International Journal of Pharmaceutics (v.426, #1-2)

Thermosensitive eyedrops containing platelet lysate for the treatment of corneal ulcers by Giuseppina Sandri; Maria Cristina Bonferoni; Silvia Rossi; Franca Ferrari; Michela Mori; Claudia Del Fante; Cesare Perotti; Carla Caramella (1-6).
Corneal lesions cause significant pain and visual impairment and, in many cases, are unresponsive to conventional treatments. Platelet lysate (PL) is an haemoderivative rich in growth factors (GFs) that are released by platelets after freeze-thawing destruction of platelet rich plasma (PRP). The aim of the present work was to develop thermosensitive and mucoadhesive eyedrops to maintain and prolong the contact of platelet lysate (PL) with cornea ulcers.A sterile vehicle based on chondroitin sulphate sodium (CS) and hydroxypropylmethyl cellulose (HPMC) was developed. An extemporaneous loading of the vehicle with PL was performed and the obtained formulation was able to quickly thermogelify at about 32 °C and was characterized by good mucoadhesive properties. ELISA evidenced that the growth factor PDGF AB was compatible with the vehicle and stable in the formulation up to 15 days of storage at 2–8 °C.In vitro wound healing and proliferation test (performed using rabbit corneal epithelial cells (RCE)) showed that the formulation enhanced cell growth and put in evidence a synergistic effect of CS and PL in stimulating cell proliferation.The overall results indicate that PL loaded in thermosensitive and mucoadhesive eyedrops can be profitably employed to treat corneal lesions.
Keywords: Growth factors; Platelet lysate; Cornea; Wound healing; Thermogelling; Mucoadhesion;

A physiologically pertinent in vitro model is urgently needed for probing interactions between inhaled drugs and the organic cation transporters (OCT) in the bronchial epithelium. This study evaluated OCT expression, functionality, inhibition by common inhaled drugs and impact on formoterol transepithelial transport in layers of human bronchial epithelial Calu-3 cells grown at an air–liquid interface. 21 day old Calu-3 layers expressed OCT1, OCT3, OCTN1 and OCTN2 whereas OCT2 could not be detected. Quantification of the cellular uptake of the OCT substrate ASP+ in presence of inhibitors suggested several OCT were functional at the apical side of the cell layers. ASP+ uptake was reduced by the bronchodilators formoterol, salbutamol (albuterol), ipratropium and the glucocorticoid budesonide. However, the OCT inhibitory properties of the two β2-mimetics were suppressed at therapeutically relevant concentrations. The absorptive permeability of formoterol across the cell layers was enhanced at a high drug concentration shown to decrease ASP+ uptake by ∼50% as well as in presence of the OCT inhibitor tetraethylammonium (TEA). Secretory transport was unaffected by the drug concentration but was reduced by TEA. Our data indicate air-interfaced Calu-3 layers offer a low-cost in vitro model suitable for assessing inhaled drug–OCT interactions in the bronchial epithelium.
Keywords: Organic cation transporters; Calu-3 cells; Bronchial epithelium; Permeability; Carrier-mediated transport; Pulmonary absorption; In vitro models;

The purpose of this work was to explore NIR spectroscopy as a PAT tool to monitor the formation of ibuprofen and nicotinamide cocrystals during extrusion based solvent free continuous cocrystallization (SFCC). Drug and co-former were gravimetrically fed into a heated co-rotating twin screw extruder to form cocrystals. Real-time process monitoring was performed using a high temperature NIR probe in the extruder die to assess cocrystal content and subsequently compared to off-line powder X-ray diffraction measurements. The effect of processing variables, such as temperature and mixing intensity, on the extent of cocrystal formation was investigated. NIR spectroscopy was sensitive to cocrystal formation with the appearance of new peaks and peak shifts, particularly in the 4800–5200 cm−1 wave-number region. PXRD confirmed an increased conversion of the mixture into cocrystal with increase in barrel temperature and screw mixing intensity. A decrease in screw rotation speed also provided improved cocrystal yield due to the material experiencing longer residence times within the process. A partial least squares analysis in this region of NIR spectrum correlated well with PXRD data, providing a best fit with cocrystal conversion when a limited range of process conditions were considered, for example a single set temperature. The study suggests that NIR spectroscopy could be used to monitor cocrystal purity on an industrial scale using this continuous, solvent-free process.
Keywords: Cocrystals; NIR spectroscopy; PAT; QbD; Process analytics; Hot melt extrusion; Twin screw extrusion; Ibuprofen; Nicotinamide;

Preparation and in vitro/in vivo evaluation of sustained-release venlafaxine hydrochloride pellets by Yang Liu; Yinghua Sun; Jin Sun; Nana Zhao; Mingyu Sun; Zhonggui He (21-28).
The objective of this study was to prepare different venlafaxine hydrochloride sustained-release products and to elucidate the influence of composition of the coating film on the in vitro drug release profiles and in vivo pharmacokinetics. Pellets were prepared by a standardized process of extrusion/spheronization. A selected fraction size (0.8–1.0 mm diameter) of pellets of each formulation was coated with Eudragit® NE30D or ethylcellulose (10 cps). Many efforts have been made to tailor drug release rate by choosing different coating materials, different percent of pore forming components and coating weight variation to achieve a desired sustained-release effect. The dissolution studies were performed and data were analyzed in terms of cumulative release as a function of time. The influence on the release of venlafaxine from sustained-release capsules was observed in dissolution media of different pH and gradient pH. Scanning electron microscope (SEM) micrographs revealed morphological changes of the pellet coating surface which were related to in vitro drug release profiles. The relative bioavailability for Formulation 1 and Formulation 2 was evaluated in six healthy beagle dogs after oral administration in a fast state using sustained-release capsules (Effexor® XR) as a reference. The results suggested that Formulation 1 and Formulation 2 both had better bioavailability compared with Effexor® XR. It could be found that there existed quite difference in the in vivo release and oral absorption performances, despite the similar in vitro drug release behavior for the two formulations. It might be attributable to complex in vivo environment and then variation in the release behavior. Thus differences in the film micro-structure and surface roughness caused by aqueous dispersion and organic solvent coating techniques strongly influence the in vivo release and oral absorption performances.
Keywords: Venlafaxine; Extrusion/spheronization; Eudragit® NE30D; Ethylcellulose; In vitro dissolution; In vivo studies;

A new expanded solubility parameter approach by Emmanuel Stefanis; Costas Panayiotou (29-43).
The partial or Hansen solubility parameters (HSP) are important properties of the various substances and very useful tools for the selection of their solvents or the prediction of their behaviour in numerous applications. Their design and evaluation relies on the basic rule of “similarity matching” for solubility. The present work attempts to enhance the capacity of HSPs by incorporating into their evaluation the other basic rule of solubility, namely, the rule of “complementarity matching”. This is done in a simple and straightforward manner by splitting the hydrogen bonding HSP into its acidic or proton donor component and its basic or proton acceptor one. The splitting is based on the third σ-moments of the screening charge distributions or sigma profiles of the quantum-mechanics based COSMO-RS theory. The whole development and application does not involve any sophisticated calculations or any strong specific background. The new method has been applied to a variety of solubility data for systems of pharmaceutical interest in order to verify the significant improvement over the classical HSP approach. The application of the new method requires, of course, the knowledge of the HSPs. For this reason, in is presented an updated version of a robust and reliable group-contribution method for the calculation of the HSPs. The key features of this combined tool are critically discussed.
Keywords: Partial solubility parameter; Hydrogen-bonding; COSMO-RS theory; Group-contribution method;

Vaginal distribution and retention of a multiparticulate drug delivery system, assessed by gamma scintigraphy and magnetic resonance imaging by Samata Mehta; Hans Verstraelen; Kathelijne Peremans; Geert Villeirs; Simon Vermeire; Filip De Vos; Els Mehuys; Jean Paul Remon; Chris Vervaet (44-53).
Retention of cream (□) and pellet (■) formulation in the vaginal area after vaginal administration to sheep (n  = 6). Spreading of the cream (□) and pellet (■) formulation after intravaginal administration to women.For any new vaginal dosage form, the distribution and retention in the vagina has to be assessed by in vivo evaluation. We evaluated the vaginal distribution and retention of starch-based pellets in sheep as live animal model by gamma scintigraphy (using Indium-111 DTPA as radiolabel) and in women via magnetic resonance imaging (MRI, using a gadolinium chelate as contrast agent). A conventional cream formulation was used as reference in both studies.Cream and pellets were administered to sheep (n  = 6) in a two period-two treatment study and to healthy female volunteers (n  = 6) via a randomized crossover trial. Pellets (filled into hard gelatin capsule) and cetomacrogol cream, both labeled with Indium-111 DTPA (for gamma scintigraphy) or with gadolinium chelate (for MRI) were evaluated for their intravaginal distribution and retention over a 24 h period. Spreading in the vagina was assessed based on the part of the vagina covered with formulation (expressed in relation to the total vaginal length). Vaginal retention of the formulation was quantified based on the radioactivity remaining in the vaginal area (sheep study), or qualitatively evaluated (women study).Both trials indicated a rapid distribution of the cream within the vagina as complete coverage of the vaginal mucosa was seen 1 h after dose administration. Clearance of the cream was rapid: about 10% activity remained in the vaginal area of the sheep 12 h post-administration, while after 8 h only a thin layer of cream was detected on the vaginal mucosa of women. After disintegration of the hard gelatin capsule, the pellet formulation gradually distributed over the entire vaginal mucosa. Residence time of the pellets in the vagina was longer compared to the semi-solid formulation: after 24 h 23 ± 7% radioactivity was detected in the vaginal area of the sheep, while in women the pellet formulation was still detected throughout the vagina.A multi-particulate system containing starch-based pellets was identified as a promising novel vaginal drug delivery system, resulting in complete coverage of the vaginal mucosa and long retention time.
Keywords: Intravaginal drug delivery; Magnetic resonance imaging; Gamma scintigraphy; Starch pellets;

Pharmaceutical evaluation of steroidal ointments by ATR-IR chemical imaging: Distribution of active and inactive pharmaceutical ingredients by Yoshihisa Yamamoto; Toshiro Fukami; Tatsuo Koide; Toyofumi Suzuki; Yukio Hiyama; Kazuo Tomono (54-60).
We recently used micro attenuated total reflection infrared (ATR-IR) spectroscopy to conduct imaging analysis of ointments and evaluate the distributions of the active pharmaceutical ingredient (API) and excipients. An alclometasone dipropionate (ALC) ointment was used as a model product. Almeta®, a brand-name product, had a domain with absorbance at 1656 cm−1 attributable to the carbonyl group of ALC, the API. Absorbances at 1040 and 3300 cm−1 were also noted in this domain, indicating the presence of the solubilizer, propylene glycol. Data also suggested the presence of benzyl alcohol in this domain. More detailed analysis showed the distribution of surfactants and other excipients in the base. Similar results were obtained for Vitra®, a generic version of Almeta®. Imaging analysis with micro ATR-IR confirmed that both ointments are liquid droplet dispersions with ALC dissolved in propylene glycol and dispersed in a base. However, minor differences in the ingredient distributions of the two ointments were detected and reflect differences in excipient concentrations and type, or manufacturing differences. In summary, we used micro ATR-IR for imaging analysis of an original ointment, Almeta®, and its generic form Vitra®, and established a method for visually evaluating the distributions of the API and excipients in these ointments.
Keywords: Alclometasone dipropionate; Excipients; Formulation; Imaging analysis; Ointments; Semi-solids;

Improved dissolution and pharmacokinetic behavior of dipyridamole formulation with microenvironmental pH-modifier under hypochlorhydria by Satomi Onoue; Ryo Inoue; Chika Taniguchi; Yohei Kawabata; Kazuhiro Yamashita; Koichi Wada; Yukinori Yamauchi; Shizuo Yamada (61-66).
The present study aimed to develop and characterize new formulations of dipyridamole (DP), a pH-dependent poorly soluble drug, employing an acidic pH-modifier for improving dissolution and absorption under hypochlorhydric condition. Granule formulations of DP (DPG) with and without fumaric acid (FA) were prepared with wet granulation, physicochemical properties of which were characterized focusing on morphology, dissolution and stability. Pharmacokinetic profiling of orally dosed DPG or DPG with 60% loading of FA (DPG/FA60) was carried out in omeprazole-treated rats as a hypochlorhydric model. Although pH-dependent dissolution behavior was observed in DPG, DPG/FA exhibited high rate and extent of dissolution in both acidic and neutral media. Complete supersaturation was achieved with a 2 h testing period in pH6.8 medium, and co-existing fumaric acid had no impact on the chemical/photochemical stability of DP in solid-state. After oral administration of DPG or DPG/FA60 (10 mg-DP/kg), there was ca. 40% reduction of AUC0–3 for DPG in omeprazole-treated rats as compared to that in normal rats; however, AUC0–3 for DPG/FA60 under hypochlorhydria was almost identical to that of DPG in normal rats. Given the improved systemic exposure early after oral administration in hypochlorhydric rats, the DPG/FA might provide better clinical outcomes in hypochlorhydric patients.
Keywords: Dipyridamole; pH-modifier; Dissolution; Bioavailability; Hypochlorhydria;

Pharmaceutical development of the novel arsenical based cancer therapeutic GSAO for Phase I clinical trial by M.A. Elliott; S.J. Ford; E. Prasad; L.J. Dick; H. Farmer; P.J. Hogg; G.W. Halbert (67-75).
GSAO; a novel organoarsenical cancer therapeutic.The novel organoarsenical GSAO, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, has potential anti-angiogenic capability with application in cancer where tumour metastasis relies on neo-vascularisation. As GSAO arsenic is trivalent, the arsenoxide moiety reacts with appropriately spaced cysteine residues on adenine nucleotide translocase (ANT) mitochondrial membrane protein. Molecular oxidation of the arsenic to the pentavalent structure, as in the degradant GSAA (4-(N-(S-glutathionylacetyl)amino) phenylarsonic acid), prevents sulphydryl interaction and risks abolition of activity. We report here on formulation studies aiming to produce a parenteral product with the primary objective of restricting GSAA transformation from GSAO to protect maximal potency of the molecule. Successful anti-oxidant strategy primarily came from pH control. The presence of glycine was proposed to form a stabilising five-membered oxazarsolidinone ring with arsenoxide and this was investigated using potentiometric assays. We report on these tritration studies identifying a pK a of 8.2 associated with an As-OH, but not confirming ring presence. An original clinical trial pharmaceutical was successfully realised by lyophilisation of 50 mg/mL GSAO in 100 mM glycine solution, pH 7 to obtain a 48-month shelf life for the freeze-dried vials. The Phase I clinical study is ongoing in patients with solid tumours refractory to standard therapy.
Keywords: GSAO; Arsenical; Cancer; Anti-neovascular;

Processing ulvan into 2D structures: Cross-linked ulvan membranes as new biomaterials for drug delivery applications by Anabela Alves; Elisabete D. Pinho; Nuno M. Neves; Rui A. Sousa; Rui L. Reis (76-81).
The polysaccharide ulvan, composed of sulphated rhamnose, glucoronic and iduronic acids was used to produce polymeric membranes by solvent casting. As ulvan is soluble in water, a cross-linking step was necessary to render the membrane insoluble in water and stable at physiological conditions. Cross-linked ulvan membranes were characterized by FTIR, SEM, swelling behaviour was investigated and the mechanical performance assessed by quasi-static tensile testing. Furthermore, the ability and mechanism of sustained release of a model drug from ulvan membranes was investigated. Produced membranes revealed remarkable ability to uptake water (up to ∼1800% of its initial dry weight) and increased mechanical performance (1.76 MPa) related with cross-linking. On the other hand, medicated ulvan dressings demonstrate the potential as drug delivery devices. Using a model drug we have observed an initial steady release of the drug – of nearly 49% – followed by slower and sustained release up to 14 days. The properties of ulvan membranes herein revealed suggest a great potential of this natural sulphated polysaccharide as a wound dressing.
Keywords: Ulvan; Membrane; Dressing; Medicated dressing; Drug delivery;

Changes in protein and mRNA expression levels of claudin family after mucosal lesion by intestinal ischemia/reperfusion by Yusuke Takizawa; Hisanao Kishimoto; Takuya Kitazato; Mikio Tomita; Masahiro Hayashi (82-89).
Ischemia/reperfusion (I/R) injury of the intestine is the leading cause of organ dysfunction after restoration of blood flow after diverse events, including shock and intestinal transplantation. I/R injury must be overcome for successful small intestinal transplantation. Tight junctions (TJ) are the most apical component of the intercellular junctional complex in epithelial cells; they establish cell polarity and functioning as major determinants of epithelial barrier function. Among the proteins that comprise TJ, the claudin family is thought to play a crucial role in homeostasis in multicellular organisms. Therefore, the aim of this study was to examine the changes in function of TJ and behavior of the claudin family during intestinal I/R. Wistar/ST rats underwent intestinal ischemia by using the spring scale and surgical suture for 1 h, followed by 24 h of reperfusion. We examined the changes in area under the blood concentration curve (AUC) after oral administration of FD-4, which is a paracellular marker, and claudin-1, -2, -4, and -7 mRNA and protein expression levels in ileum. The structure of ileal mucosa was partly damaged and its function was diminished by intestinal I/R until 3 h after reperfusion, but were almost recovered 24 h after reperfusion. However, a time difference was shown between the recoveries of mucosal structure and function. Furthermore, a difference in the expression among various kinds of claudin was found. It was suggested that claudin-4 and multi-PDZ domain protein, which is a scaffolding protein, regulate intestinal paracellular permeability during intestinal I/R. Moreover, the changes in the expression level of claudin-2 were unique.
Keywords: In vivo intestinal ischemia/reperfusion; Tight junction; Claudin family; Paracellular permeability; Barrier dysfunction;

Copolymers based on 2-hydroxyethyl methacrylate with 3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5]-undecane were loaded with indomethacin as model drug. The spiroacetal comonomer brought improvements upon the copolymer characteristics as network as well as from the viewpoint of the controlled release from the copolymer structure.Networks based on poly(2-hydroxyethyl methacrylate-co-3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5]-undecane), synthesized through radical dispersion polymerization, were used as template for indomethacin (INN) as model drug. The copolymers were characterized by swelling studies at three pH values (2.4, 5.5 and 7.4) and two temperatures (room temperature 24 °C and physiological temperature 37 °C). Fourier transform infrared (FTIR) spectroscopic analysis was used to sustain the copolymer structures. Scanning electron microscopy (SEM) and thermogravimetric (TG) investigations were used to examine microstructure and appreciate the thermal stability of the polymer samples. The studies of the INN drug release from the copolymer networks were in vitro performed. The in vivo study results (biocompatibility tests, somatic nociceptive experimental model (tail flick test) and visceral nociceptive experimental model (writhing test)) are also reported in this paper.
Keywords: 2-Hydroxyethyl methacrylate; Spiroacetal moiety; Copolymer network; Indomethacin; Controlled release; In vitro and In vivo;

Dry powder inhalers of gentamicin and leucine: formulation parameters, aerosol performance and in vitro toxicity on CuFi1 cells by R.P. Aquino; L. Prota; G. Auriemma; A. Santoro; T. Mencherini; G. Colombo; P. Russo (100-107).
The high hygroscopicity of gentamicin (G) as raw material hampers the production of respirable particles during aerosol generation and prevents its direct use as powder for inhalation in patients suffering from cystic fibrosis (CF). Therefore, this research aimed to design a new dry powder formulation of G studying dispersibility properties of an aminoacid, l-leucine (leu), and appropriate process conditions. Spray-dried powders were characterized as to water uptake, particle size distribution, morphology and stability, in correlation with process parameters. Aerodynamic properties were analyzed both by Single Stage Glass Impinger and Andersen Cascade Impactor. Moreover, the potential cytotoxicity on bronchial epithelial cells bearing a CFTR F508/F508 mutant genotype (CuFi1) were tested. Results indicated that leu may improve the aerosol performance of G-dried powders. The maximum fine particle fraction (FPF) of about 58.3% was obtained when water/isopropyl alcohol 7:3 system and 15–20% (w/w) of leu were used, compared to a FPF value of 13.4% for neat G-dried powders. The enhancement of aerosol efficiency was credited both to the improvement of the powder flowability, caused by the dispersibility enhancer (aminoacid), and to the modification of the particle surface due to the influence of the organic co-solvent on drying process. No significant degradation of the dry powder was observed up to 6 months of storage. Moreover, particle engineering did not affect either the cell viability or cell proliferation of CuFi1 over a 24 h period.
Keywords: Cystic fibrosis; Gentamicin sulfate; l-leucine; Spray drying; Dry powder inhaler; CF airway epithelial cells;

Distribution of aerosols in mouse lobes by fluorescent imaging by Dandan Yi; Amir Naqwi; Angela Panoskaltsis-Mortari; Timothy Scott Wiedmann (108-115).
Better methods are needed to quantify the distribution of drug among the airways of the lungs of small animals to facilitate the development of agents that can target specific airways. Mice were exposed to aerosols of aluminum phthalocyanine tetrasulfonic acid (AlPCS) that ranged in concentration and size (0.2–2.8 μm). The trachea and lobes were removed and placed between glass slides, and fluorescent images were obtained at two different compression thicknesses. The intensity, normalized by the area, exposure time, and thickness, was then plotted as a function of compression thickness, from which the concentration and attenuation coefficient were estimated for each lobe and then for each pixel of the image. The latter was then used to generate an image reflective of the concentration. The lobe volume, concentration, and tissue attenuation of AlPCS was consistent among the lobes. The deposition fraction increased with decreasing particle size. The network of lines in the concentration image indicated that connective tissue has a lower concentration. The central airways were clearly evident in the images of mice exposed to the very small and large aerosols. This approach provides a rapid, economical means to obtain high resolution images of mouse lungs from which detailed analysis of the distribution of deposited aerosol particles can be obtained.
Keywords: Fluorescent imaging; Lungs; Aerosol; Aluminum phthalocyanine tetrasulfonic acid; Aerosol distribution;

Instrumented roll technology for the design space development of roller compaction process by Vishwas V. Nesarikar; Nipa Vatsaraj; Chandrakant Patel; William Early; Preetanshu Pandey; Omar Sprockel; Zhihui Gao; Robert Jerzewski; Ronald Miller; Michael Levin (116-131).
Instrumented roll technology on Alexanderwerk® WP120 roller compactor was developed and utilized successfully for the measurement of normal stress on ribbon during the process. The effects of process parameters such as roll speed (4–12 rpm), feed screw speed (19–53 rpm), and hydraulic roll pressure (40–70 bar) on normal stress and ribbon density were studied using placebo and active pre–blends. The placebo blend consisted of 1:1 ratio of microcrystalline cellulose PH102 and anhydrous lactose with sodium croscarmellose, colloidal silicon dioxide, and magnesium stearate. The active pre-blends were prepared using various combinations of one active ingredient (3–17%, w/w) and lubricant (0.1–0.9%, w/w) levels with remaining excipients same as placebo.Three force transducers (load cells) were installed linearly along the width of the roll, equidistant from each other with one transducer located in the center. Normal stress values recorded by side sensors and were lower than normal stress values recorded by middle sensor and showed greater variability than middle sensor. Normal stress was found to be directly proportional to hydraulic pressure and inversely to screw to roll speed ratio. For active pre-blends, normal stress was also a function of compressibility. For placebo pre-blends, ribbon density increased as normal stress increased. For active pre-blends, in addition to normal stress, ribbon density was also a function of gap.Models developed using placebo were found to predict ribbon densities of active blends with good accuracy and the prediction error decreased as the drug concentration of active blend decreased. Effective angle of internal friction and compressibility properties of active pre blend may be used as key indicators for predicting ribbon densities of active blend using placebo ribbon density model. Feasibility of on-line prediction of ribbon density during roller compaction was demonstrated using porosity–pressure data of pre-blend and normal stress measurements. Effect of vacuum to de-aerate pre blend prior to entering the nip zone was studied. Varying levels of vacuum for de-aeration of placebo pre blend did not affect the normal stress values. However, turning off vacuum completely caused an increase in normal stress with subsequent decrease in gap.Use of instrumented roll demonstrated potential to reduce the number of DOE runs by enhancing fundamental understanding of relationship between normal stress on ribbon and process parameters.
Keywords: Roller compaction; Instrumented roll; Process design space; Ribbon normal stress; Ribbon density; Vacuum de-aeration; Roll gap;

In situ forming implant for controlled delivery of an anti-HIV fusion inhibitor by Deepak N. Kapoor; Om Prakash Katare; Sanju Dhawan (132-143).
Different approaches applied to prepare in situ forming implants for controlled delivery of enfuvirtide, an anti HIV fusion inhibitor.An injectable, phase sensitive, in situ forming, implantable delivery system was developed for enfuvirtide, a therapeutic peptide used in the treatment of HIV infection. The development studies were carried out using poly (d,l-lactide-co-glycolide), a smart, biodegradable polymer. Different formulations were designed, prepared and evaluated by employing response surface, optimal design of experiment technique. The optimized formulation was identified and validated for its performance by using numerical optimization technique. The in vitro evaluation parameters included rheology, compatibility studies, drug release as well as conformational and physicochemical stability studies. In vivo pharmacokinetic parameters and biocompatibility studies were determined in rat models and were statistically analyzed. It was found that the optimized formulation extended the enfuvirtide release and maintained the drug plasma concentration within therapeutically effective range up to 48 h. The optimized formulation maintained physicochemical and conformational stability for at least 6 months and was biocompatible with the animal tissue.
Keywords: Enfuvirtide; Fusion inhibitors; Anti-HIV; Therapeutic peptides; Smart polymers; PLGA; Implants;

The rheology of self-emulsifying drug delivery systems (SEDDS) is not thoroughly characterized these days. Since mechanical rheometers are often not well suited to study this kind of systems, there is need for novel physical methods. Several new optical techniques based on microrheology have recently made significant progress. We apply for the first time a specific microrheological technique called diffusing wave spectroscopy (DWS) to study different SEDDS. The obtained data were then correlated with the dosing precision of automated capsule filling.As a result, the dynamic viscosities obtained from microrheology were in accordance with data from capillary viscosimetry. The DWS measurements revealed that all formulations had a clearly measurable storage modulus at frequencies >200 rad/s. Thus, all samples were low-viscous, while exhibiting non-Newtonian flow behavior. Obtained values of storage and loss modulus were then successfully correlated with the weight variability of capsules that were filled on a machine. In conclusion, the DWS technique enabled rheological analysis of self-emulsifying systems in a broad frequency range. The good data correlation with a capsule quality attribute was especially promising, since microrheological techniques are typically contact-free. Thus, they have a high potential in a quality by design framework of formulation development and production.
Keywords: Diffusing wave spectroscopy; Microrheology; Visco-elastic; Self-emulsifying drug delivery systems; Capsules; Quality by design;

Molecular modelling and multisimplex optimization of tocotrienol-rich Self Emulsified Drug Delivery Systems by Alaadin Alayoubi; Seetharama D. Satyanarayanajois; Paul W. Sylvester; Sami Nazzal (153-161).
The objective of this study was to optimize a novel tocotrienol (TRF)-rich Self Emulsified Drug Delivery System (SEDDS). In the first part, an unusual phenomenon was investigated. It was observed that by substituting Tween® 80 with Cremophor® EL in the SEDDS it was possible to emulsify > 55% TRF (by weight of the formulation) into submicron (<200 nm) emulsion. With Tween®, only 17.5% of the loaded TRF could be emulsified into crude emulsion. The superiority of Cremophor® was attributed to the special arrangement of the surfactant at the oil/water interface, which was confirmed by modelling and docking studies. In the second part of this study, the composition of the secondary ingredients in the TRF-rich SEDDS were optimized by the modified Multisimplex® approach. SEDDS were manufactured at pre-defined step-size and tested for their dissolution behavior. Testing was performed sequentially until the optimum composition that can emulsify 50% of the loaded TRF into a stable < 150 nm submicron emulsion was obtained. Optimization end-point was identified when the “membership value” approached 1, which was confirmed by a second Multisimplex® run. Overall, this study demonstrated the utility of docking studies and the Multisimplex® approach in product development when little is known about the experimental “design space”.
Keywords: Surfactants; Nanoemulsions; Physical characterization; Tocotrienol; Multisimplex®; SEDDS;

A novel ultra cryo-milling micronization technique has been established using dry ice beads and liquid nitrogen (LN2). Drug particles were co-suspended with dry ice beads in LN2 and ground by stirring. Dry ice beads were prepared by storing dry ice pellets in LN2. A poorly water-soluble drug, phenytoin, was micronized more efficiently using either dry ice beads or zirconia beads compared to jet milling. Dry ice beads retained their granular shape without pulverizing and sublimating in LN2 as the milling operation progressed. Longer milling times produced smaller-sized phenytoin particles. The agitation speed for milling was optimized. Analysis of the glass transition temperature revealed that phenytoin particles co-ground with polyvinylpyrrolidone (PVP) by dry ice milling were crystalline, whereas a planetary ball-milled mixtures process with zirconia beads contained the amorphous form. The dissolution rate of phenytoin milled with PVP using dry ice beads or zirconia beads was significantly improved compared to jet-milled phenytoin or the physical mixture. Dry ice beads together with LN2 were spontaneously sublimated at ambient condition after milling. Thus, the yield was significantly improved by dry ice beads compared to zirconia beads since the loss arisen from adhering to the surface of dry ice beads could be completely avoided, resulting in about 85–90% of recovery. In addition, compounds milled using dry ice beads are free from abraded contaminating material originating from the beads and internal vessel wall.
Keywords: Cryo-milling; Dry ice beads; Liquid nitrogen; Submicron; Zirconia beads; Jet milling;

PEG/RGD-modified magnetic polymeric liposomes for controlled drug release and tumor cell targeting by Wenya Su; Hanjie Wang; Sheng Wang; Zhenyu Liao; Shiyin Kang; Yao Peng; Lei Han; Jin Chang (170-181).
Polymeric liposomes (PEG/RGD-MPLs), composed of amphiphilic polymer octadecyl-quaternized modified poly (γ-glutamic acid) (OQPGA), PEGylated OQPGA, RGD peptide grafted OQPGA and magnetic nanoparticles, was prepared successfully. These PEG/RGD-MPLs could be used as a multifunctional platform for targeted drug delivery. The results showed that PEG/RGD-MPLs were multilamellar spheres with nano-size (50–70 nm) and positive surface charge (28–42 mV). Compared with magnetic conventional liposomes (MCLs), PEG/RGD-MPLs exhibited sufficient size and zeta potential stability, low initial burst release and less magnetic nanoparticles leakage. The cell uptake results suggested that the PEG/RGD-MPLs (with RGD and magnetic particles) exhibited more drug cellular uptake than non RGD and non magnetism carriers in MCF-7 cells. MTT assay revealed that PEG/RGD-MPLs showed lower in vitro cytotoxicity to GES-1 cells at ≤100 μg/mL. These data indicated that the multifunctional PEG/RGD-MPLs may be an alternative formulation for drug delivery system.
Keywords: Drug delivery; Polymeric liposomes; Magnetic nanoparticles; Tumor Targeting; RGD peptide;

Folic acid conjugated mPEG-PEI600 as an efficient non-viral vector for targeted nucleic acid delivery by Zhenhua Xu; Jiefu Jin; Leo K.S. Siu; Hong Yao; Johnny Sze; Hongzhe Sun; Hsiang-fu Kung; Wai Sang Poon; Samuel S.M. Ng; Marie C. Lin (182-192).
In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. 1H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine™2000 and Linear PEI 22kDa (L-PEI 22kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier.
Keywords: Polymer; Polyethylenimine; Tumor gene delivery; PEG;

Reversion of multidrug resistance by co-encapsulation of doxorubicin and curcumin in chitosan/poly(butyl cyanoacrylate) nanoparticles by Jinghua Duan; Heidi M. Mansour; Yangde Zhang; Xingming Deng; Yuxiang Chen; Jiwei Wang; Yifeng Pan; Jinfeng Zhao (193-201).
Co-encapsulated doxorubicin (DOX) and curcumin (CUR) in poly(butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared with emulsion polymerization and interfacial polymerization. The mean particle size and mean zeta potential of CUR–DOX–PBCA-NPs were 133 ± 5.34 nm in diameter and +32.23 ± 4.56 mV, respectively. The entrapment efficiencies of doxorubicin and curcumin were 49.98 ± 3.32% and 94.52 ± 3.14%, respectively. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX–PBCA-NPs + CUR–PBCA-NPs), which was slightly higher than that of the free drug combination (DOX + CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX + CUR–PBCA-NPs or CUR + DOX–PBCA-NPs). The simultaneous administration of doxorubicin and curcumin achieved the highest reversal efficacy and down-regulation of P-glycoprotein in MCF-7/ADR cell lines, an MCF-7 breast carcer cell line resistant to adriamycin. Multidrug resistance can be enhanced by combination delivery of encapsulated cytotoxic drugs and reversal agents.
Keywords: Curcumin; Doxorubicin; Poly(butyl cyanoacrylate) nanoparticles; Multidrug resistance;

Preparation of high solubilizable microemulsion of naproxen and its solubilization mechanism by Qingping Tian; Fude Ren; Zhen Xu; Ying Xie; Shuqiu Zhang (202-210).
To improve the skin permeation of naproxen with larger dosage, microemulsion with high content of naproxen was investigated for transdermal delivery and its solubilization mechanism was studied. Naproxen micoremulsions composed of 4% isopropyl myristate, 18% Tween 80, 18% ethanol and water were prepared and phase inversion temperature (PIT) method was used to increase drug content. The using of PIT method resulted in the maximum content of naproxen in microemulsion increased from 1.98 ± 0.13% to 4.12 ± 0.07%, accordingly the permeation rate of naproxen from microemulsion through excised mice skin increased from 135.13 ± 5.50 to 214.46 ± 7.53 μg cm−2  h−1. The analyses of Natural Bond Orbital and interaction energy using the B3LYP and MP2 (fc) methods suggested that the solubilization mechanism of microemulsion for naproxen mainly might be the formation of complex between the hydrogen atom of hydroxyl in Tween 80 and the oxygen atom of carbonyl group in naproxen, as is in accordance with the result from 1H NMR experiments. The change of thermodynamic function with temperature confirmed that, because the complex was easy to be formed in high temperature and that formed at PIT became more stable when the temperature decreased to below PIT, the solubilization ability of microemulsion for naproxen could be improved by the PIT method. The powerful permeation enhancing ability of microemulsion induced by the solubilization of PIT method makes it a promising vehicle for the transdermal delivery of naproxen.
Keywords: Naproxen; Microemulsion; Solubilization; Phase inversion temperature; Intermolecular interaction;

A novel two-stage reverse dialysis method has been developed for in vitro release testing of liposomal drug product with passive targeting characteristics. The first stage of the test is to mimic the circulation of liposomes in the body, whereas the second stage is to imitate the drug release process at the target. Buffer and surfactant solution were used during the first and second stages, respectively. For formulations containing high phase transition temperature lipids and high cholesterol content, no drug leakage was observed during the first stage of test. In the second stage, however, formulations with different compositions showed significant differences in terms of drug release rate, and discriminatory ability of the method was demonstrated. On comparing two different membrane diffusion techniques, dialysis and reverse dialysis methods, the reverse dialysis method showed significantly lower variation, and therefore is the preferred method. The developed in vitro release testing method should help to distinguish formulations with varied compositions for quality control testing purposes. This two-stage reverse dialysis method may pave the way to the development of more bio-relevant release testing methods for liposomal drug products.
Keywords: Liposome; In vitro release; Tenofovir; Dialysis; Reverse dialysis; Membrane diffusion;

The objectives of the present study were to circumvent the moisture-associated instability, enhance bioavailability and achieve enhanced passive targeting of melphalan to the ovaries. Solubility of the drug was determined in various excipients to select the components of nanoemulsion. Pseudoternary phase diagrams were constructed using aqueous titration method. Formulations selected from the pseudoternary phase diagram were subjected to thermodynamic stability and dispersibility studies to select the final test formulations which were characterized for average globule size, polydispersity index (PDI), zeta potential, viscosity, refractive index, in-vitro drug release and percentage transmittance to optimize the final formulation. Pharmacokinetic and biodistribution studies of the optimized formulation in comparison to the pure drug suspension were done using γ-scintigraphy on female Balb/c mice. In-vitro cytotoxicity study on Hela cervical cancer cell lines was also done to compare the anticancer activity of the developed formulation with respect to the pure drug solution. In vitroin vivo correlation was established for the amount of drug released and the amount of drug absorbed using suitable deconvolution. Stability studies on the final formulation were performed at 40 ± 2 °C and 75 ± 5% RH for 3 months and the shelf life was determined. Capmul MCM, Tween 80 and Transcutol P® (S mix) were selected as the oil, surfactant and co-surfactant respectively on the basis of solubility studies. Out of 17 formulations prepared, six formulations were selected as the final test formulations on the basis of thermodynamic stress and dispersibility tests. The optimized formulation composed of oil (10%, v/v), S mix (35%, v/v), and double distilled water (55%, v/v). Bioavailability studies revealed 4.83 folds enhancement in bioavailability of the drug from nanoemulsion as compared to that from suspension. Biodistribution studies revealed more than 2 folds increase in uptake of the drug from nanoemulsion by ovaries as compared to that from the suspension. In vitro cytotoxicity studies demonstrated augmented anticancer potential of the drug in the form of nanoemulsion formulation in comparison to the drug solution. Level A correlation was established between the amount of drug released and the amount of drug absorbed. The shelf life of the formulation was found to be 1.30 years. The results demonstrate surface modified nanoemulsion to be a promising approach so as to increase stability, bioavailability and cellular uptake of the drug.
Keywords: Nanoemulsion; Oral; Bioavailability; Melphalan; Pseudoternary phase diagram;

Serum-stable, long-circulating paclitaxel-loaded colloidal carriers decorated with a new amphiphilic PEG derivative by Livia Basile; Catherine Passirani; Ngoc-Trinh Huynh; Jérôme Béjaud; Jean-Pierre Benoit; Giovanni Puglisi; Rosario Pignatello (231-238).
The paper describes sterically stabilized lipid nanocapsules (LNC) and multilamellar liposomes (MLV) coated using a new amphiphilic conjugate of PEG2000 with a 2-alkyl-lipoamino acid (LAA). A complement activation assay (CH50) and uptake experiments by THP-1 macrophage cells were used to assess in vitro the effectiveness of the PEG-LAA derivative of modifying the surface behavior of nanocarriers. Administered to rats or Swiss mice, respectively, the PEG2000-LAA—modified LNC and MLV showed plasma half-lives longer than the corresponding naked carriers.To assess the ability of nanocarriers to specifically reach tumor sites, paclitaxel (PTX)—loaded LNC and MLV were administered subcutaneously to rats implanted with a 9L glioma. Animals treated with saline or naked LNC and MLV underwent a quick expansion of tumor mass, up to a volume of 2000 mm3 25 days after the injection of tumor cells. On the contrary, treatment with a PEG-LAA modified LNC carrier reduced the growth of the tumor volume, which did not exceed 1000 mm3 by day 25. Analogous positive results were obtained with the liposomal systems. The experimental findings confirmed that these new PEG-LAA conjugates allow to obtain sterically stable nanocarriers that behave effectively and in a comparable or even better way than the (phospho)lipid PEG derivatives commercially available.
Keywords: Liposomes; Lipid nanocapsules (LNC); Lipoamino acids; Stealth® carriers; CH50 assay; Lipid PEG derivatives;

Possible enhancing mechanisms for gene transfer activity of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate by Takayuki Anno; Taishi Higashi; Keiichi Motoyama; Fumitoshi Hirayama; Kaneto Uekama; Hidetoshi Arima (239-247).
We previously reported that glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with polyamidoamine starburst dendrimer (GUG-β-CDE conjugate) with the average degree of substitution (DS) of cyclodextrin (CyD) of 1.8 (GUG-β-CDE conjugate (DS 1.8)), showed remarkably higher gene transfer activity than α-CyD/dendrimer conjugate (α-CDE conjugate (DS 1.2)) and β-CyD/dendrimer conjugate (β-CDE conjugate (DS 1.3)) in vitro and in vivo. In this study, to clarify the enhancing mechanism for high gene transfer activity of GUG-β-CDE conjugate (DS 1.8), we investigated the physicochemical properties, cellular uptake, endosomal escape and nuclear translocation of the plasmid DNA (pDNA) complexes as well as pDNA release from the complexes. The particle size, ζ-potential and cellular uptake of GUG-β-CDE conjugate (DS 1.8)/pDNA complex were mostly comparable to those of α-CDE conjugate (DS 1.2) and β-CDE conjugate (DS 1.3). Meanwhile, GUG-β-CDE conjugate (DS 1.8)/pDNA complex was likely to have high endosomal escaping ability and nuclear localization ability in A549 and RAW264.7 cells. In addition, the pDNA condensation and decondensation abilities of GUG-β-CDE conjugate (DS 1.8) were lower and higher than that of α-CDE conjugate (DS 1.2) or β-CDE conjugate (DS 1.3), respectively. These results suggest that high gene transfer activity of GUG-β-CDE conjugate (DS 1.8) could be, at least in part, attributed to high endosomal escaping ability, nuclear localization ability and suitable pDNA release from its complex.
Keywords: PAMAM starburst dendrimer; Glucuronylglucosyl-β-cyclodextrin; Conjugate; Cyclodextrins; Non-viral vector; Enhancing mechanism;

Novel superparamagnetic iron oxide nanoparticles for tumor embolization application: Preparation, characterization and double targeting by Xiaoli Chen; Haiyan Lv; Min Ye; Shengyu Wang; Erru Ni; Fanwei Zeng; Chang Cao; Fanghong Luo; Jianghua Yan (248-255).
The goal of this study was to develop novel embolic nanoparticles for targeted tumor therapy with dual targeting: magnetic field-guided and peptide-directed targeting. The embolic nanoparticles SP5.2/tTF-OCMCs-SPIO-NPs were prepared by surface-modifying of superparamagnetic iron oxide nanoparticles (SPIO-NPs) with o-carboxymethylchitosans (OCMCs) and SP5.2/tTF (SP5.2: a peptide binding to VEGFR-1; tTF: truncated tissue factor) to improve their stability and to target over-expressing VEGFR-1 cells. The physicochemical characterization results showed that the OCMCs-SPIO-NPs have a spherical or ellipsoidal morphology with an average diameter of 10–20 nm. And they possess magnetism with a saturation magnetization of 66.1 emu/g, negligible coercivity and remanence at room temperature. In addition, the confocal microscopy, Prussian blue staining and FX activation analysis respectively demonstrated the peptide-directed targeting, magnetic field-guided targeted and blood coagulation activity of the SP5.2/tTF-OCMCs-SPIO-NPs. These properties separately belong to SP5.2, Fe3O4 and tTF moieties of the SP5.2/tTF-OCMCs-SPIO-NPs. Thus these SP5.2/tTF-OCMCs-SPIO-NPs with double-targeting function should have a potential application in embolization therapy of tumor blood vessels.
Keywords: Magnetic nanoparticles; Superparamagnetic; o-Carboxymethylchitosan; Truncated tissue factor; Tumor targeting;

Low molecular weight heparin gels, based on nanoparticles, for topical delivery by C. Loira-Pastoriza; A. Sapin-Minet; R. Diab; J.L. Grossiord; P. Maincent (256-262).
A commercial suspension of nanoparticles (Eudragit® RS 30D) was used to manufacture a gel for topical application. Gels were prepared by mixing a polycationic polymer (Eudragit® RS 30D) and a low molecular weight heparin (LMWH), an antithrombotic agent. Gels formed spontaneously at a ratio of 1:1 as a result of electrostatic interactions between the polyanionic drug and the polycationic polymer. Different types of heparin were used: Bemiparin, Enoxaparin (Lovenox®), Nadroparin (Fraxiparin®) and Tinzaparin (Innohep®). Several LMWH concentrations were tested. Rheological measurements were performed to investigate the gel behavior. Gel formation was confirmed by dynamic rheological measurements as the elastic modulus (G′) was higher than the viscous one (G″). The amount of heparin incorporated into the gel matrix was determined. A maximum of incorporation (100%) was reached using a heparin solution of 600 IU/mL. The release kinetics of LMWH from the gel were also studied. Regardless of the LMWH used in the formulation, a biphasic release profile was observed. Accordingly, a burst effect was observed. Afterwards, the release rate became steady. The penetration of the LMWH through the dermal barrier was also investigated.
Keywords: Low molecular weight heparin; Gel; Topical delivery; Nanoparticles;

Combination therapy with metronomic S-1 dosing and oxaliplatin-containing PEG-coated cationic liposomes in a murine colorectal tumor model: Synergy or antagonism? by Amr S. Abu Lila; Tomoko Okada; Yusuke Doi; Masako Ichihara; Tatsuhiro Ishida; Hiroshi Kiwada (263-270).
Combination therapy with 2 or more drugs with different mechanisms of action has been considered a promising strategy for the effective treatment of advanced and metastatic cancers. However, the rational design of combination therapy represents a potential prerequisite for its effectiveness. Recently, we showed that the combination of oral metronomic S-1 dosing with oxaliplatin (l-OHP)-containing PEG-coated “neutral” liposomes exerted excellent antitumor activity. In addition, we recently designed a PEG-coated “cationic” liposome for dual-targeting delivery of l-OHP to tumor endothelial cells and tumor cells in a solid tumor. This targeted liposomal l-OHP formulation showed efficient antitumor activity in a murine tumor model, compared with l-OHP-containing PEG-coated “neutral” liposomes. In the present study, we investigated the issue of whether metronomic S-1 dosing with l-OHP-containing PEG-coated “cationic” liposomes creates synergy. Unfortunately, metronomic S-1 dosing resulted in impaired delivery of PEG-coated “cationic” liposomes into tumor tissue, presumably by decreasing the binding sites on tumor blood vessels available for the liposomes. The anticipated cytotoxic synergistic effect of the combination treatment was not achieved. Instead, the combination treatment showed lower antitumor efficacy than l-OHP-containing PEG-coated “cationic” liposomes alone. These results suggest that the combined treatment of S-1 and l-OHP-containing PEG-coated “cationic” liposomes seems to be antagonistic rather than synergistic.
Keywords: Antitumor activity; Combination therapy; Metronomic S-1 dosing; Oxaliplatin; PEG-coated cationic liposomes;

Hemocompatibility of poly(ɛ-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan by Eduardo A. Bender; Márcia D. Adorne; Letícia M. Colomé; Dulcinéia S.P. Abdalla; Sílvia S. Guterres; Adriana R. Pohlmann (271-279).
The hemocompatibility of nanoparticles is of critical importance for their systemic administration as drug delivery systems. Formulations of lipid-core nanocapsules, stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan (LNC and LNC-CS), were prepared and characterized by laser diffraction (D[4,3]: 129 and 134 nm), dynamic light scattering (119 nm and 133 nm), nanoparticle tracking (D50: 124 and 139 nm) and particle mobility (zeta potential: −15.1 mV and +9.3 mV) analysis. In vitro hemocompatibility studies were carried out with mixtures of nanocapsule suspensions in human blood at 2% and 10% (v/v). The prothrombin time showed no significant change independently of the nanocapsule surface potential or its concentration in plasma. Regarding the activated partial thromboplastin time, both suspensions at 2% (v/v) in plasma did not influence the clotting time. Even though suspensions at 10% (v/v) in plasma decreased the clotting times (p  < 0.05), the values were within the normal range. The ability of plasma to activate the coagulation system was maintained after the addition of the formulations. Suspensions at 2% (v/v) in blood showed no significant hemolysis or platelet aggregation. In conclusion, the lipid-core nanocapsules uncoated or coated with chitosan are hemocompatible representing a potential innovative nanotechnological formulation for intravenous administration.
Keywords: Hemocompatibility; Polymeric nanoparticles; Lipid-core nanocapsules; Polysorbate 80; Lecithin; Chitosan;

Effect of polyamidoamine dendrimer G3 and G4 on skin permeation of 8-methoxypsoralene—In vivo study by Katarzyna Borowska; Stanisław Wołowiec; Andrzej Rubaj; Kazimierz Głowniak; Elwira Sieniawska; Sebastian Radej (280-283).
In the present study we have assessed the ability of (PAMAM) dendrimers G3 and G4 to facilitate transdermal delivery of 8-methoxypsoralen (8-MOP) in vivo. In vitro study using Franz diffusion cell revealed an enhanced transdermal flux for 8-MOP in complex with G3 and G4 dendrimer in relation to standard 8-MOP solution. In present study in vivo skin permeation potential of 8-MOP complex with G3 and G4 PAMAM dendrimer was assessed using confocal laser scanning microscopy (CLSM), which revealed an enhanced permeation of the 8-MOP to the deeper layers of the skin and significantly higher concentration in comparison with standard 8-MOP solution. Skin tissue 8-MOP concentration, evaluated by HPLC indicates that G3 and G4 PAMAM application significantly increase 8-MOP skin deposition in comparison with standard 8-MOP solutions after 1 and 2 h. G4 appeared to be a more effective 8-MOP penetration enhancer than G3 PAMAM. Our results suggest the feasibility of G3 and G4 PAMAM dendrimers for transdermal delivery of 8-MOP resulting in better skin permeation and higher concentration of 8-MOP in epidermis and dermis of the drug that could help to improve effectiveness and safety of PUVA therapy.
Keywords: PAMAM dendrimers; 8-MOP; PUVA; Rat skin permeation; Confocal microscopy;

Anti-NogoA is a promising anti-inhibitory molecule that has been shown to enhance functional recovery after spinal cord injury when delivered in rat and primate models over the span of weeks. To achieve this sustained release, anti-NogoA is typically delivered by osmotic minipumps; however, external minipumps are susceptible to infection. To address this issue, we developed a drug delivery system that consists of anti-NogoA-loaded poly(lactic-co-glycolic acid) nanoparticles dispersed in a hydrogel of hyaluronan and methylcellulose (composite HAMC). To optimize in vitro release, we screened formulations for improved anti-NogoA bioactivity and sustained release based on combinations of co-encapsulated trehalose, hyaluronan, MgCO3, and CaCO3. Co-encapsulated MgCO3 and CaCO3 slowed the rate of anti-NogoA release and did not influence anti-NogoA bioactivity. Co-encapsulated trehalose significantly improved anti-NogoA bioactivity at early release time points by stabilizing the protein during lyophilization. Co-encapsulated trehalose with hyaluronan improved bioactivity up to 28 d and dramatically increased the rate and duration of sustained delivery. The sustained release of bioactive anti-NogoA from composite HAMC is a compelling formulation for in vivo evaluation in a model of spinal cord injury.
Keywords: Anti-NogoA (11c7); Poly(lactic-co-glycolic acid) (PLGA); Nanoparticle; Hydrogel; Drug delivery;

Preparation of E-selectin-targeting nanoparticles and preliminary in vitro evaluation by Emile Jubeli; Laurence Moine; Valérie Nicolas; Gillian Barratt (291-301).
Targeted delivery aims to concentrate therapeutic agents at their site of action and thereby enhance treatment and limit side-effects. E-selectin on endothelial cells is markedly up-regulated by cytokine stimulation of inflamed and some tumoral tissues, promoting the adhesion of leukocytes and metastatic tumor cells, thus making it an interesting molecular target for drug delivery systems.We report here the preparation of targeted nanoparticles from original amphiphilic block copolymers functionalized with an analog of sialyl Lewis X (SLEx), the physiological ligand of E-selectin. Nanoparticles, prepared by nanoprecipitation, caused no significant cytotoxicity. Ligand-functionalized nanoparticles were specifically recognized and internalized better by tumor necrosis factor α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs) than control nanoparticles or HUVECs with low E-selectin expression. These nanoparticles are designed to carry the ligand at the end of a PEG spacer to improve accessibility. This system has potential for the treatment of inflammation, inhibition of tumor metastasis, and for molecular imaging.
Keywords: Carbohydrate ligand conjugated polymers; E-selectin; HUVECs; Nanoparticles;

Comparative studies on physicochemical stability of cyclosporine A-loaded amorphous solid dispersions by Hideyuki Sato; Yohei Kawabata; Kayo Yuminoki; Naofumi Hashimoto; Yukinori Yamauchi; Kumiko Ogawa; Takahiro Mizumoto; Shizuo Yamada; Satomi Onoue (302-306).
The present study aimed to evaluate the physical stability on amorphous solid dispersion (SD) of cyclosporine A (CsA) employing hydroxypropyl cellulose (HPC). SD formulations (5–30% CsA) of CsA such wet-milled SD (WM/SD) and freeze-dried SD (FD/SD) were prepared, and both SD formulations were stored at 40 °C/75% relative humidity for 8 weeks. Transitions in morphology, dissolution behavior, crystallinity and thermal behavior of CsA were evaluated. There was at least 84-fold improvement in initial dissolution rate of SD formulations compared with that of amorphous CsA powder, although their dissolution rate was gradually decreased under accelerated conditions. In particular, aged FD/SD with a drug load of 30% exhibited highly limited dissolution as evidenced by 40% reduction of solubility after 8 weeks of storage. In contrast, aged WM/SD exhibited less reduction in dissolution rate compared with FD/SD. No significant changes were seen in crystallinity and thermal behavior after aging of SD formulations for 8 weeks; however, electron microscopic observations revealed aggregation of drug molecules/particles in the aged FD/SD, possibly leading to the reduced dissolution. From these findings, stability on CsA-loaded SD might be variable depending on the preparation methodology, and the wet-milling approach could be a viable option for preparing efficacious SD formulations with improved stability.
Keywords: Cyclosporine A; Amorphous; Solid dispersion; Solid solution; Stability;