International Journal of Pharmaceutics (v.423, #2)

Development of udenafil-loaded microemulsions for intranasal delivery: In vitro and in vivo evaluations by Hyun-Jong Cho; Wan-Sung Ku; Ubonvan Termsarasab; Insoo Yoon; Chung-Wook Chung; Hyun Tae Moon; Dae-Duk Kim (153-160).
Novel microemulsion system for the intranasal delivery of udenafil achieved rapid onset of action (T max  = 1 min) and improved bioavailability (85.7%) compared to oral administration (6.6%) in rats.To achieve rapid onset of action and improved bioavailability of udenafil, a microemulsion system was developed for its intranasal delivery. Phase behavior, particle size, transmission electron microscope (TEM) images, and the drug solubilization capacity of the microemulsion were investigated. A single isotropic region was found in pseudo-ternary phase diagrams developed at various ratios with CapMul MCM L8 as an oil, Labrasol as a surfactant, and Transcutol or its mixture with ethanol (1:0.25, v/v) as a cosurfactant. Optimized microemulsion formulations with a mean diameter of 120–154 nm achieved enhanced solubility of udenafil (>10 mg/ml) compared with its aqueous solubility (0.02 mg/ml). An in vitro permeation study was performed in human nasal epithelial (HNE) cell monolayers cultured by the air–liquid interface (ALI) method, and the permeated amounts of udenafil increased up to 3.41-fold versus that of pure udenafil. According to the results of an in vivo pharmacokinetic study in rats, intranasal administration of udenafil-loaded microemulsion had a shorter T max value (1 min) compared with oral administration and improved bioavailability (85.71%) compared with oral and intranasal (solution) administration. The microemulsion system developed for intranasal administration may be a promising delivery system of udenafil, with a rapid onset of action and improved bioavailability.
Keywords: Udenafil; Microemulsion; Intranasal delivery; Erectile dysfunction;

Controlling lipolysis through steric surfactants: New insights on the controlled degradation of submicron emulsions after oral and intravenous administration by Miguel Wulff-Pérez; Juan de Vicente; Antonio Martín-Rodríguez; María J. Gálvez-Ruiz (161-166).
In this work we have investigated how steric surfactants influence the metabolic degradation of emulsions (lipolysis). To do so, we have prepared submicron emulsions stabilized with Pluronic F68, Pluronic F127, Myrj 52 or Myrj 59, four non-ionic surfactants with key differences on their structure. Submicron emulsions have been prepared also with mixtures of these surfactants with different proportions between them. Then, in vitro methods have been applied to analyze the lipolysis of these emulsions, both under duodenal and intravenous conditions, to simulate lipolysis after oral and intravenous administration. Our results show that the properties of the surfactant influence dramatically the lipolysis rates observed both under duodenal and intravenous conditions, e.g., intravenous lipolysis was completely blocked when Pluronic F127 was used, while it was almost complete within 6 h when using Myrj 52. The reason for this seems to be the steric hindrance that the surfactant produces around the droplet and at the interface. As a result, we can modify the lipolysis patterns by changing some characteristics of the surfactant, or by varying the proportion between two surfactants in a mixture. These findings may be applied in the development of novel strategies to rationally design submicron emulsions as lipophilic drug carriers.
Keywords: Submicron emulsion; Lipolysis; Lipoprotein lipase; Pancreatic lipase; Pluronic; Steric surfactants;

Quality by design approach for formulation development: A case study of dispersible tablets by Naseem A. Charoo; Areeg A.A. Shamsher; Ahmed S. Zidan; Ziyaur Rahman (167-178).
The focus of the current investigations was to apply quality by design (QbD) approach to the development of dispersible tablets. Critical material and process parameters are linked to the critical quality attributes of the product. Variability is reduced by product and process understanding which translates into quality improvement, risk reduction and productivity enhancement. The risk management approach further leads to better understanding of the risks, ways to mitigate them and control strategy is proposed commensurate with the level of the risk. Design space in combination with pharmaceutical quality management system provide for flexible regulatory approaches with opportunity for continuous improvement that benefit patient and manufacturer alike. The development of dispersible tablet was proposed in the current study through a QbD paradigm for a better patient compliance and product quality. The quality target product profile of a model biopharmaceutical class II drug was identified. Initial risk analysis led to the identification of the critical quality attributes. Physicochemical characterization and compatibility studies of the drug with commonly used excipients were performed. Experiments were designed with focus on critical material and process attributes. Design space was identified and risk factors for all the possible failure modes were below critical levels after the implementation of control strategy. Compliance to the design space provides an opportunity to release batches in a real time. In conclusion, QbD tools together with risk and quality management tools provided an effective and efficient paradigm to build the quality into dispersible tablet.
Keywords: Quality by design; Dispersible tablets; Critical quality attributes; Risk assessment; Design space; Control strategy;

The human skin sandwich technique was used to explore the effect of brief ultrasound exposure on the transfollicular pathway of absorption. Hydrocortisone was used as a model drug. In order to calculate the permeability coefficient of hydrocortisone, its concentration at saturation in the PBS donor solution was determined. Skin samples were prepared by sandwich technique with total hydration of the epidermal and sandwich membranes. The skin was sonicated for 0 s (control), 30 s or 45 s using a pulsed mode (10% duty cycle) with the spatial and temporal average intensity (SATA) of 3.7 W/cm2. The transducer was then removed and permeation was allowed to proceed for 52 h. Then the percentage follicular contribution was determined. It was determined that without ultrasound, drug entry into follicles accounted for 46% of total penetration. As the duration of sonication increased, the follicular contribution fell to zero even though total transepidermal flux dramatically increased. This is explained by ultrasound exposure causing sloughing off of the uppermost stratum corneum. This permeabilises the continuous surface but at the same time the disturbed cornceocytes will plug hair follicle orifices.
Keywords: Sonophoresis; Low frequency ultrasound; Hair follicles, Skin sandwich technique;

The purpose of this study was to evaluate the relationships between physicochemical properties and aerosolisation performance of different grades of lactose. In order to get a wide range of physicochemical properties, various grades of lactose namely Flowlac ® 100 (FLO), Lactopress anhydrous ® 250 (LAC), Cellactose ® 80 (CEL), Tablettose ® 80 (TAB), and Granulac ® 200 (GRA) were used. The different lactose grades were carefully sieved to separate 63–90 μm particle size fractions and then characterised in terms of size, shape, density, flowability, and solid state. Formulations were prepared by blending each lactose with salbutamol sulphate (SS) at ratio of 67.5:1 (w/w), and then evaluated in terms of SS content uniformity, lactose–SS adhesion properties, and in vitro aerosolisation performance delivered from the Aerolizer®. Sieved lactose grades showed similar particle size distributions (PSDs) and good flow properties but different particle shape, particle surface texture, and particle solid state. Content uniformity assessments indicated that lactose particles with rougher surface produced improved SS homogeneity within DPI formulation powders. Lactose–SS adhesion assessments indicated that lactose particles with more elongated shape and the rougher surface showed smaller adhesion force between lactose and salbutamol sulphate. Lactose powders with higher bulk density and higher tap density produced smaller emission (EM) and higher drug loss (DL) of SS. In vitro aerosolisation for various lactose grades followed the following rank order in terms of deposition performance: GRA > TAB > LAC ≈ CEL > FLO. Linear relationships were established showing that in order to maximize SS delivery to lower airway regions, lactose particles with more elongated shape, more irregular shape, and rougher surface are preferred. Therefore, considerable improvement in DPI performance can be achieved by careful selection of grade of lactose included within DPI formulations.
Keywords: Inhalation; Commercial lactose; Salbutamol sulphate; Pulmonary delivery; Aerolizer®; Shape; Surface roughness; Content uniformity; Adhesion;

Polyelectrolyte complex nanoparticles of amino poly(glycerol methacrylate)s and insulin by Xueyou Lu; Hui Gao; Cui Li; Ying-Wei Yang; Yinong Wang; Yunge Fan; Guolin Wu; Jianbiao Ma (195-201).
Amino poly(glycerol methacrylate)s (PGOHMAs) were synthesized from linear or star-shaped poly(glycidyl methacrylate)s (PGMA)s via ring opening reactions with 1,2-ethanediamine, 1,4-butanediamine and diethylenetriamine, respectively. The resulting cationic polymers were employed to form polyelectrolyte complexes (PECs) with insulin. Parameters influencing complex formation were investigated by dynamic light scattering (DLS). PECs in the size range of 100–200 nm were obtained under optimal conditions, i.e., the pH value of PECs was 5.58–6.27, the concentration of NaCl was 0.02 mol/L, and insulin–polymer weight ratio was 0.8. The insulin association efficiency (AE) of current system increased with zeta potentials of PECs. Circular dichroism (CD) analysis corroborated that the structure of insulin in the PEC nanoparticles was preserved after lyophilization. Fourier transform infrared (FT-IR) and X-ray diffraction (XRD) experiments demonstrated that weak physical interactions between insulin and amino PGOHMAs play an important role in the formation of PECs. The release of insulin depends on both structure and architecture of amino PGOHMAs. These PECs would be potentially useful for mucosal administration.
Keywords: Amino poly(glycerol methacrylate); Insulin; Polyelectrolyte complexes; Nanoparticles;

The aim of this study was to investigate the influence of binder content, binder particle size, granulation time and inlet air flow rate on granule size and size distribution, granule shape and flowability, as well as on drug release rate. Hydrophilic (polyetilenglycol 2000) and hydrophobic meltable binder (glyceryl palmitostearate) were used for in situ fluidized hot melt granulation. Granule size was mainly influenced by binder particle size. Binder content was shown to be important for narrow size distribution and good flow properties. The results obtained indicate that conventional fluid bed granulator may be suitable for production of highly spherical agglomerates, particularly when immersion and layering is dominant agglomeration mechanism. Granule shape was affected by interplay of binder content, binder particle size and granulation time. Solid state analysis confirmed unaltered physical state of the granulate components and the absence of interactions between the active and excipients. Besides the nature and amount of binder, the mechanism of agglomerate formation seems to have an impact on drug dissolution rate. The results of the present study indicate that fluidized hot melt granulation is a promising powder agglomeration technique for spherical granules production.
Keywords: Melt agglomeration; Fluid bed; In situ melt granulation; Granule shape; Binder particle size;

Improvement of flow and bulk density of pharmaceutical powders using surface modification by Laila J. Jallo; Chinmay Ghoroi; Lakxmi Gurumurthy; Utsav Patel; Rajesh N. Davé (213-225).
Improvement in flow and bulk density, the two most important properties that determine the ease with which pharmaceutical powders can be handled, stored and processed, is done through surface modification. A limited design of experiment was conducted to establish a standardized dry coating procedure that limits the extent of powder attrition, while providing the most consistent improvement in angle of repose (AOR). The magnetically assisted impaction coating (MAIC) was considered as a model dry-coater for pharmaceutical powders; ibuprofen, acetaminophen, and ascorbic acid. Dry coated drug powders were characterized by AOR, particle size as a function of dispersion pressure, particle size distribution, conditioned bulk density (CBD), Carr index (CI), flow function coefficient (FFC), cohesion coefficient using different instruments, including a shear cell in the Freeman FT4 powder rheometer, and Hansen flowability index. Substantial improvement was observed in all the measured properties after dry coating relative to the uncoated powders, such that each powder moved from a poorer to a better flow classification and showed improved dispersion. The material intrinsic property such as cohesion, plotted as a function of particle size, gave a trend similar to those of bulk flow properties, AOR and CI. Property improvement is also illustrated in a phase map of inverse cohesion (or FFC) as a function of bulk density, which also indicated a significant positive shift due to dry coating. It is hoped that such phase maps are useful in manufacturing decisions regarding the need for dry coating, which will allow moving from wet granulation to roller compaction or to direct compression based formulations.
Keywords: Dry coating; Design of experiment; Flow properties; Pharmaceutical powders; Cohesion;

Enhancement of nose-to-brain delivery of basic fibroblast growth factor for improving rat memory impairments induced by co-injection of β-amyloid and ibotenic acid into the bilateral hippocampus by Chengcheng Feng; Chi Zhang; Xiayan Shao; Qingfeng Liu; Yong Qian; Liang Feng; Jie Chen; Yuan Zha; Qizhi Zhang; Xinguo Jiang (226-234).
Basic fibroblast growth factor (bFGF) delivery to the brain of animals appears to be an emerging potential therapeutic approach to neurodegenerative diseases, such as Alzheimer's disease (AD). The intranasal route of administration could provide an alternative to intracerebroventricular infusion. A nasal spray of bFGF had been developed previously and the objective of the present study was to investigate whether bFGF nasal spray could enhance brain uptake of bFGF and ameliorate memory impairment induced by co-injection of β-amyloid25–35 and ibotenic acid into bilateral hippocampus of rats. The results of brain uptake study showed that the AUC0–12 h of bFGF nasal spray in olfactory bulb, cerebrum, cerebellum and hippocampus was respectively 2.47, 2.38, 2.56 and 2.19 times that of intravenous bFGF solution, and 1.11, 1.95, 1.40 and 1.93 times that of intranasal bFGF solution, indicating that intranasal administration of bFGF nasal spray was an effective means of delivering bFGF to the brain, especially to cerebrum and hippocampus. In Morris water maze tasks, intravenous administration of bFGF solution at high dose (40 μg/kg) showed little improvement on spatial memory impairment. In contrast, bFGF solution of the same dose following intranasal administration could significantly ameliorate spatial memory impairment. bFGF nasal spray obviously improved spatial memory impairment even at a dose half (20 μg/kg) of bFGF solution, recovered their acetylcholinesterase and choline acetyltransferase activity to the sham control level, and alleviated neuronal degeneration in rat hippocampus, indicating neuroprotective effects on the central nerve system. In a word, bFGF nasal spray may be a new formulation of great potential for treating AD.
Keywords: Basic fibroblast growth factor; Intranasal administration; Alzheimer's disease; Brain uptake; Morris water maze;

Evaluation of metered dose inhaler spray velocities using Phase Doppler Anemometry (PDA) by Xiaofei Liu; William H. Doub; Changning Guo (235-239).
Droplet velocity is an important parameter which can significantly influence inhalation drug delivery performance. Together with the droplet size, this parameter determines the efficiency of the deposition of MDI products at different sites within the lungs. In this study, Phase Doppler Anemometry (PDA) was used to investigate the instantaneous droplet velocity emitted from MDIs as well as the corresponding droplet size distribution. The nine commercial MDI products surveyed showed significantly different droplet velocities, indicating that droplet velocity could be used as a discriminating parameter for in vitro testing of MDI products. The droplet velocity for all tested MDI products decreased when the testing distance was increased from 3 cm to 6 cm from the front of mouthpiece, with CFC formulations showing a larger decrease than HFA formulations. The mean droplet diameters of the nine MDIs were also significantly different from one-another. Droplet size measurements made using PDA (a number-based technique) could not be directly compared to results obtained using laser light scattering measurements (a volume-based technique). This work demonstrates that PDA can provide unique information useful for characterizing MDI aerosol plumes and evaluating MDI drug delivery efficiency. PDA could also aid the evaluation of in vitro equivalence in support of formulation or manufacturing changes and in evaluation of abbreviated new drug applications (ANDAs) for MDIs.
Keywords: Metered dose inhaler; MDI; Droplet velocity; Droplet size; Phase Doppler Anemometry;

The Refined Integral Method has been successfully applied to moving boundary problems encountered in the diffusional release of solutes from torus-shaped single-layer devices.A mathematical modeling of controlled release of drug from one-layer torus-shaped devices is presented. Analytical solutions based on Refined Integral Method (RIM) are derived. The validity and utility of the model are ascertained by comparison of the simulation results with matrix-type vaginal rings experimental release data reported in the literature. For the comparisons, the pair-wise procedure is used to measure quantitatively the fit of the theoretical predictions to the experimental data. A good agreement between the model prediction and the experimental data is observed. A comparison with a previously reported model is also presented. More accurate results are achieved for small A/C s ratios.
Keywords: Intravaginal ring; Refined Integral Method; Mathematical modeling; Dispersed drug; Moving front;

Relationship between aggregation properties and antimicrobial activities of alkylphosphocholines with branched alkyl chains by Miloš Lukáč; Mária Garajová; Martin Mrva; Marián Bukovský; František Ondriska; Eszter Máriássy; Ferdinand Devínsky; Ivan Lacko (247-256).
Synthesis of five alkylphosphocholines with branched alkyl chains (Isophol-PCs) with different length of alkyl chains was described. Isophol8-PC and Isophol12-PC represent new compounds. The physico-chemical properties of Isophol-PCs were determined, critical micelle concentration and types of formed aggregates in aqueous solutions were investigated. The biological activities of Isophol-PCs have been studied for the first time in the present study. Antimicrobial activities of alkylphosphocholines were studied against bacteria (Staphylococcus aureus, Escherichia coli), yeast (Candida albicans) and pathogenic free-living amoebae (Acanthamoeba lugdunensis and Acanthamoeba quina). A. lugdunensis and A. quina are relatively insusceptible to action of miltefosine (standard compound of alkylphosphocholines) and therefore they are good models for studies of amoebicidal action of the investigated compounds. Relationship between structure, physico-chemical and biological activities of Isophol-PCs was discussed. S. aureus and C. albicans were sensitive to action of Isophol16-PC, Isophol20-PC. E. coli was not sensitive to action of all studied alkylphosphocholines in the concentrations equal to, or less than 10 mM. Among all the synthesized compounds, Isophol16-PC had the highest level of activity against both strains of Acanthamoeba. The minimum trophocidal concentrations of Isophol16-PC against A. lugdunensis and A. quina are about four times lower than the minimum trophocidal concentrations of miltefosine against booth strains.
Keywords: Alkylphosphocholines; Acanthamoeba spp.; Critical micelle concentration; Miltefosine; Trophozoite;

Delivery of salmon calcitonin using a microneedle patch by Cetin Tas; Saffar Mansoor; Haripriya Kalluri; Vladimir G. Zarnitsyn; Seong-O Choi; Ajay K. Banga; Mark R. Prausnitz (257-263).
Peptides and polypeptides have important pharmacological properties but only a limited number have been exploited as therapeutics because of problems related to their delivery. Most of these drugs require a parenteral delivery system which introduces the problems of pain, possible infection, and expertise required to carry out an injection. The aim of this study was to develop a transdermal patch containing microneedles (MNs) coated with a peptide drug, salmon calcitonin (sCT), as an alternative to traditional subcutaneous and nasal delivery routes. Quantitative analysis of sCT after coating and drying onto microneedles was performed with a validated HPLC method. In vivo studies were carried out on hairless rats and serum levels of sCT were determined by ELISA. The AUC value of MNs coated with a trehalose-containing formulation (250 ± 83 ng/mL min) was not significantly different as compared to subcutaneous injections (403 ± 253 ng/mL min), but approximately 13 times higher than nasal administration (18.4 ± 14.5 ng/mL min). T max (7.5 ± 5 min) values for MN mediated administration were 50% shorter than subcutaneous injections (15 min), possibly due to rapid sCT dissolution and absorption by dermal capillaries. These results suggest that with further optimization of coating formulations, microneedles may enable administration of sCT and other peptides without the need for hypodermic injections.
Keywords: Microneedle; Peptide delivery; Salmon calcitonin; Transdermal drug delivery;

Computational fluid dynamics (CFD) insights into agitation stress methods in biopharmaceutical development by Ge Bai; Jared S. Bee; James G. Biddlecombe; Quanmin Chen; W. Thomas Leach (264-280).
Agitation of small amounts of liquid is performed routinely in biopharmaceutical process, formulation, and packaging development. Protein degradation commonly results from agitation, but the specific stress responsible or degradation mechanism is usually not well understood. Characterization of the agitation stress methods is critical to identifying protein degradation mechanisms or specific sensitivities. In this study, computational fluid dynamics (CFD) was used to model agitation of 1 mL of fluid by four types of common laboratory agitation instruments, including a rotator, orbital shaker, magnetic stirrer and vortex mixer. Fluid stresses in the bulk liquid and near interfaces were identified, quantified and compared. The vortex mixer provides the most intense stresses overall, while the stir bar system presented locally intense shear proximal to the hydrophobic stir bar surface. The rotator provides gentler fluid stresses, but the air–water interfacial area and surface stresses are relatively high given its low rotational frequency. The orbital shaker provides intermediate-level stresses but with the advantage of a large stable platform for consistent vial-to-vial homogeneity. Selection of experimental agitation methods with targeted types and intensities of stresses can facilitate better understanding of protein degradation mechanisms and predictability for “real world” applications.
Keywords: Agitation stress; Shear; Protein aggregation; Computational fluid dynamics (CFD);

Physical properties of pectin–high amylose starch mixtures cross-linked with sodium trimetaphosphate by Fernanda M. Carbinatto; Ana Dóris de Castro; Beatriz S.F. Cury; Alviclér Magalhães; Raul C. Evangelista (281-288).
Pectin–high amylose starch mixtures (1:4; 1:1; 4:1) were cross-linked at different degrees and characterized by rheological, thermal, X-ray diffraction and NMR analyses. For comparison, samples without cross-linker addition were also prepared and characterized. Although all samples behaved as gels, the results evidenced that the phosphorylation reaction promotes the network strengthening, resulting in covalent gels (highest critical stress, G′ and recovery %). Likewise, cross-linked samples presented the highest thermal stability. However, alkaline treatment without cross-linker allowed a structural reorganization of samples, as they also behaved as covalent gels, but weaker than those gels from cross-linked samples, and presented higher thermal stability than the physical mixtures. X-ray diffractograms also evidenced the occurrence of physical and chemical modifications due to the cross-linking process and indicated that samples without cross-linker underwent some structural reorganization, resulting in a decrease of crystallinity. The chemical shift of resonance signals corroborates the occurrence of structural modifications by both alkaline treatment and cross-linking reaction.
Keywords: High amylose starch; Pectin; Cross-linking; Thermal analysis; Rheology; X-ray diffraction;

Low frequency sonophoresis mediated transdermal and intradermal delivery of ketoprofen by Anushree Herwadkar; Vishal Sachdeva; Leslie F. Taylor; Herb Silver; Ajay K. Banga (289-296).
The objective of this study was to test low frequency sonophoresis at 20 kHz for delivery of ketoprofen into and across the skin. Permeation studies were carried out in vitro on excised hairless rat skin over a period of 24 h using Franz diffusion cells after which, skin samples were subjected to skin extraction to quantify the amount of drug present in skin. Parameters like ultrasound application time, duty cycle coupling medium and distance of ultrasound horn from skin were optimized. Transepidermal water loss (TEWL) was measured to indicate the extent of barrier disruption following sonophoresis. Confocal microscopy was used to visualize dye penetration through sonophoresis treated skin. Application of ultrasound significantly enhanced permeation of ketoprofen from 74.87 ± 5.27 μg/cm2 for passive delivery to 491.37 ± 48.78 μg/cm2 for sonophoresis. Drug levels in skin layers increased from 34.69 ± 7.25 μg following passive permeation to 212.62 ± 45.69 μg following sonophoresis. TEWL increased from 31.6 ± 0.02 (passive) to 69.5 ± 12.60 (sonophoresis) indicating disruption of barrier properties. Confocal microscopy images depicted enhanced dye penetration through sonophoresis treated skin confirming barrier disruption. Low frequency sonophoresis with optimized ultrasound parameters can be effectively used to actively enhance transdermal and topical delivery of ketoprofen.
Keywords: Ketoprofen; Transdermal; Sonophoresis; Phonophoresis; Intradermal; Ultrasound;

The aggregation of tetanus toxoid leads to reduced bioavailability of the vaccine and failure of immunization programmes in many parts of the globe. One of the main reasons for denaturation and aggregation of tetanus toxoid formulations is agitation of the protein during transport. We have identified that agitation leads to collapse of the gel matrix of aluminium hydroxide which is used as an adjuvant in these preparations. This results in desorption of the toxoid from the matrix, which then loses its antigenicity due to agitation-induced denaturation of the protein. We show that incorporation of some compatible osmolytes like sorbitol, glucose and arginine, but not trehalose, is able to protect the adjuvant matrix from degradation, and retain the integrity of the vaccine preparation in terms of its antigenicity.
Keywords: Agitation; Denaturation; Osmolyte; Physical stability; Stabilization; Vaccine adjuvants;

Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid by Varaporn Buraphacheep Junyaprasert; Pratyawadee Singhsa; Jiraphong Suksiriworapong; Doungdaw Chantasart (303-311).
Ellagic acid (EA) is a potent antioxidant phytochemical substance which has limitation to use due to its poor biopharmaceutical properties, low solubility and low permeability. The aim of the present study was to develop niosomal formulations obtained from the mixture of Span 60 and Tween 60 that could encapsulate EA for dermal delivery. The EA-loaded niosomes were prepared with 1:0, 2:1, 1:1, 0:1 Span 60 and Tween 60, using polyethylene glycol 400 (PEG 400), propylene glycol (PG) or methanol (MeOH) as a solubilizer. The influence of formulations on vesicle size, entrapment efficiency and stability of EA-loaded niosomes was investigated. It was found that all ratios of surfactants could produce EA-loaded niosomes when using 15% (v/v) PG, 15% (v/v) PEG 400 or 20% (v/v) MeOH. The niosomes were spherical multilamellar vesicles showing the localization of EA in the vesicles. The vesicle sizes of the niosomes after extrusion were 124–752 nm with PI less than 0.4. The percentages of entrapment efficiency (% E.E.) of all EA-loaded niosomes varied between 1.35% and 26.75% while PEG 400 niosomes gave the highest % E.E. The most stable and highest entrapped formulation was 2:1 Span 60 and Tween 60 niosomes. Additionally, the in vitro skin permeation revealed that penetration of EA from the niosomes depended on vesicle size, the amount of EA entrapped and the added solubilizers which could act as a permeation enhancer. From skin distribution study, the EA-loaded niosomes showed more efficiency in the delivery of EA through human epidermis and dermis than EA solution. The results indicated that the Span 60 and Tween 60 niosomes may be a potential carrier for dermal delivery of EA.
Keywords: Ellagic acid; Niosomes; Non-ionic surfactants; Skin permeation; Skin distribution;

Drug solubilization and in vitro toxicity evaluation of lipoamino acid surfactants by Nathalie Ménard; Nicolas Tsapis; Cécile Poirier; Thomas Arnauld; Laurence Moine; François Lefoulon; Jean-Manuel Péan; Elias Fattal (312-320).
To improve solubilization of a water insoluble anticancer drug, novel surfactants were synthesized. All surfactants derived from lysine, with a so-called nitrilo triacetic acid (NTA) polar head, and differed from the length and saturation degree of their hydrophobic moieties: C19:0-NTA, C20:4-NTA, C25:0-NTA and C25:4-NTA. Self-assembling properties and critical micellar concentration (CMC) values were determined using pyrene fluorescence and cytotoxicity using MTT and LDH assays on endothelial cells. Surfactant haemolytic activity and drug solubilization capacity were also evaluated. All surfactants self-assemble with low CMC values from 0.012 to 0.430 mg/mL. Cytotoxicity assays showed that C20:4-NTA and C25:0-NTA were less cytotoxic than polysorbate 80. Unsaturations and alkane chain length have a marked influence on toxicity. Saturated surfactants had a similar haemolytic activity, explained by their low CMC values and the linear configuration of their hydrophobic tail. C20:4-NTA and C25:4-NTA were less haemolytic than polysorbate 80. Furthermore, C19:0-NTA, C25:0-NTA and C25:4-NTA increased drug solubility from <0.15 μg/mL up to 7 mg/mL, with 46% (w/w) drug loading, due to their linear and flexible hydrophobic chain configuration, as evidenced by molecular modelling. Although these solubilizers are promising, a compromise between drug solubilization and toxicity remains to be found.
Keywords: Amino acid; Lipid; Surfactant; Micelle; Self-assembly; Drug solubilization;

Investigation on the need of multiple dose bioequivalence studies for prolonged-release generic products by Alfredo García-Arieta; Susana Morales-Alcelay; Marta Herranz; José María de la Torre-Alvarado; Antonio Blázquez-Pérez; Mª Luisa Suárez-Gea; Covadonga Álvarez (321-325).
In the European Union multiple dose bioequivalence studies are required for the approval of generic prolonged-release products, but they are not required by the US-FDA. In order to investigate if the multiple dose bioequivalence studies are necessary, the bioequivalence studies assessed in the Spanish Agency for Medicines and Health Care Products in the last 10 years were searched to find all reasons for rejection and identify those cases where the multiple dose study had failed to show bioequivalence and the single dose study had shown bioequivalence. In these latter cases, the plasma concentration at the end of the dosing interval (C τ ) in the single dose study was assessed to investigate its sensitivity to predict non-bioequivalence in the steady state.The search identified six cases where the non-equivalence in the multiple dose study was not detected by the corresponding single dose study. C τ was not able to detect the difference in five cases and in general it was more variable than conventional metrics. In conclusion, the multiple dose bioequivalence study is necessary to ensure therapeutic equivalence and the use of C τ would be counterproductive, increasing the sample size of the studies without enough sensitivity to detect differences in the steady state.
Keywords: Generic drugs; Therapeutic equivalency; Bioequivalence; Sustained-release; Single dose; Multiple dose;

Physical characterisation and component release of poly(vinyl alcohol)–tetrahydroxyborate hydrogels and their applicability as potential topical drug delivery systems by Diarmaid J. Murphy; Mayur G. Sankalia; Ryan G. Loughlin; Ryan F. Donnelly; Mark G. Jenkins; Paul A. McCarron (326-334).
Poly(vinyl alcohol)–tetrahydroxyborate (PVA–THB) hydrogels are dilatant formulations with potential for topical wound management. To support this contention, the physical properties, rheological behaviour and component release of candidate formulations were investigated. Oscillatory rheometry and texture profile analysis were used at room temperature and 37 °C. Results showed that it was possible to control the rheological and textural properties by altering component concentration and modifying the type of PVA polymer used. Hydrogels made using PVA grades with higher degrees of hydrolysis displayed favourable characteristics from a wound healing perspective. In vitro release of borate and PVA were assessed in order to evaluate potential clinical dosing of free species originating from the hydrogel structure. Component diffusion was influenced by both concentration and molecular weight, where relevant, with up to 5% free PVA cumulative release observed after 30 min. The results of this study demonstrated the importance of poly(vinyl alcohol) selection for ensuring appropriate gel formation in PVA–THB hydrogels. The benefits of higher degrees of hydrolysis, in particular, included lower excipient release and reduced bioadhesion. The unique physical characteristics of these hydrogels make them an appealing delivery vehicle for chronic and acute wound management purposes.
Keywords: Local anaesthesia; Topical; Hydrogels; Lacerations; Wound care;

Quantifying crystallisation rates of amorphous pharmaceuticals with dynamic mechanical analysis (DMA) by Nina Soutari; Asma B.M. Buanz; Mine Orlu Gul; Catherine Tuleu; Simon Gaisford (335-340).
One of the stability concerns for amorphous pharmaceuticals is phase transformation to a crystalline form. Since conversion from an amorphous matrix to a crystalline lattice should result in a change in mechanical modulus of the material dynamic mechanical analysis (DMA) offers potential as a stability-indicating assay for what are often complex formulations. Amorphous indomethacin glasses were used as model samples. Pockets made of a metal weave allowed the glass to be mounted in the instrument while ensuring exposure to RH. Crystallisation was manifest as an increase in the storage modulus signal with time. Conversion of the data to fraction crystallisation allowed quantitative determination of the rate and mechanism of crystallisation by application of the Urbanovici–Segal model. Rates of crystallisation were seen to increase with temperature and humidity while temperature and humidity affected the mechanism of crystallisation. High temperature and humidity resulted in three dimensional crystal growth. Reducing the humidity caused a switch in mechanism to growth from edges. Reducing temperature resulted in a mixed mechanism of growth from surfaces and edges. The DMA was also sensitive to crystallisation of phenobarbital sodium formulated in an oral film, but quantitative analysis was not possible as the onset of crystallisation was not recorded.
Keywords: Dynamic mechanical analysis (DMA); Indomethacin; Crystallisation; Amorphous materials; Stability assay;

Crystallinity evaluation of tacrolimus solid dispersions by chemometric analysis by Ahmed S. Zidan; Ziyaur Rahman; Vilayat Sayeed; Andre Raw; Lawrence Yu; Mansoor A. Khan (341-350).
Different destructive and nondestructive analytical methods, namely powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), Raman and near-infrared (NIR) spectroscopy and imaging, to detect and characterize tacrolimus trace crystallinity in an amorphous solid dispersion (SD) using chemometric analysis were developed. The SD was spiked with different percentages of the crystalline drug to construct an array of SDs with different crystallinity percentages. Partial least square (PLS) regression analysis was employed to compare the performance of the calibration models created using these analytical methods. The obtained results indicated a significant interaction between tacrolimus and the employed polymer and a drug dissolution dependency on the crystalline fraction within the SDs. Using two PLS factors, these analytical methods were ranked according to its specificity to detect the trace crystallinity of SDs as NIR > PXRD > Raman > DSC. Through the application of PLS, root-mean-squared error of calibration values of 2.91%, 5.36%, 7.07% and 11.58% were calculated for the calibration models constructed by NIR, PXRD, Raman and DSC, respectively. Having a prediction error of 2.1% and a correlation coefficient of 0.99, it is demonstrated that combined NIR imaging and chemometric analysis outperformed the other methods in detecting trace crystallinity in tacrolimus amorphous systems. The spatial distributions of amorphous and crystalline drug were also obtained in order to allow for studying the crystallization dissemination in the solid dispersions. Consequently, NIR and NIR imaging coupled with chemometry was shown to be a powerful tool for the prediction of drug crystallinity within SDs.
Keywords: Tacrolimus; Solid dispersions; Crystallinity; Near-infrared; Raman;

Further improvement of orally disintegrating tablets using micronized ethylcellulose by Yutaka Okuda; Yosuke Irisawa; Kazuto Okimoto; Takashi Osawa; Shinji Yamashita (351-359).
The aim of this study is to design a new orally disintegrating tablet (ODT) containing micronized ethylcellulose (MEC). The new ODT was prepared by physical mixing of rapidly disintegrating granules (RDGs) with MEC. To obtain RDGs, mannitol was spray-coated with a suspension of corn starch and crospovidone (9:1, w/w ratio) using a fluidized-bed granulator (suspension spray-coating method). The new ODTs were evaluated for their hardness, friability, thickness, internal structure (X-ray-CT scanning), in vivo disintegration time, and water absorption rate. Since MEC increases tablet hardness by increasing the contact frequency between the granules, the new ODTs could obtain high hardness (>50 N) and low friability (<0.5%) with relatively low compression force. In addition, fine capillary channels formed in ODTs facilitated the wicking action and enabled rapid disintegration in vivo (<30 s). On the other hand, since MEC has low hygroscopicity, the tablet hardness of ODTs containing MEC remained high for 1 month in high-humidity conditions.In conclusion, the new ODTs containing MEC developed in this study possessed superior properties for clinical use and are expected to be applied for a wide range of functionally released drugs for bitter taste masking, sustained release, and controlled release (pH-dependent film coating, matrix, and microcapsule).
Keywords: Orally disintegrating tablet; Micronized ethylcellulose; Physical strength; Capillary channels; Wicking action; Suspension spray-coating method;

Design of a pediatric oral formulation with a low proportion of hydrochlorothiazide by A. Santoveña; Z. Hernández-Paiz; J.B. Fariña (360-364).
It is a normal pediatric practice in community and hospital pharmacies to prepare a new drug formulation when no commercial forms of it are available. Any dose or stability control is usually done for these types of compounding formulations due to the effort which means to develop these types of tests in pharmacies. We have studied five different hydrochlorothiazide oral formulations prepared with traditional compounding techniques in pharmacies to treat heart failure and edemas in babies. A Standard Operating Procedure (SOP) was done for every suspension. After the strictly monitoring of the SOP, every suspension was subjected to quality control tests (pH, particle size, viscosity, dose content and stability). There is only one studied formulation that guarantees the correct dose administering and stability after 3 weeks stored at 5 °C and light protected. Both, the percentage of wetting agent and the viscosity of the suspensor vehicle in this formulation make the correct dose administering possible after the formulation is shaken.
Keywords: Hydrochlorothiazide oral suspension; Dose control; Stability;

The lack of suitable vectors for efficient nucleic acid delivery into target cells represents a major hurdle for the successful application of gene therapy. Cationic liposomes exhibit attractive features for gene delivery, but their efficacy is still unsatisfactory, particularly for in vivo applications, which justifies the drive to further improve their performance by developing novel and efficient formulations. In the present study, we generated a new formulation of lipoplexes through electrostatic association of folate (FA) to 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes, prepared at different lipid/DNA charge ratios, and explored their potential to mediate gene delivery. The optimal FA-lipoplex formulation was evaluated for its efficacy to mediate antitumoral activity upon application of HSV-tk suicide gene therapy, both in vitro and in an animal model of oral cancer. Our results demonstrate that FA-EPOPC:Chol/DNA lipoplexes were able to promote a great enhancement of transfection and high in vitro antitumoral activity compared to plain lipoplexes in two different cancer cell lines. Most importantly, a considerable reduction of tumor growth was achieved with the developed FA-lipoplexes as compared to that observed for control FA-lipoplexes or plain lipoplexes. Overall, our study shows that FA-EPOPC:Chol/DNA lipoplexes constitute a promising system for the successful application of suicide gene therapy aiming at treating solid tumors.
Keywords: Folate-associated lipoplexes; Cationic liposomes; Gene delivery; Suicide gene therapy; Antitumoral activity;

In this study, hyperbranched polyamidoamine (hPAMAM) was developed as a novel non-viral gene vector for the first time. The hPAMAM was synthesized using a modified “one-pot” method. DNA was then bound to hPAMAM at different weight ratios (whPAMAM/wDNA). The higher weight ratio could bring larger particle size and higher zeta potential of hPAMAM–DNA complexes. The encapsulated DNA was protected by hPAMAM from degradation for over 3 h. Under the optimal condition, high gene transfection efficiency could be achieved in COS7 (47.47 ± 1.42%) and HEK293 (40.8 ± 0.98%) cell lines. And hPAMAM showed rather minor cytotoxicity in vitro (cell viability = 91.38 ± 0.46% in COS7 and 92.38 ± 0.61% in HEK293). The hPAMAM mediated human vascular endothelial growth factor 165 (hVEGF165) gene transfected cells could express hVEGF165 stably for 14 days, with the peak expression at day 2. In conclusion, hPAMAM based gene delivery was economical, effective and biocompatible, and may serve as a promising non-viral vehicle for gene therapy.
Keywords: Hyperbranched polyamidoamine; Nanoparticle; Gene delivery; Cytotoxicity;

Solid-state nanoparticle coated emulsions for encapsulation and improving the chemical stability of all-trans-retinol by Nasrin Ghouchi-Eskandar; Spomenka Simovic; Clive A. Prestidge (384-391).
Submicron oil-in-water (o/w) emulsions stabilised with conventional surfactants and silica nanoparticles were prepared and freeze-dried to obtain free-flowing powders with good redispersibility and a three-dimensional porous matrix structure. Solid-state emulsions were characterised for visual appearance, particle size distribution, zeta potential and reconstitution properties after freeze-drying with various sugars and at a range of sugar to oil ratios. Comparative degradation kinetics of all-trans-retinol from freeze-dried and liquid emulsions was investigated as a function of storage temperatures. Optimum stability was observed for silica-coated oleylamine emulsions at 4 °C in their wet state. The half-life of all-trans-retinol was 25.66 and 22.08 weeks for silica incorporation from the oil and water phases respectively. This was ∼4 times higher compared to the equivalent solid-state emulsions with drug half-life of 6.18 and 6.06 weeks at 4 °C. Exceptionally, at a storage temperature of 40 °C, the chemical stability of the drug was 3 times higher in the solid-state compared to the wet emulsions which confirmed that freeze-drying is a promising approach to improve the chemical stability of water-labile compounds provided that the storage conditions are optimised.
Keywords: O/W emulsion; All-trans-retinol; Silica nanoparticles; Chemical stability; Freeze drying; Redispersibility;

Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro by Namrata Jain; Valérie Goldschmidt; Sule Oncul; Youri Arntz; Guy Duportail; Yves Mély; Andrey S. Klymchenko (392-400).
The development of new nonviral vectors characterized by high transfection efficiency and low cytotoxicity remains an important challenge in the field of gene delivery. Unsymmetrical bolaamphiphiles (bolas) appear as new emerging candidates for this application. In this work, new unsymmetrical bolas, bearing neutral lactonic acid and cationic ornithine residues at the two ends of a hydrophobic spacer, were synthesized and their properties were compared to analogues bearing a gluconic acid residue. The new bolas showed DNA binding and condensation at higher N/P ratios than their gluconic analogues, probably due to their larger neutral head group. Whereas the size of the complexes of the new bolas with DNA (bolaplexes) increased with N/P, as a result of charge neutralization, their formulations with DOPE at high N/P were of small size (ca. 200 nm). These DOPE formulations showed high transfection efficiency in different cell lines (HeLa, COS-7 and HepG2), close to that of jetPEI. Their cytotoxicity was relatively low, which allowed repetitive transfection in vitro. Fluorescence imaging showed that the bolaplexes bind rapidly to cell surface and internalize mainly through endocytosis. This work suggests a new type of efficient nonviral vectors based on bolaamphiphiles.
Keywords: Nonviral vectors; Bolaamphiphiles; Sugar head group; Gene delivery; Endocytosis;

Evaluation of intracellular trafficking and clearance from HeLa cells of doxorubicin-bound block copolymers by Kumiko Sakai-Kato; Keiko Ishikura; Yuki Oshima; Minoru Tada; Takuo Suzuki; Akiko Ishii-Watabe; Teruhide Yamaguchi; Nobuhiro Nishiyama; Kazunori Kataoka; Toru Kawanishi; Haruhiro Okuda (401-409).
New technologies are needed to deliver medicines safely and effectively. Polymeric nanoparticulate carriers are one such technology under investigation. We examined the intracellular trafficking of doxorubicin-bound block copolymers quantitatively and by imaging doxorubicin-derived fluorescence using confocal microscopy. The polymers were internalized by endocytosis and distributed in endosomal/lysosomal compartments and the endoplasmic reticulum; unlike free doxorubicin, the polymers were not found in the nucleus. Moreover, the ATP-binding cassette protein B1 (ABCB1) transporter may be involved in the efflux of the polymer from cells. This drug delivery system is attractive because the endogenous transport system is used for the uptake and delivery of the artificial drug carrier to the target as well as for its efflux from cells to medium. Our results show that a drug delivery system strategy targeting this endogenous transport pathway may be useful for affecting specific molecular targets.
Keywords: Doxorubicin-bound block copolymers; Intracellular trafficking; Confocal microscopy; Transporter; Endocytosis;

Predicting hydrophilic drug encapsulation inside unilamellar liposomes by Xiaoming Xu; Mansoor A. Khan; Diane J. Burgess (410-418).
Mathematical model (bottom) and schematic drawing of a unilamellar vesicle (right) and vesicles of various sizes distributed in sample medium (left). d, bilayer thickness; a, average lipid molecular area; 2r i , mean particle size; V in , internal volume; V 2, external volume.A mathematical model has been developed to predict the encapsulation efficiency of hydrophilic drugs in unilamellar liposomes, and will be useful in formulation development to rapidly achieve optimized formulations. This model can also be used to compare drug encapsulation efficiencies of liposomes prepared via different methods, and will assist in the development of suitable process analytical technologies to achieve real-time monitoring and control of drug encapsulation during liposome manufacturing for hydrophilic molecules. Liposome particle size as well as size distribution, lipid concentration, lipid molecular surface area, and bilayer thickness were used in constructing the model. Most notably, a Log-Normal probability function was utilized to account for sample particle size distribution. This is important to avoid significant estimation error. The model-generated predictions were validated using experimental results as well as literature data, and excellent correlations were obtained in both cases. A Langmuir balance study provided insight regarding the effect of media on the liposome drug encapsulation process. The results revealed an inverse correlation between media ionic strength and lipid average molecular area, which helps to explain the phenomenon of inverse correlation between media ionic strength and drug encapsulation efficiency. Finally, a web application has been written to facilitate use of the model allowing calculations to be easily performed. This model will be useful in formulation development to rapidly achieve optimized formulation.
Keywords: Liposome; Encapsulation efficiency; Lipid molecular area; Mathematical model; Langmuir balance; Log-Normal distribution;

Vitamin E or α-tocopherol is widely used as a strong antioxidant in many medical and cosmetic applications, but is rapidly degraded, because of its light, heat and oxygen sensitivity. In this study, we applied the nanoprecipitation method to prepare vitamin E-loaded nanocapsules, at laboratory-scale and pilot-scale. We scaled-up the preparation of nanocapsule with the membrane contactor technique. The effect of several formulation variables on the vitamin E-loaded nanocapsules properties (mean diameter, zeta potential, and drug entrapment efficiency) was investigated. The optimized formulation at laboratory-scale and pilot-scale lead to the preparation of vitamin E-loaded nanocapsules with mean diameter of 165 and 172 nm, respectively, and a high encapsulation efficiency (98% and 97%, respectively).
Keywords: Nanocapsules; Nanoprecipitation; Membrane contactor; α-Tocopherol; Vitamin E;

Collagenase-1 injection improved tumor distribution and gene expression of cationic lipoplex by Mako Kato; Yoshiyuki Hattori; Manami Kubo; Yoshie Maitani (428-434).
Elevated interstitial fluid pressure (IFP) in a tumor is a barrier to tumor accumulation of systemic delivery of nanocarriers. In this study, we investigated whether intravenous injection of type I collagenase (collagenase-1) reduced IFP in tumors and increased the accumulation and gene expression of cationic liposome/plasmid DNA complex (lipoplex) in tumors after intravenous injection into mice bearing mouse lung carcinoma LLC tumors. Collagenase-1 reduced the amount of type I collagen in the tumor, and significantly decreased IFP by 65% at 1 h after injection. Therefore, collagenase-1 induced 1.5-fold higher accumulation and 2-fold higher gene expression of lipoplex in tumors after intravenous injection. These findings indicated that intravenous injection of collagenase-1 improved the accumulation of lipoplex by decreasing IFP in tumors. These results support the potential use of collagen digestion as a strategy to improve systemic gene delivery into tumors.
Keywords: Collagenase; Collagen; Interstitial fluid pressure; Nonviral gene delivery; Lipoplex;

Nano-sized flake carboxymethyl cassava starch as excipient for solid dispersions by Xiepeng Lin; Wenxia Gao; Chenen Li; Jiuxi Chen; Chen Yang; Huayue Wu (435-439).
(a) SEM images of nano-size CMCS (DS = 1.15); (b) SEM image of solid dispersion composed of nano-size CMCS; (c) dissolution profiles of solid dispersion composed of nano-size CMCS.Nano-sized excipients were used in solid dispersions (SD) to enhance the dissolution rate of poorly water-soluble drug in this study. Nano-sized flake carboxymethyl cassava starch (CMCS) was firstly synthesized under ultrasonic irradiation. Then acetylsalicylic acid (ASA) was selected as water insoluble drug model to prepare solid dispersions using three different kinds of excipients. SD1 was prepared using native cassava starch as carrier. SD2 and SD3 were prepared using nano-sized CMCS (degree substitution, DS = 1.15, 100–400 nm) and micro-sized CMCS (DS = 0.36, 8–28 μm), respectively. These solid dispersions were characterized by powder X-ray diffractometry, scanning electron micrographs and dissolution. The results suggested that the SD2 prepared by nano-sized CMCS had much better dispersion capability for the drug than the other two solid dispersions. And the dissolution rate of SD2 was considerably higher than that of pure drug. These results indicated that the nanoscale CMCS was a kind of good carrier for solid dispersion to improve the solubility of poorly water-soluble drugs.
Keywords: Solid dispersion; Nano-size; Carboxymethyl cassava starch (CMCS);

Physicochemical characterization of curcuminoid-loaded solid lipid nanoparticles by Andreas Noack; Gerd Hause; Karsten Mäder (440-451).
Curcuminoid-loaded solid lipid nanoparticles (SLN) were produced by melt-homogenization. The used lipid matrices were medium chain triglycerides, trimyristin and tristearin. The resulting nanoparticles had an anisometric shape and a platelet-like structure. Curcuminoid-loaded trimyristin particles did not solidify when stored at room temperature. The supercooled state of trimyristin was studied by DSC and 1H NMR experiments. A partial recrystallization of the lipid matrix was detected but no change of the mobility of the lipid was noted. Nanoparticles based on tristearin had an α- and β-modification which was subsequently converted into the stable β-phase. Curcuminoids did neither influence the melting behavior nor the crystalline or geometric structure of the particles. The interactions between the curcuminoids and the lipid matrix were investigated by Raman and fluorescence spectroscopy. The shape of the curcuminoid bands in the Raman spectra suggested that the drug was in an amorphous state. The fluorescence spectra showed an effect of the lipid matrix on fluorescence properties of the curcuminoids. It was further demonstrated that the drug was not secluded by the solid lipid matrix, but it was influenced by the surrounding aqueous environment. Fluorescence anisotropy measurements revealed a decreased mobility of the curcuminoids within the nanodispersions. From the results of Raman and fluorescence measurements it was concluded that the drug was mainly located on the surface of the crystalline particles.
Keywords: Nanoparticles; SLN; Curcumin; High pressure homogenization; Raman spectroscopy; Fluorescence;

Tocol modified glycol chitosan for the oral delivery of poorly soluble drugs by Nicolas Duhem; Julien Rolland; Raphaël Riva; Pierre Guillet; Jean-Marc Schumers; Christine Jérome; Jean-François Gohy; Véronique Préat (452-460).
The aim of this study was to develop tocol derivatives of chitosan able (i) to self-assemble in the gastrointestinal tract and (ii) to enhance the solubility of poorly soluble drugs. Among the derivatives synthesized, tocopherol succinate glycol chitosan (GC-TOS) conjugates spontaneously formed micelles in aqueous solution with a critical micelle concentration of 2 μg mL−1. AFM and TEM analysis showed that spherical micelles were formed. The GC-TOS increased water solubility of 2 model class II drugs. GC-TOS loading efficiency was 2.4% (w/w) for ketoconazole and 0.14% (w/w) for itraconazole, respectively. GC-TOS was non-cytotoxic at concentrations up to 10 mg mL−1. A 3.4-fold increase of the apparent permeation coefficient of ketoconazole across a Caco-2 cell monolayer was demonstrated. Tocol polymer conjugates may be promising vehicles for the oral delivery of poorly soluble drugs.
Keywords: Chitosan; Amphiphilic chitosan; Tocopherol; Micelles; Oral drug delivery; Poorly soluble drugs;

Baicalein loaded in tocol nanostructured lipid carriers (tocol NLCs) for enhanced stability and brain targeting by Ming-Jun Tsai; Pao-Chu Wu; Yaw-Bin Huang; Jui-Sheng Chang; Chin-Lin Lin; Yi-Hung Tsai; Jia-You Fang (461-470).
The objective of the present work was to investigate the specific brain targeting of baicalein by intravenous injection after incorporation into nanostructured lipid carriers (NLCs). The NLC system, composed of tripalmitin, Gelucires, vitamin E, phospholipids, and poloxamer 188 (referred to as tocol NLCs), was characterized in terms of its physicochemical properties, differential scanning calorimetry (DSC), stability, in vivo pharmacokinetics, and brain distribution. The lipid nanoparticles were spherical with an average size of ∼100 nm. The zeta potential of the nanoparticles was about −50 mV. DSC studies suggested that the majority of the inner cores of tocol NLCs had a slightly disordered crystal arrangement. The nanoparticulate dispersions demonstrated good physical stability during storage for 6 days. The incorporation of vitamin E in the formulations greatly reinforced baicalein's stability. The aqueous control and tocol NLCs were intravenously administered to rats. The plasma level of baicalein in NLCs was much higher and the half-life much longer than those in the free control. In the experiment on the brain distribution, NLCs respectively revealed 7.5- and 4.7-fold higher baicalein accumulations compared to the aqueous solution in the cerebral cortex and brain stem. Greater baicalein accumulations with NLCs were also detected in the hippocampus, striatum, thalamus, and olfactory tract. A 2–3-fold increase in baicalein amounts were achieved in these regions. Tocol NLCs improved baicalein's stability and the ability of baicalein to penetrate the brain; thus, this is a promising drug-targeting system for the treatment of central nervous system disorders.
Keywords: Nanostructured lipid carriers; Baicalein; Vitamin E; Gelucire; Brain targeting;

Preparation, characterization and in vivo assessment of the bioavailability of glycyrrhizic acid microparticles by supercritical anti-solvent process by Xiaoyu Sui; Wei Wei; Lei Yang; Yuangang Zu; Chunjian Zhao; Lin Zhang; Fengjian Yang; Zhonghua Zhang (471-479).
In this study, glycyrrhizic acid (GA) microparticles were successfully prepared using a supercritical anti-solvent (SAS) process. Carbon dioxide and ethanol were used as the anti-solvent and solvent, respectively. The influences of several process parameters on the mean particle size (MPS), particle size distribution (PSD) and total yield were investigated. Processed particle sizes gradually decreased as temperature and solution flow rate increased. In addition, processed particle sizes increased from 119 to 205 nm as GA concentration increased. However, CO2 flow rate did not significantly affect particle size. The optimized process conditions were applied, those included temperature (65 °C), pressure (250 bar), CO2 and drug solution flow rate (15 and 8 mL min−1), drug concentration in ethanol (20 mg mL−1). Microparticles with a span of PSD ranging from 95 and 174 nm, MPS of 128 nm were obtained, and total yield was 63.5%. The X-ray diffraction patterns of glycyrrhizic acid microparticles show apparent amorphous nature. Fourier transform infrared (FT-IR) spectroscopy results show that no chemical structural changes occurred. The in vitro dissolution tests showed that the GA microparticles exhibited great enhancement of dissolution performance when compared to GA original drug. Furthermore, the in vivo studies revealed that the microparticles provided improved pharmacokinetic parameter after oral administration to rats as compared with original drug.
Keywords: Glycyrrhizic acid; Microparticles; Supercritical anti-solvent; Oral bioavailability;

α-Tocopherol succinate hydrophobically modified chitosan (CS-TOS) containing 17 α-tocopherol groups per 100 anhydroglucose units was synthesized by coupling reaction. The formation of CS-TOS was confirmed by 1H NMR and FT-IR analysis. In aqueous medium, the polymer could self-aggregate to form micelles, and the critical micelle concentration (CMC) was determined to be 5.8 × 10−3  mg/ml. Transmission electron microscopy (TEM) observation revealed that both bare and paclitaxel-loaded micelles were near spherical in shape. The mean particle size and zeta potential of drug-loaded micelles were about 78 nm and +25.7 mV, respectively. The results of DSC and XRD analysis indicated that paclitaxel was entrapped in the micelles in molecular or amorphous state. In vitro cytotoxicity and hemolysis study revealed the effectiveness and safety of this delivery system, which was further confirmed by the in vivo antitumor evaluations. It can be concluded that the CS-TOS was a potential micellar carrier for paclitaxel.
Keywords: Chitosan; α-Tocopherol succinate; Hydrophobically modified; Polymeric micelle; Paclitaxel;

Boron nitride nanotubes radiolabeled with 99mTc: Preparation, physicochemical characterization, biodistribution study, and scintigraphic imaging in Swiss mice by Daniel Crístian Ferreira Soares; Tiago Hilário Ferreira; Carolina de Aguiar Ferreira; Valbert Nascimento Cardoso; Edésia Martins Barros de Sousa (489-495).
In the present study, boron nitride nanotubes (BNNTs) were synthesized from an innovative process and functionalized with a glycol chitosan polymer in CDTN (Centro de Desenvolvimento da Tecnologia Nuclear) laboratories. As a means of studying their in vivo biodistribution behavior, these nanotubes were radiolabeled with 99mTc and injected in mice. Their size, distribution, and homogeneity were determined by photon correlation spectroscopy (PCS), while their zeta potential was determined by laser Doppler anemometry. The morphology and structural organization were evaluated by scanning electron microscopy (SEM). The functionalization in the nanotubes was evaluated by thermogravimetry analysis (TGA) and Fourier transformer infrared spectroscopy. The results showed that BNNTs were obtained and functionalized successfully, reaching a mean size and dispersity deemed adequate for in vivo studies. The BNNTs were also evaluated by ex vivo biodistribution studies and scintigraphic imaging in healthy mice. The results showed that nanostructures, after 24 h, having accumulated in the liver, spleen and gut, and eliminated via renal excretion. The findings from this study reveal a potential application of functionalized BNNTs as new potential drugs or radioisotope nanocarriers to be applied in therapeutic procedures.
Keywords: Functionalized boron nitride nanotubes; Physicochemical characterization; Biodistribution study and scintigraphic image;

Preparation and characterization of hydroxypropyl methyl cellulose films containing stable BCS Class II drug nanoparticles for pharmaceutical applications by Lucas Sievens-Figueroa; Anagha Bhakay; Jackeline I. Jerez-Rozo; Natasha Pandya; Rodolfo J. Romañach; Bozena Michniak-Kohn; Zafar Iqbal; Ecevit Bilgili; Rajesh N. Davé (496-508).
The design and feasibility of a simple process of incorporating stable nanoparticles into edible polymer films is demonstrated with the goal of enhancing the dissolution rate of poorly water soluble drugs. Nanosuspensions produced from wet stirred media milling (WSMM) were transformed into polymer films containing drug nanoparticles by mixing with a low molecular weight hydroxylpropyl methyl cellulose (HPMC E15LV) solution containing glycerin followed by film casting and drying. Three different BCS Class II drugs, naproxen (NPX), fenofibrate (FNB) and griseofulvin (GF) were studied. The influence of the drug molecule on the film properties was also investigated. It was shown that film processing methodology employed has no effect on the drug crystallinity according to X-ray diffraction (XRD) and Raman spectroscopy. Differences in aggregation behavior of APIs in films were observed through SEM and NIR chemical imaging analysis. NPX exhibited the strongest aggregation compared to the other drugs. The aggregation had a direct effect on drug content uniformity in the film. Mechanical properties of the film were also affected depending on the drug–polymer interaction. Due to strong hydrogen bonding with the polymer, NPX exhibited an increase in Young's Modulus (YM) of approximately 200%, among other mechanical properties, compared to GF films. A synergistic effect between surfactant/polymer and drug/polymer interactions in the FNB film resulted in an increase of more than 600% in YM compared to the GF film. The enhancement in drug dissolution rate of films due to the large surface area and smaller drug particle size was also demonstrated.
Keywords: Nanosuspensions; Media milling; BCS Class II; Pharmaceutical films; Hydroxylpropyl methyl cellulose;

Cancer therapy improvement with mesoporous silica nanoparticles combining targeting, drug delivery and PDT by Magali Gary-Bobo; Ouahiba Hocine; David Brevet; Marie Maynadier; Laurence Raehm; Sébastien Richeter; Virginie Charasson; Bernard Loock; Alain Morère; Philippe Maillard; Marcel Garcia; Jean-Olivier Durand (509-515).
The synthesis of Mesoporous Silica Nanoparticles (MSN) covalently encapsulating fluoresceine or a photosensitizer, functionalized with galactose on the surface is described. Confocal microscopy experiments demonstrated that the uptake of galactose-functionalized MSN by colorectal cancer cells was mediated by galactose receptors leading to the accumulation of the nanoparticles in the endosomal and lysosomal compartments. The MSN functionalized with a photosensitizer and galactose were loaded with the anti-cancer drug camptothecin. Those MSN combining drug delivery and photodynamic therapy were tested on three cancer cell lines and showed a dramatic enhancement of cancer cell death compared to separate treatments.
Keywords: Mesoporous; Silica; Nanoparticles; Photodynamic therapy; Targeting; Drug delivery; Carbohydrates;

Biodegradable nanoparticles mimicking platelet binding as a targeted and controlled drug delivery system by Soujanya Kona; Jing-Fei Dong; Yaling Liu; Jifu Tan; Kytai T. Nguyen (516-524).
This research aims to develop targeted nanoparticles as drug carriers to the injured arterial wall under fluid shear stress by mimicking the natural binding ability of platelets via interactions of glycoprotein Ib-alpha (GPIbα) of platelets with P-selectin of damaged endothelial cells (ECs) and/or with von Willebrand factor (vWF) of the subendothelium. Drug-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles were formulated using a standard emulsion method and conjugated with glycocalicin, the external fraction of platelet GPIbα, via carbodiimide chemistry. Surface-coated and cellular uptake studies in ECs showed that conjugation of PLGA nanoparticles, with GPIb, significantly increased nanoparticle adhesion to P-selectin- and vWF-coated surfaces as well as nanoparticle uptake by activated ECs under fluid shear stresses. In addition, effects of nanoparticle size and shear stress on adhesion efficiency were characterized through parallel flow chamber studies. The observed decrease in bound nanoparticle density with increased particle sizes and shear stresses is also explained through a computational model. Our results demonstrate that the GPIb-conjugated PLGA nanoparticles can be used as a targeted and controlled drug delivery system under flow conditions at the site of vascular injury.
Keywords: Biodegradable nanoparticles; Endothelial cells; Platelets; Glycoprotein Ib; Parallel plate flow system;

Stability of lyophilized siRNA nanosome formulations by Anup K. Kundu; Partha K. Chandra; Sidhartha Hazari; Grace Ledet; Yashoda V. Pramar; Srikanta Dash; Tarun K. Mandal (525-534).
Compare to non-transfected control cells and non-targeted siRNA (i.e. si-74 nanosome) or IFN treated cells, the stored siRNA nanosomes along with freshly prepared siRNA nanosomes (L) and siRNA nanosomes (P) showed significant knock-down efficiency of IFN-α resistant HCV during long term storage period upto 3 months.The goal of this study is to evaluate the stability of lyophilized siRNA formulations. The gene silencing efficiency of a stored lyophilized siRNA formulation (i.e. siRNA nanosomes) was evaluated in interferon-α (IFN-α) resistant hepatitis C virus (HCV) at different time points up to three months in an in vitro cell culture model and compared with freshly prepared siRNA formulations. Novel siRNA sequences were encapsulated within nanosize liposomes following condensation with protamine sulfate. The siRNA encapsulated nanosomes were lyophilized and stored at 4 °C for 3 months, along with liquid liposomes (L) and lyophilized liposome powder (P) which were subsequently used to prepare siRNA nanosomes (L) and siRNA nanosomes (P), respectively at different time points. Physiochemical and biological properties of all three formulations were compared at different time points up to 3 months. The particle size of the stored siRNA nanosomes (642 ± 25 nm) was considerably larger initially in comparison with the liquid liposomes (134 ± 5 nm) and lyophilized liposomes (118 ± 3). However, the particle size gradually became smaller over time (413 ± 128 nm by the third month). The zeta potential of all three formulations was initially very high (> +40 mV), followed by a gradual decrease over time. The amount of siRNA in the stored siRNA nanosomes decreased ∼18% during the 3 month storage period (1.16 ± 0.03 nmol initially on day 1 vs. 0.95 ± 0.04 nmol after 3 months). With respect to biological potency, all three formulations were significantly effective to knock-down HCV throughout the storage time. The cell viability was well-maintained throughout this period. Thus, this study indicates that the stored lyophilized siRNA formulation is as effective as the fresh preparation and that long-term storage could be a viable option to treat deadly diseases such as cancer and viral infection.
Keywords: siRNA delivery; Hepatitis C virus; Nanosome; Lyophilization; Stability;

Numerous reports on the enhancement effect of cyclodextrins (CDs) on the skin permeation of dermally applied drugs exist, the majority of which is based on in vitro diffusion cell studies. The specific experimental setup of such studies may skew the obtained results, which is rarely discussed in the context of CD studies. Thus, the aim of this work was to conduct a systematic in vitro investigation of the permeation enhancement potential of γ-CD on a steroidal drug from a nanoemulsion. The role of critical diffusion cell parameters such as the dose of application, occlusive conditions, the nature of the receptor medium and the skin thickness were investigated. The results showed that significantly enhanced skin permeation rates of fludrocortisone acetate were indeed caused by 1% (w/w) of γ-CD at both finite and infinite dose conditions. At 0.5% (w/w) of γ-CD, significant enhancement was only achieved at infinite dose application. Additional in vitro tape stripping experiments confirmed these tendencies, but the observed effects did not reach statistical significance. It may be concluded that the full permeation enhancement potential of the CD as observed in the franz-cell setup can only be realised at infinite dose conditions while preserving the formulation structure.
Keywords: γ-Cyclodextrin; Skin penetration; Skin permeation; Fludrocortisone acetate; Permeation enhancement; Franz-type diffusion cell;

Contour plot for EE% with respect to lipid concentration and drug concentration. Red star points are central composite design points. Within the circle (red star points) is the design space for prediction of EE%. Additional data points (white disks) are included to evaluate the accuracy and robustness of the design space.Two statistical designs were used in this case study as part of an investigation into the feasibility and the advantages of applying QbD concepts to liposome-based complex parenteral controlled release systems containing a hydrophilic active pharmaceutical ingredient (API). The anti-viral drug Tenofovir was used as a model compound. First design (Plackett–Burman) was used to screen eight high-risk variables obtained from risk analysis and assess their impact on liposome characteristics (drug encapsulation efficiency, particle size, and physical stability). It was discovered that out of eight high-risk variables only lipid and drug concentration had significant effects on the drug encapsulation efficiency. This allowed the use of a central composite design (CCD) (with more predictive capability) to fully elucidate the relationship between lipid concentration, drug concentration and encapsulation efficiency. On comparing the CCD model generated response surface with additional data points, the accuracy and robustness of the model was confirmed. Using this developed model, the design space for Tenofovir liposomes preparation has been established in a laboratory setting, within which the preparation variability is minimized. With regard to sample storage stability, it was shown that at 4 °C the prepared Tenofovir liposomes, dispersed in aqueous phase, achieved stability for at least 2 years. These principles can be applied to liposomes containing other hydrophilic APIs, and can provide time and cost saving to industrial formulation scientists, and result in a more robust liposome preparation process.
Keywords: Liposome; QbD; Tenofovir; Encapsulation efficiency; Plackett–Burman; Central composite design; Response surface method (RSM); Design space;

Lipomer of doxorubicin hydrochloride for enhanced oral bioavailability by Derajram M. Benival; Padma V. Devarajan (554-561).
The present study discusses design of doxorubicin hydrochloride (Dox) loaded lipid based nanocarrier (LIPOMER) for oral delivery. High entrapment (>90%) and high loading (38.11 ± 0.37% w/w) of hydrophilic Dox in lipid nanocarrier of polyglyceryl-6-distearate was achieved using poly(methyl vinyl ether-co-maleic anhydride) (Gantrez® AN 119) and a modified nanoprecipitation method. Dox-LIPOMER revealed nanosize (314 ± 16.80 nm) and negative zeta potential (−25.00 ± 2.41 mV). Dox-LIPOMER exhibits sustained release in vitro and was influenced by ionic strength of dissolution medium. DSC and XRD studies suggested amorphous nature of Dox in LIPOMER. TEM revealed spherical morphology of Dox-LIPOMER. Dox-LIPOMER was stable up to 12 months at 25 °C/60% RH. A 384% enhancement in oral bioavailability compared to Dox solution was observed following Dox-LIPOMER administration at 10 mg/kg body weight. Superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) assay data of heart and kidney tissues of rats treated with Dox-LIPOMER were comparable with untreated rats. Dox-LIPOMER represents a potential safe drug delivery system for oral administration.
Keywords: Doxorubicin hydrochloride; Lipid nanocarrier; Gantrez; Lipomer; Drug loading; Oral bioavailability;

Functionalized (poly(ɛ-caprolactone))2-poly(ethylene glycol) nanoparticles with grafting nicotinic acid as drug carriers by Jiraphong Suksiriworapong; Kittisak Sripha; Jörg Kreuter; Varaporn Buraphacheep Junyaprasert (562-570).
Nicotinic acid was grafted on (poly(ɛ-caprolactone))2-poly(ethylene glycol) copolymers that were used for the preparation of nanoparticles with the objectives to monitor particle size and to optimize the drug loading capacity as well as the release profile of the particles. Increasing amounts of grafting nicotinic acid increased the particle size as a result of an enhanced hydrophobicity of the copolymer. Ibuprofen and indomethacin with two different molecular characteristics were selected as model drugs to be bound to the nanoparticles. The presence of grafting nicotinic acid enhanced the loading capacity for both drugs compared to the nanoparticles without nicotinic acid. However, no correlation between amount of grafting nicotinic acid and loading capacity was observed. The release characteristic of both drugs was fitted to the Higuchi model indicating Fickian diffusion. The release characteristic of indomethacin mainly depended on the crystalline property of the copolymer whereas that of ibuprofen was additionally influenced by the hydrogen bonding between drug and grafted copolymer.
Keywords: Ibuprofen; Indomethacin; Nanoparticle; Nicotinic acid; Poly(ɛ-caprolactone); Poly(ethylene glycol);

Molecular-level characterization of probucol nanocrystal in water by in situ solid-state NMR spectroscopy by Junying Zhang; Kenjirou Higashi; Waree Limwikrant; Kunikazu Moribe; Keiji Yamamoto (571-576).
The molecular state of colloidal probucol nanoparticles with additives was evaluated by 13C in situ solid-state NMR spectroscopy. The nanoparticles were obtained by dispersing a ternary co-ground mixture of probucol/polyvinylpyrrolidon (PVP)/sodium dodecyl sulfate (SDS) in water. Their mean particle size was found to be approximately 150 nm by dynamic light scattering and cryogenic-scanning electron microscopy measurements. The results of the 13C in situ solid-state NMR spectroscopy showed that probucol existed in the crystalline state (form I) in water. 13C liquid-state NMR results indicated that PVP and SDS interacted with probucol in water. Their broad signals suggested that the surface interaction of the probucol nanocrystal with PVP and SDS stabilized the suspension. In addition, a freeze-dried sample of the suspension was studied by 13C solid-state NMR and powder X-ray diffraction experiments, which confirmed the presence of the probucol nanocrystals. The combination of the in situ solid-state, solid-state, and liquid-state NMR measurement results provided molecular-level insights about the role of intermolecular interactions in the design of nanoformulations.
Keywords: In situ solid-state NMR; Cryo-SEM; Nanocrystal; PXRD; Co-grinding;

Optimization of a spray drying process to prepare dry powder microparticles containing plasmid nanocomplex by N. Mohajel; A. Roholamini Najafabadi; K. Azadmanesh; A. Vatanara; E. Moazeni; A. Rahimi; K. Gilani (577-585).
Successful gene delivery to the lung depends not only on precise and effective design of a nanosized nucleic acid delivery system but also on well engineered liquid or solid microparticles. In present work, we tried to statistically optimize spray dried formulations of low molecular weight chitosan–plasmid nanocomplexes via a D-optimal design with respect to five critical responses: yield of the process, microparticle sizes, nanocomplex sizes, DNA stability and relative transfection efficiency. Nonocomplex formulations prepared by different amounts of solid contents and leucine ratio, and spray dried immediately with varying inlet temperature, feed rate and spray air flow rate. Mean results fitted to 2FI models except for relative transfection efficiency, which fitted in a quadratic model. According to the fitted models, the most important pure factors influencing each response determined to be feed rate for yield and DNA stability, feed fluid concentration for microparticle size, inlet temperature for nanoparticle size and leucine concentration for relative transfection efficiency. However, two-factor interactions have more important roles in microparticle size, nanocomplex size and DNA stability. It was concluded that the optimized formulation could be obtained when all the independent variables were at their maximum tested values, except for feed fluid concentration, which should be in its middle point.
Keywords: Gene delivery; Dry powder formulation; Spray drying; Nanocomplex;

In vitro and in vivo evaluation of camptothecin nanosuspension: A novel formulation with high antitumor efficacy and low toxicity by Liping Yao; Xiuhua Zhao; Qingyong Li; Yuangang Zu; Yujie Fu; Baishi Zu; Xiangdong Meng; Chen Liu (586-588).
The purpose of this study was to evaluate the in vitro and in vivo antitumor efficacy and the dose dependent toxicity of camptothecin nanosuspension (Nano-CPT) comparing with that of topotecan (TPT). A novel supercritical antisolvent (SAS) process-high pressure homogenization technique has been developed to prepare Nano-CPT. The cytotoxicity of Nano-CPT and TPT was investigated against MCF-7, HCT-8, and PC-3 cell lines using MTT assay, antitumor activity in vivo were evaluated against HCT-8 xenograft model, and the dose dependent toxicity in vivo during the treatment were investigated by body weight changes and relative organ weight variations. The Nano-CPT presents about 6 times in vitro cytotoxicity active than TPT against cell lines MCF-7, nearly the same in vivo antitumor activity with TPT and lower toxicity. The results confirm that Nano-CPT is a novel potential formulation with high antitumor efficacy and low toxicity.
Keywords: Camptothecin nanosuspension; Topotecan; Cytotoxicity; Antitumor activity; Dose dependent toxicity;

Letter to the Editor by Brian E. Jones; Fridrun Podczeck (589-592).

Corrigendum to “Delivery systems for natural antioxidant compounds: Archaeosomes and archaeosomal hydrogels characterization and release study” [Int. J. Pharm. 421 (2011) 321–331] by Ana González-Paredes; Beatriz Clarés-Naveros; Ma Adolfina Ruiz-Martínez; Juan José Durbán-Fornieles; Alberto Ramos-Cormenzana; Mercedes Monteoliva-Sánchez (593-594).