was relatively increased and IMC existed as the molecular phase or in an amorphous state. The in vitro adhesive force of the tablets consisting of hard fat, EC, considerable amounts of PEG and IMC was significantly increased as compared with the tablets consisting of hard fat and IMC. A significantly high tissue concentration and significantly low plasma concentration were observed after buccal administration of this matrix type mucoadhesive tablet as compared with that after oral administration of IMC. Thus, the matrix type mucoadhesive tablet has good potential as a preparation for the treatment of pain due to oral aphtha.
Keywords: Mucosal delivery; Buccal; Formulation; Solid dosage form; Controlled release; X-ray powder diffractometry;

Rheological properties of gamma-irradiated antimicrobial wafers and in vitro efficacy against Pseudomonas aeruginosa by Olga Labovitiadi; Noelle H. O’Driscoll; Andrew J. Lamb; Kerr H. Matthews (462-472).
Lyophilised polysaccharide solutions and gels incorporating the broad spectrum antimicrobial compound, chlorhexidine digluconate (CHD), have potential application as self-adhering, topical delivery systems for the prophylaxis and treatment of wound infections. It is desirable to sterilise these dosage forms but they do not possess suitable thermal properties to be sterilised by autoclaving or dry heat procedures. Ionising radiation may offer a solution hence ‘antimicrobial wafers’ fabricated from guar (GG), xanthan (XG), karaya (KAG), sodium alginate (SA) and a 50:50 blend of SA:KAG, were gamma-irradiated (25 and 40 kGy). Wafer sterility was qualitatively determined and validated for bacterial and fungal species. The rheological properties of gels reconstituted from irradiated discs (‘wafers’) were measured and compared with control samples (non-irradiated) to assess changes to the flow properties. Diffusion of CHD from the resultant gels and viscous solutions, and efficacy against Pseudomonas aeruginosa, was determined using a disc diffusion test and release studies conducted in a diffusion cell apparatus. Although there was no change to cast shape, the rheological properties were drastically depleted for all irradiated samples except XG which showed no effective change in consistency, yield stress or efficacy, from the non-irradiated control. SA and SA-KAG samples displayed ‘anomalous’ release according to Korsmeyer–Peppas.
Keywords: Antimicrobial wafers; Sterilisation; Gamma-irradiation; Rheology; Chlorhexidine digluconate; Pseudomonas aeruginosa;

Networking and rheology of concentrated clay suspensions “matured” in mineral medicinal water by Carola Aguzzi; Rita Sánchez-Espejo; Pilar Cerezo; José Machado; Cristina Bonferoni; Silvia Rossi; Inmaculada Salcedo; César Viseras (473-479).
This work studied the influence of “maturation” conditions (time and agitation) on aggregation states, gel structure and rheological behaviour of a special kind of pharmaceutical semisolid products made of concentrated clay suspensions in mineral medicinal water. Maturation of the samples was carried out in distilled and sulphated mineral medicinal water, both in static conditions (without agitation) and with manual stirring once a week, during a maximum period of three months. At the measured pH interval (7.5–8.0), three-dimensional band-type networks resulting from face/face contacts were predominant in the laminar (disc-like) clay suspensions, whereas the fibrous (rod-like) particles formed micro-aggregates by van der Waals attractions. The high concentration of solids in the studied systems greatly determined their behaviour. Rod-like sepiolite particles tend to align the major axis in aggregates promoted by low shearing maturation, whereas aggregates of disc-like smectite particles did not have a preferential orientation and their complete swelling required long maturation time, being independent of stirring. Maturation of both kinds of suspensions resulted in improved rheological properties. Laminar clay suspensions became more structured with time, independently from static or dynamic maturation conditions, whereas for fibrous clay periodic agitation was also required. Rheological properties of the studied systems have been related to aggregation states and networking mechanisms, depending on the type of clay minerals constituents. Physical stability of the suspensions was not impaired by the specific composition of the Graena medicinal water.
Keywords: Clay pastes; Rheology; Pelotherapy; Maturation; Mineral medicinal water;

Establishing the optimal nebulization system for paclitaxel, docetaxel, cisplatin, carboplatin and gemcitabine: Back to drawing the residual cup by Paul Zarogoulidis; Dimitris Petridis; Christos Ritzoulis; Kaid Darwiche; Dionysis Spyratos; Haidong Huang; Eugene P. Goldberg; Lonny Yarmus; Qiang Li; Lutz Freitag; Konstantinos Zarogoulidis (480-487).
Chemotherapy drugs have still the major disadvantage of non-specific cytotoxic effects. Although, new drugs targeting the genome of the tumor are already in the market, doublet chemotherapy regimens still remain the cornerstone of lung cancer treatment. Novel modalities of administration are under investigation such as; aerosol, intratumoral and intravascular.In the present study five chemotherapy drugs; paclitaxel, docetaxel, gemcitabine, carboplatin and cisplatin were nebulized with three different jet nebulizers (Maxineb®, Sunmist®, Invacare®) and six different residual cups at different concentrations. The purpose of the study was to identify the “ideal” combination of nebulizer-residual cup design-drug–drug loading for a future concept of aerosol chemotherapy in lung cancer patients. The Mastersizer® 2000 was used to evaluate the aerosol droplet mass median aerodynamic diameter.The drug, nebulizer and residual cup design greatly influences the producing droplet size (p  < 0.005, in each case). However; the design of the residual cup is the most important factor affecting the produced droplet size (F  = 834.6, p  < 0.001). The drug loading plays a vital role in the production of the desired droplet size (F  = 10.42, p  < 0.001). The smallest droplet size was produced at 8 ml loading (1.26 μm), while it remained the same at 2, 4 and 6 mls of drug loading.The ideal nebulizer would be Maxineb®, with a large residual cup (10 ml maximum loading capacity) and 8 mls loading and the drug with efficient pulmonary deposition would be docetaxel.
Keywords: Nebulizers; Chemotherapy; Lung cancer;

Behavior of printable formulations of loperamide and caffeine on different substrates—Effect of print density in inkjet printing by Natalja Genina; Daniela Fors; Mirja Palo; Jouko Peltonen; Niklas Sandler (488-497).
The primary goal of the current work was to study the applicability of precision inkjet printing in fabrication of personalized doses of active pharmaceutical ingredients (APIs). Loperamide hydrochloride (LOP) and caffeine (CAF) were used as model compounds. Different doses of the drugs in a single dosage unit were produced, using a drop-on-demand inkjet printer by varying printing parameters such as the distance between jetted droplets (drop spacing) and the physical dimensions of the printed dosage forms. The behavior of the formulated printable inks for both APIs was investigated on the model substrates, using different analytical tools. The obtained results showed that printed LOP did not recrystallize on any substrates studied, whereas at least partial recrystallization of printed CAF was observed on all carrier surfaces. Flexible doses of both APIs were easily obtained by adjusting the drop spacing of the depositing inks, and the results were relevant with regards to the theoretical content. Adapting the dose by varying physical dimensions of single dosage units was less successful than the approach in which drop spacing was altered. In conclusion, controlled printing technology, by means of adjusting the distance between jetted droplets, offers a means to fabricate dosage forms with individualized doses.
Keywords: Inkjet printing; Flexible doses; Personalized medicine; Loperamide hydrochloride; Caffeine;

The majority of patients with advanced ovarian cancer will experience a relapse and ultimately die from refractory diseases. Targeted therapy shows promise for these patients. Novel therapeutic strategies should be developed on the basis of the molecular mechanisms involved in ovarian cancer and the steroid hormone environment of ovaries. The ovary is the main target organ of follicle-stimulating hormone (FSH), which bind to its receptor with high affinity. In this study a FSH receptor-targeting ligand, FSH β 81–95 peptide, was used as a targeting moiety to synthesize an FSH receptor-mediated drug delivery system. FSH β 81–95 peptide-conjugated nanoparticles (FSH81-NPs) and paclitaxel-loaded FSH81-NPs (FSH81-NP-PTXs) were synthesized. In vitro studies showed that FSH β 81–95 peptide enabled the specific uptake of cytotoxic drugs and increased the intracellular paclitaxel concentration in FSH receptor-expressing cancer cells, resulting in enhanced cytotoxic effects. In vivo studies showed that FSH81-NP-PTXs possessed higher antitumor efficacy against FSH receptor-expressing tumors without any clinical signs of adverse side effects or body weight loss due to modification with FSH β 81–95 peptide. Therefore, FSH binding peptide-targeted drug delivery system exhibited high potential in the treatment of ovarian cancer, and tumor targeting via reproductive hormone receptors might improve the outcome of diseases.
Keywords: Ovarian carcinoma; Targeted therapy; Follicle stimulating hormone; Paclitaxel; Nanoparticle;

The internalization of fluorescence-labeled PLA nanoparticles by macrophages by Fengjuan Li; Aiping Zhu; Xiaoli Song; Lijun Ji; Juan Wang (506-513).
A novel method was used to label RhB onto PLA nanoparticles through glycidyl methacrylate (GMA) grafted on the PLA nanoparticles. PLA nanoparticles with different surface and size were also constructed. As a result, the surface and the size of PLA nanoparticles dramatically effect their interaction with macrophages.Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The “stealth” function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology
Keywords: RhB-labeled PLA nanoparticles; Surface grafting copolymerization; Macrophages internalization; Electrochemical impedance;

This study examines the performance of novel hyaluronidase enzyme core-5-fluorouracil-loaded chitosan-polyethylene glycol-gelatin polymer nanocomposites, which were prepared using an ionic gelation technique, as targeted and controlled drug delivery vehicles. These hyaluronidase-loaded nanoparticles have recently been proposed as targeted and controlled drug delivery vehicle systems to tissues due to their ability to loosen the intercellular connective matrix of hyaluronic acid. The encapsulation efficiency and loading capacities of the nanoparticles demonstrated that these nanocomposites displayed sufficient binding ability, which depends on the pH and initial concentration of the drug. The cytotoxic effects of the chitosan-hyaluronidase-5-fluorouracil (CS-HYL-5-FU), chitosan-hyaluronidase-5-fluorouracil polyethylene glycol (CS-HYL-5-FU-PEG), and chitosan-hyaluronidase-5-fluorouracil polyethylene glycol-gelatin (CS-HYL-5-FU-PEG-G) nanoparticles were assessed using MTT assays, and the nanovectors were found to be less cytotoxic than the chemotherapeutic 5-FU after incubation for 3–12 h. The particle sizes of the CS-HYL-5-FU, CS-HYL-5-FU-PEG and CS-HYL-5-FU-PEG-G polymer composites were between 300 and 580 nm, as determined by a Zetasizer. Scanning electron microscopy (SEM) analysis indicated that the nanocomposites exhibit a clear, smooth surface and fine morphology. Linkages of the polymers, enzyme, and drug were confirmed by FTIR spectroscopy. Atomic fluorescence microscopy (AFM) analysis confirmed the size of the polymer composite nanoparticles. Therefore, this work established that the drug can be successfully encapsulated in chitosan-polyethylene glycol-gelatin-accompanied hyaluronidase nanoparticles with a homogeneous distribution. These nanoparticles can be potential carriers for targeted and controlled drug delivery to cancer cells.
Keywords: Chitosan; Drug delivery; Encapsulation; 5-Fluorouracil; Gelatin; Hyaluronidase;

We attempted to prepare sustained release fine particles using a two-step mechanical powder processing method; particle-shape modification and dry particle coating. First, particle shape of bulk drug was modified by mechanical treatment to yield drug crystals suitable for the coating process. Drug crystals became more rounded with increasing rotation speed, which demonstrates that powerful mechanical stress yields spherical drug crystals with narrow size distribution. This process is the result of destruction, granulation and refinement of drug crystals. Second, the modified drug particles and polymer coating powder were mechanically treated to prepare composite particles. Polymer nanoparticle agglomerate obtained by drying poly(meth)acrylate aqueous dispersion was used as a coating powder. The porous nanoparticle agglomerate has superior coating performance, because it is completely deagglomerated under mechanical stress to form fine fragments that act as guest particles. As a result, spherical drug crystals treated with porous agglomerate were effectively coated by poly(meth)acrylate powder, showing sustained release after curing. From these findings, particle-shape modification of drug crystals and dry particle coating with nanoparticle agglomerate using a mechanical powder processor is expected as an innovative technique for preparing controlled-release coated particles having high drug content and size smaller than 100 μm.
Keywords: Mechanical powder processing; Particle shape modification; Dry particle coating; Sustained release fine particle; Polymethacrylate nanoparticle agglomerate; Theophylline;

N-terminal mono-PEGylation of growth hormone antagonist: Correlation of PEG size and pharmacodynamic behavior by Ling Wu; Sa V. Ho; Wei Wang; Jianping Gao; Guifeng Zhang; Zhiguo Su; Tao Hu (533-540).
Growth hormone antagonist (GHA), an analog of growth hormone (GH), can inhibit GH action and treat acromegaly. However, GHA suffers from a short plasma half-life of 15–20 min that has limited its clinical application. PEGylation, conjugation with polyethylene glycol (PEG), can increase the plasma half-life of GHA. Single PEG attachment (mono-PEGylation) at N-terminus of GHA has the advantages of product homogeneity and minimization of the bioactivity loss. Conjugation of large PEG molecule may increase the plasma half-life but could potentially decrease the bioactivity of GHA, due to the steric shielding effect of PEG. Thus, N-terminal mono-PEGylation of GHA with 20 kDa and 40 kDa PEG were used to look for a balance of the two competing factors. Sedimentation velocity analysis suggested that 40 kDa PEG was more efficient than 20 kDa PEG to elongate the molecular shape of the conjugate. As reflected by marginal suppression of insulin-like growth factor I (IGF-I), GHA conjugated with 40 kDa PEG was statistically indistinguishable from the saline solution that could not inhibit GH action. In contrast, GHA conjugated with 20 kDa PEG can apparently inhibit GH action, as reflected by IGF-I suppression of 30–43%. Thus, our work demonstrated the effective therapeutic potency of N-terminally mono-PEGylated GHA.
Keywords: PEGylation; Growth hormone antagonist; N-terminus; Polyethylene glycol; Acromegaly;

Acid-cleavable ketal containing poly(β-amino ester) for enhanced siRNA delivery by Kyeonghye Guk; Hyungsuk Lim; Byungkuk Kim; Minsung Hong; Gilson Khang; Dongwon Lee (541-550).
The safe and effective intracellular delivery of nucleic acids remains the most challenging obstacle to the broad application of gene therapy in clinic. Endosomal escape of nucleic acids is also a major barrier for efficient gene delivery. Ketal linkage is known to readily cleave at the acidic pH of endosomal compartments. Here, we report ketal containing poly(β-amino ester) (KPAE) as an acid-cleavable non-viral siRNA delivery system. KPAE efficiently condensed siRNA into nanocomplexes with a diameter of ∼150 nm, which are stable under neutral conditions but rapidly dissociate to release siRNA at acidic pH. KPAE had a buffering capacity due to the presence of secondary amines in its backbone, confirmed by acid–base titration. Moreover, the studies of confocal fluorescence imaging using calcein and LysoTracker Red revealed that KPAE disrupted endosomes by colloid osmotic mechanism and “proton sponge” effects. Cell culture studies demonstrated that KPAE can deliver tumor necrosis factor-α (TNF-α) siRNA to lipopolysaccharide (LPS)-stimulated macrophages and significantly inhibit the expression of TNF-α. The results demonstrate that acid-cleavable KPAE has great potential as gene delivery systems based on its excellent biocompatibility, pH sensitivity and high gene delivery efficiency.
Keywords: Gene silencing; Biodegradable polymer; siRNA; Poly(β-amino esters); pH-sensitive;

Synthesis of surfactant free PCL–PEG brushed nanoparticles with tunable degradation kinetics by Raffaele Ferrari; Claudio Colombo; Cristina Casali; Monica Lupi; Paolo Ubezio; Francesca Falcetta; Maurizio D’Incalci; Massimo Morbidelli; Davide Moscatelli (551-559).
A delivery system based on polymer nanoparticles (NPs) is developed and tested in relevant biological conditions for breast cancer treatment. ɛ-Caprolactone (CL) and polyethylene glycol (PEG) copolymers have been used for the one pot synthesis of surfactant free PEGylated NPs which are monodispersed, stable in physiological conditions and have size in the range 90–250 nm. The degradation behavior of these NPs has been investigated in cell medium and a relation between degradation time and molecular weight of the starting CL-based material has been established. This allows producing NPs with controlled degradation kinetics. Finally, selected NPs have been tested in 4T1 breast cancer cells to check their toxicity and to investigate the uptake process, in order to validate their use as targeted vectors for breast cancer treatment.
Keywords: Nanoparticles; Degradation; Emulsion polymerization; Poly(ɛ-caprolactone); PEG;

Interactions of N′-acetyl-rifabutin and N′-butanoyl-rifabutin with lipid bilayers: A synchrotron X-ray study by Marina Pinheiro; Cláudia Nunes; João M. Caio; Cristina Moiteiro; Gerald Brezesinski; Salette Reis (560-568).
This work focuses on the interaction of N′-acetyl-rifabutin (RFB2) and N′-butanoyl-rifabutin (RFB3) with human and bacterial cell membrane models under physiological conditions. The effect of RFB2 and RFB3 on human cell membrane models was assessed using multilamellar vesicles (MLVs) composed of 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC). In order to mimic the bacterial cell membrane, MLVs of 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG) and a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) (8:2 molar ratio) were chosen. Small and wide-angle X-ray scattering (SAXS and WAXS) were used to study the effect of these antimycobacterial compounds on the structure formed in aqueous lipid dispersions. This study contributes to understanding the molecular mechanisms of the drugs delivery through the human and bacterial cells and the effect of these antimycobacterial compounds on the membrane lipids organization, which is related with their antibiotic efficacy and toxic effects.
Keywords: Cubic phases; Membrane models; Rifabutin; Small-angle X-ray scattering; Tuberculosis; Wide-angle X-ray scattering;

Microemulsion (ME) and poloxamer microemulsion-based gel (PMBG) were developed and optimized to enhance transport of diclofenac epolamine (DE) into the skin forming in-skin drug depot for sustained transdermal delivery of drug. D-optimal mixture experimental design was applied to optimize ME that contains maximum amount of oil, minimum globule size and optimum drug solubility. Three formulation variables; the oil phase X1 (Capryol®), Smix X2 (a mixture of Labrasol®/Transcutol®, 1:2 w/w) and water X3 were included in the design. The systems were assessed for drug solubility, globule size and light absorbance. Following optimization, the values of formulation components (X1, X2, and X3) were 30%, 50% and 20%, respectively. The optimized ME and PMBG were assessed for pH, drug content, skin irritation, stability studies and ex vivo transport in rat skin. Contrary to PMBG and Flector® gel, the optimized ME showed the highest cumulative amount of DE permeated after 8 h and the in vivo anti-inflammatory efficacy in rat paw edema was sustained to 12 h after removal of ME applied to the skin confirming the formation of in-skin drug depot. Our results proposed that topical ME formulation, containing higher fraction of oil solubilized drug, could be promising for sustained transdermal delivery of drug.
Keywords: Diclofenac epolamine; In-skin depot; Sustained transdermal delivery; D-optimal design; Microemulsion;

Anticancer drug delivery of PEG based micelles with small lipophilic moieties by Ying Lei; Yusi Lai; Yuanlin Li; Sai Li; Gang Cheng; Dong Li; Haiping Li; Bin He; Zhongwei Gu (579-586).
Herein, we reported a new type of self-assembly micelles based on amphiphilic polymers of cinnamate and coumarin derivatives modified PEG for drug delivery applications. Lipophilic cinnamic acid (CIN) and 7-carboxyl methoxycoumarin (COU) were immobilized on the terminal groups of poly(ethylene glycol) (PEG) to prepare amphiphiles. The amphiphiles self-assembled into micelles. The amphiphiles and micelles were characterized by 1H NMR, FT-IR, DLS and TEM. Doxorubicin (DOX) was used as a model drug to investigate the lipophilic moieties effects on the drug release behaviors. The DOX loaded micelles were incubated with HepG2 liver cancer cells to study the in vitro anticancer activities. The results showed that DOX could be encapsulated in the micelles efficiently. The mean diameter of the drug loaded micelles was around 100 nm. Drug release profile revealed that the release rate of DOX loaded COU-PEG-COU micelles was significantly slower than that of CIN-PEG-CIN micelles. The DOX loaded micelles could be internalized in HepG2 cells. Both CLSM and flow cytometry results showed that the DOX loaded CIN-PEG-CIN micelles exhibited better anticancer efficacy.
Keywords: Polymeric micelles; Drug delivery; Lipophilic moiety; Doxorubicin;

Enhancement of water solubility of indomethacin by complexation with protein hydrolysate by Asuka Inada; Tatsuya Oshima; Hiromi Takahashi; Yoshinari Baba (587-593).
Complex formation between indomethacin (Indo) and casein hydrolysate was developed as a novel technique for enhancing the water solubility of Indo. The complex (Indo-Pep) was prepared by mixing an ethanol solution of Indo and an aqueous solution of peptide mixture, followed by lyophilization. The water solubility of Indo-Pep under weakly acidic and neutral conditions is much higher than that of Indo alone. The water solubility of Indo increased with increasing quantity of peptide. Characterization of Indo-Pep using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction showed that Indo was incorporated in Indo-Pep in an amorphous state. The fluorescence quenching of Indo-Pep also suggested complexation between Indo and the peptides. An aqueous solution of Indo-Pep was fractioned by centrifugation followed by filtration using membrane filters and ultrafilters. Analysis of the fractions by dynamic light scattering and ultraviolet–visible spectroscopy showed that Indo-Pep consisted of small particles and was not a hydrocolloidal material.
Keywords: Indomethacin; Peptide; Complex; Solubility; Dissolution;

Effect of particle size on the biodistribution of lipid nanocapsules: Comparison between nuclear and fluorescence imaging and counting by Samuli Hirsjärvi; Lucie Sancey; Sandrine Dufort; Camille Belloche; Claire Vanpouille-Box; Emmanuel Garcion; Jean-Luc Coll; François Hindré; Jean-Pierre Benoît (594-600).
In vivo biodistribution of nanoparticles depends on several physicochemical parameters such as size. After intravenous injection of 25, 50 and 100 nm lipid nanocapsules (LNC) in nude mice bearing HEK293(β3) tumour xenografts, biodistribution was evaluated by γ-scintigraphy and by γ-counting. The small LNC 25 nm disappeared faster than the larger LNC 50 and 100 nm from the blood circulation due to faster elimination and wider tissue distribution. At 24 h, biodistribution profiles of all these LNC were similar. Low LNC quantities were found in this weak EPR (enhanced permeability and retention) tumour regardless the particle size. Co-injected 50 nm fluorescent DiD-LNC and 99mTc-LNC allowed direct comparison of biodistribution as evaluated by the two methods. Optical imaging underestimated LNC quantity especially in dark-colored organs that were observed to capture extensive quantities of the particles by γ-counting (i.e. liver, spleen, and kidney).
Keywords: Lipid nanocapsules; Biodistribution; γ-Scintigraphy; Optical imaging; Fluorescence;

Determination of haloperidol in biological samples using molecular imprinted polymer nanoparticles followed by HPLC-DAD detection by Homeira Ebrahimzadeh; Zahra Dehghani; Ali Akbar Asgharinezhad; Nafiseh Shekari; Karam Molaei (601-609).
In this study an extraction procedure using molecular imprinted polymer nanoparticles for the determination of haloperidol in biological samples is proposed. The haloperidol imprinted polymer nanoparticles were synthesized successfully by precipitation polymerization in a flask containing haloperidol as a template, ethyleneglycoldimethacrylate as a crosslinking agent, methacrylic acid as a functional monomer, and 2,2′-azobisisobutyronitrile as an initiator. The leached and unleached polymer nanoparticles have been characterized by infrared spectroscopy and scanning electron microscopy. The effect of different variables such as the pH of solution, uptake and elution time, type, and the least amount of eluent for elution of haloperidol from polymer was evaluated. Extraction efficiencies more than 97% were obtained by elution of the polymer with 1.5 mL of methanol–acetic acid–trifluoroacetic acid 79.9:20:0.1. Under optimal conditions maximum adsorption capacity was obtained 153.84 mg g−1. The detection limit of the proposed procedure was between 0.2 and 0.35 μg L−1. Finally this method was applied to the determination of haloperidol in plasma and urine samples and satisfactory results were achieved (RSD < 6.9%).
Keywords: Molecular imprinted polymer nanoparticles; Precipitation polymerization; Haloperidol;

Synthesis, in vitro and in vivo evaluation of new norcantharidin-conjugated hydroxypropyltrimethyl ammonium chloride chitosan derivatives as polymer therapeutics by Xiaofen Xu; Yanggong Li; Feihu Wang; Li Lv; Jieying Liu; Mingna Li; Aijie Guo; Jinjun Jiang; Yuanyuan Shen; Shengrong Guo (610-619).
New norcantharidin-conjugated hydroxypropyltrimethyl ammonium chloride chitosan derivatives (NCTD-HACCs) were synthesized and characterized by 1H NMR, Fourier-transform infrared spectroscopy (FT-IR), and wide-angle X-ray diffraction (WAXD). Two NCTD-HACCs with different degrees of substitution (DS) (12.2% and 24.8%) were obtained, which had good water solubility. NCTD was released from the NCTD-HACCs via hydrolysis, faster in pH 5.0 than pH 7.4 and presenting one biphasic drug release pattern with rapid release at the initial stage and slow release later. Fluorescence microscope and flow cytometry analysis demonstrated that the NCTD-HACC was endocytosized into MGC80-3 cells and the uptaken amount increased as incubation time. Compared with free NCTD, the NCTD-HACCs showed lower in vitro anti-tumor activity against human gastric cancer MGC80-3 cells, but higher in vivo tumor growth inhibition in S180 tumor-bearing mice. The in vivo near-infrared (NIR) fluorescence real-time imaging result showed the fluorescence intensity in tumor was much higher than that in heart, liver, spleen and lung (except kidney) after i.v. injection of the FITC-labeled NCTD-HACC2, indicating specific accumulation of the NCTD-HACC in tumor.
Keywords: NCTD-HACC; Cytotoxicity; Cellular uptake; In vivo anti-tumor activity;

Application of different analytical methods for the characterization of non-spherical micro- and nanoparticles by Roman Mathaes; Gerhard Winter; Julia Engert; Ahmed Besheer (620-629).
Non-spherical micro- and nanoparticles have recently gained considerable attention due to their surprisingly different interaction with biological systems compared to their spherical counterparts, opening new opportunities for drug delivery and vaccination. Up till now, electron microscopy is the only method to quantitatively identify the critical quality attributes (CQAs) of non-spherical particles produced by film-stretching; namely size, morphology and the quality of non-spherical particles (degree of contamination with spherical ones). However, electron microscopy requires expensive instrumentation, demanding sample preparation and non-trivial image analysis. To circumvent these drawbacks, the ability of different particle analysis methods to quantitatively identify the CQA of spherical and non-spherical poly(1-phenylethene-1,2-diyl (polystyrene) particles over a wide size range (40 nm, 2 μm and 10 μm) was investigated. To this end, light obscuration, image-based analysis methods (Microflow imaging, MFI, and Vi-Cell XR Coulter Counter) and flow cytometry were used to study particles in the micron range, while asymmetric flow field fractionation (AF4) coupled to multi-angle laser scattering (MALS) and quasi elastic light scattering (QELS) was used for particles in the nanometer range, and all measurements were benchmarked against electron microscopy. Results show that MFI can reliably identify particle size and aspect ratios of the 10 μm particles, but not the 2 μm ones. Meanwhile, flow cytometry was able to differentiate between spherical and non-spherical 10 or 2 μm particles, and determine the amount of impurities in the sample. As for the nanoparticles, AF4 coupled to MALS and QELS allowed the measurement of the geometric (r g ) and hydrodynamic (r h ) radii of the particles, as well as their shape factors (r g /r h ), confirming their morphology. While this study shows the utility of MFI, flow cytometry and AF4 for quantitative evaluation of the CQA of non-spherical particles over a wide size range, the limitations of the methods are discussed. The use of orthogonal characterization methods can provide a complete picture about the CQA of non-spherical particles over a wide size range.
Keywords: Non-spherical particles; Flow cytometry (FACS); Microflow imaging (MFI); Asymmetric flow field flow fractionation (AF4); Nano-rods;

Temperature- and pH-responsive poly(N-isopropylacrylamide) (polyNIPAM) copolymerised with 5% (w/v) of acrylic acid (AAc), termed as poly(NIPAM-co-AAc) nanogel was investigated as a novel multi-responsive topical drug delivery carrier, using caffeine as a model permeant. The role of a pH modulator (citric acid) on the nanogel system was also studied. The loading was carried out in deionised water at two different temperatures, which were 2–4 °C and 25 °C (room temperature, RT) over 3 days. The loading of caffeine into the poly(NIPAM-co-AAc) nanogel was found to be significantly higher at 2–4 °C than at RT (p  = 0.0072). As for the control nanogel (polyNIPAM), a similar pattern of loading level can be observed (p  = 0.0005). This enhanced loading at low temperatures could be attributed to the hydrophilic behaviour of the polyNIPAM network in response to temperatures lower than its lower critical solution temperature (LCST). In vitro diffusion studies across epidermis porcine skin were carried out at 32 °C for the saturated solution of caffeine as well as caffeine-loaded poly(NIPAM-co-AAc) and polyNIPAM nanogels. The in vitro permeation data of caffeine-loaded poly(NIPAM-co-AAc) at 2–4 °C were shown to enhance the delivery of the loaded caffeine across the epidermis in comparison to the saturated solution of caffeine, by 3.5 orders of magnitude. Additionally, the study demonstrated that the effect of pH modulator on the release of loaded permeant was insignificant.
Keywords: Nanogel; NIPAM; Acrylic acid; Stimulus responsive; pH; Temperature;

The aim of this work was to find relationships between critical bioactive glass characteristics and their antibacterial behaviour using an artificial intelligence tool. A large dataset including ingredients and process variables of the bioactive glasses production, bacterial characteristics and microbiological experimental conditions was generated from literature and analyzed by neurofuzzy logic technology. Our findings allow an explanation on the variability in antibacterial behaviour found by different authors and to obtain general conclusions about critical parameters of bioactive glasses to be considered in order to achieve activity against some of the most common skin and implant surgery pathogens.
Keywords: Bioactive glass; Antibacterial behaviour; Modelling; Artificial intelligence; Neurofuzzy logic; Machine learning;

Long term stability of lyophilized plasmid DNA pDERMATT by Iris van der Heijden; Jos H. Beijnen; Bastiaan Nuijen (648-650).
In this short note we report on the shelf-life stability of pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) 2 mg lyophilized powder for reconstitution for intradermal administration, used in an in-house, investigator-initiated clinical phase I study. pDERMATT was stored at 25 °C/60% relative humidity (6 months), 2–8 °C (24 months), and −20 °C (66 months) in the dark and analyzed at several timepoints during the conduct of the clinical study for appearance, identity, purity (plasmid topology), content and residual water content. pDERMATT appeared stable at all storage conditions for the periods tested which, although patient inclusion in the study was significantly delayed, ensured the clinical supply needs. This study shows that lyophilization is an useful tool to preserve the quality of the pDNA and can prevent the need for costly and time-consuming additional manufacture of drug product in case of study delays, not uncommon at the early stage of drug development. To our knowledge, this is the first study reporting shelf life stability of a pDNA formulation for more than 5 years.
Keywords: Plasmid DNA; Lyophilization; Stability; Topology;

A peptide-morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model by Andrew J. Sawyer; Donna Wesolowski; Neeru Gandotra; Alexander Stojadinovic; Mina Izadjoo; Sidney Altman; Themis R. Kyriakides (651-655).
Management of skin wound infections presents a serious problem in the clinic, in the community, and in both civilian and military clinical treatment centers. Staphylococcus aureus is one of the most common microbial pathogens in cutaneous wounds. Peptide-morpholino oligomer (PMO) conjugates targeted to S. aureus gyrase A mRNA have shown the ability to reduce bacterial viability by direct site-specific mRNA cleavage via RNase P. As a treatment, these conjugates have the added advantages of not being susceptible to resistance due to genetic mutations and are effective against drug resistant strains. While this strategy has proven effective in liquid culture, it has yet to be evaluated in an animal model of infected surface wounds. In the present study, we combined PMO conjugates with a thermoresponsive gel delivery system to treat full-thickness mouse cutaneous wounds infected with S. aureus. Wounds treated with a single dose of PMO conjugate displayed improved healing that was associated with increased epithelialization, reduced bacterial load, and increased matrix deposition. Taken together, our findings demonstrate the efficacy and flexibility of the PMO conjugate drug delivery system and make it an attractive and novel topical antimicrobial agent.
Keywords: Staphylococcus aureus; RNase P; Skin wounds; PMOs;