International Journal of Pharmaceutics (v.515, #1-2)

Application of RPMI 2650 as a cell model to evaluate solid formulations for intranasal delivery of drugs by Vanessa S.S. Gonçalves; Ana A. Matias; Joana Poejo; Ana T. Serra; Catarina M.M. Duarte (1-10).
Display OmittedDuring the development of intranasal drug delivery systems for local/systemic effect or brain targeting, it is necessary to assess its cytotoxicity and drug transport through nasal epithelium. In order to avoid animal experiments or the use of excised tissues, in vitro cell models, such as RPMI 2650 cells, are being preferred during recent years. Nevertheless, the deposition of solid formulations into nasal cell layers with further transepithelial transport rate of drugs has been poorly studied or reported.Thus, the purpose of this work is to further investigate RPMI 2650 cell line as an effective alternative to animal tissues for solid drug-loaded formulations cytotoxicity and drug permeation studies in order to become an option as a tool for drug discovery. Furthermore, we wanted to determine the extent to which the administration of drugs in particulate forms would differ in relation to the permeability of the same compounds applied as solutions. RPMI 2650 cells were cultured in submersed or at air-liquid interface conditions and characterized regarding transepithelial electrical resistance (TEER) and production of mucus. Pure ketoprofen (used as model compound) and five formulations loaded with same drug, namely solid lipid particles (Gelucire 43/01™), structured lipid particles (Gelucire 43/01™:Glyceryl monooleate) and aerogel microparticles (Alginate, Alginate:Pectin, Alginate:Carrageenan), were evaluated with RPMI 2650 model in terms of cytotoxicity and permeability of drug (applied as solution, dispersion or powder + buffer).RPMI 2650 cells were capable to grow in monolayer and multilayer, showing the same permeability as excised human nasal mucosa for sodium fluorescein (paracellular marker), with analogous TEER values and production of mucus, as referred by other authors. None of the powders showed cytotoxicity when applied to RPMI 2650 cells. Regarding permeation of drug through cell layers, not only the form of application of powders but also their physical and chemical properties affected the final permeation of active pharmaceutical ingredient. Aerogel microparticles administered directly to the cell layer (powder + buffer) exhibited the highest permeation-enhancing effect compared to the pure drug, which can be attributed to the mucoadhesive properties of the materials composing the carriers, proving to be an attractive formulation for nasal drug delivery.According to these results, RPMI 2650 showed to be a promising alternative to ex vivo or in vivo nasal models for cytotoxicity and evaluation of drug permeability of nasal drug-loaded formulations.
Keywords: RPMI 2650; Nasal epithelial cell model; Transepithelial electrical resistance; Mucus production; Air–liquid interface; Cytotoxicity; Drug permeation;

Graphical abstract of inducing apoptosis and altering the release of some cytokines in breast cancer cell lines followed by entry of the CpG-ODN molecules loaded on magnetic PAMAM nanoparticles and their interaction with TLR9 protein.Display OmittedCpG-oligodeoxynucleotide (CpG-ODN) can function as an immune adjuvant. Previously, we showed that stimulation of breast cancer cells with CpG-ODN conjugated with PAMAM dendrimer-coated magnetic nanoparticles (DcMNPs) has induced apoptosis. The aim of the current study was to evaluate the expression levels of some apoptosis-regulating genes in several human breast cancer cells treated with CpG/DcMNPs. Treated MDA-MB231 cells showed an increase in Noxa and Bax gene expression levels, whereas the expression level of Survivin decreased. Similarly, Noxa gene was overexpressed in treated MCF7 cells. In treated SKBR3 cells, a decline in the c-Flip mRNA level was determined. Furthermore, release of cytokines, IL-6, IL-10, and TNF-α, was determined in cell culture supernatants. CpG/DcMNP treatment leads to an increase in the release of IL-6 in MDA-MB231 and SKBR3 cells, whereas release of IL-10 and TNF-α did not change significantly. It is indicated that CpG-ODN may show its cytotoxic effect by regulating the expression of apoptosis-related genes and the release of cytokine in breast cancer cells.
Keywords: CpG-ODN; Apoptosis-related genes; Cytokine release; Dendrimeric nanoparticles; Magnetic nanoparticles;

Display OmittedThe potential in vivo application of liposome for polycationic colistin has been hindered by the poor entrapment efficiency (EE) due to their phospholipid membrane permeability. The objective of this study is to investigate the loading mechanism and validity of applying electrostatic attraction for the colistin entrapment and delivery in liposomes. Anionic lipids with various structures were used for colistin entrapment, and the properties of resulting liposomes (i.e. zeta-potential, EE and release rate) were highly dependent on the structure of anionic lipids. Based on consideration of intermolecular interactions, the retention of electrostatically entrapped colistin is essentially determined by the balance of interfacial hydrophobic attraction and electrostatic repulsion. The liposomal colistin showed the reduced bacterial killing rate, but did not compromise the in vitro antibacterial activity. Specially, the PEGylated liposomal colistin of sodium cholesteryl sulfate (Chol-SO4) showed the best drug retention, resulting in the significantly increased maximum-tolerated dose, prolonged blood circulation and decreased colistin distribution in kidney after intravenous administration in mice. These results highlight the potential utility of electrostatically entrapped liposome for polycationic colistin delivery.
Keywords: Liposome; Electrostatic interaction; Colistin; Biodistribution; Anionic lipid; Loading mechanism;

Tunable doxorubicin release from polymer-gated multiwalled carbon nanotubes by Alessandro Pistone; Daniela Iannazzo; Shabana Ansari; Candida Milone; Marina Salamò; Signorino Galvagno; Santa Cirmi; Michele Navarra (30-36).
Display OmittedTwo pH and temperature controlled drug delivery systems for cancer therapy are here reported by using vapour phase and liquid phase functionalized multiwalled carbon nanotubes (MWCNT). Both oxidized MWCNT were functionalized at the carboxyl groups with a short hydrophilic polyethylene glycol (PEG) chain. The nanosystems were loaded with doxorubicin and covered with the biocompatible polymer polylactide, able to form hydrogen bonding with PEG and to entrape the drug inside the two polymeric chains. The different oxidative reaction conditions of MWCNT have demonstrated to deeply affect their agglomeration ability and the available reactive surface area for drug loading which in turn, affected the drug release abilities of the synthesized polymer-gated drug delivery systems. The in vitro release abilities as well as their antiproliferative effect on three different human cancer cell lines were evaluated and compared, highlighting the possibility to tune the amount of drug released by controlling the functionalization degree of the carbon nanotube based material. Biological tests highlighted the high biocompatibility of both systems and their ability to deliver doxorubicin to cancer cells.
Keywords: Multi walled carbon nanotubes; Drug delivery systems; Doxorubicin; Polymer-gated nanosystems; Drug release studies; Antiproliferative effect;

LARETH-25 and β-CD improve central transitivity and central pharmacological effect of the GLP-2 peptide by Yusuke Nakao; Michiko Horiguchi; Ryuji Nakamura; Sachie Sasaki-Hamada; Chihiro Ozawa; Taichi Funane; Ryo Ozawa; Jun-Ichiro Oka; Chikamasa Yamashita (37-45).
Display OmittedDepression is a common mental disorder. More than 350 million people of all ages suffer from depression worldwide. Although a number of antidepressants are available, >20% of patients with major depressive disorder suffer from treatment-resistant depression. Therefore, development of novel therapeutics to overcome this condition is required. We reported that intracerebroventricular administration of glucagon-like peptide-2 (GLP-2) exerts antidepressant-like effects treated with or without adrenocorticotropic hormone. In the present study, we developed a nasal formulation of GLP-2 containing 5% polyoxyethylene (25) lauryl ether and 1% β-cyclodextrin that enhanced the resistance of GLP-2 to inactivation by dipeptidyl peptidase-4. Intranasal administration of this formulation (60 μg/kg) increased the delivery of GLP-2 to the brain and had antidepressant-like effects on rats. These results suggest the potential of the GLP-2 nasal formulation for use as a novel antidepressant.
Keywords: Glucagon-like peptide-2; Nasal formulation; Brain drug delivery and targeting; Nano micelle; Polycxyethylene (25) lauryl ether (LAURETH-25); β-cyclodextrin (β-CD);

Mechanisms of cellular uptake and endosomal escape of calcium-siRNA nanocomplexes by Matan Goldshtein; Efrat Forti; Emil Ruvinov; Smadar Cohen (46-56).
Display OmittedCa2+-siRNA nanocomplexes represent a simple yet an effective platform for siRNA delivery into the cell cytoplasm, with subsequent successful siRNA-induced target gene silencing. Herein, we aimed to elucidate the roles played by calcium ions in siRNA nanocomplex formation, cell uptake, and endosomal escape. We investigated whether the replacement of Ca2+in the nanocomplex by other bivalent cations would affect their cell entry and subsequent gene silencing. Our results indicate that Mg2+ and Ba2+ lead to the formation of nanocomplexes of similar physical features (size = 100 nm, surface charge ζ = −8 mV) as the Ca2+-siRNA nanocomplexes. Yet, these nanocomplexes were not uptaken by the cells to the same extent as those prepared with Ca2+, and siRNA-induced target gene silencing was not obtained. Cell internalization of Ca2+−-siRNA nanocomplexes, examined by employing chemical inhibitors to clathrin-, caveolin- and dynamin-mediated endocytosis pathways, indicated the involvement of all mechanisms in the process. Inhibition of endosome acidification by bafilomycin completely abolished the siRNA-mediated silencing by Ca2+-siRNA nanocomplexes. Collectively, our results indicate that Ca2+ promotes cell internalization and rapid endosomal escape, thus leading to the efficient siRNA-induced target gene silencing elicited by the Ca2+-siRNA nanocomplexes.
Keywords: Nano particles; siRNA; Calcium; Cellular uptake;

Pharmaceutical film dosage forms have recently become of interest to pharmaceutical formulation development, particularly for patients who experience difficulty in swallowing tablets or capsules. Furthermore, formulation scientists require a reliable analytical approach to reveal vital insight and investigate the drying process of these films to consolidate suitable quality control.Since most of the polymer-based films containing a drug are produced via solution or dispersion states, an estimation of the physicochemical properties of drugs during drying and dissolution is critical to design novel formulations with the consideration to control drug release, i.e. safety and efficacy to patients. This work presents the novel application of attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectroscopic imaging to study the drying process and dissolution behaviour of polymer-based films. Two types of the ibuprofen containing films, hydroxypropyl methylcellusose (HPMC) based films for immediate release and polyvinylpyrrolidone (PVP) based films for extended release, were studied in modified pH environments and changing hydrophobicity. ATR-FTIR imaging has revealed important information on water ingress into the films and the presence, distribution, and physicochemical state of the drug. ATR-FTIR imaging is a powerful technique to investigate and to deeply understand physicochemical processes for pharmaceutical polymer-based films.
Keywords: Drug delivery; Polymer films; Interactions; ATR; FT-IR spectroscopy; Imaging; Dissolution;

Can lipid nanoparticles improve intestinal absorption? by M. Mendes; H.T Soares; L.G Arnaut; J.J Sousa; A.A.C.C. Pais; C. Vitorino (69-83).
Display OmittedLipid nanoparticles and their multiple designs have been considered appealing nanocarrier systems. Bringing the benefits of these nanosystems together with conventional coating technology clearly results in product differentiation.This work aimed at developing an innovative solid dosage form for oral administration based on tableting nanostructured lipid carriers (NLC), coated with conventional polymer agents. NLC dispersions co-encapsulating olanzapine and simvastatin (Combo-NLC) were produced by high pressure homogenization, and evaluated in terms of scalability, drying procedure, tableting and performance from in vitro release, cytotoxicity and intestinal permeability stand points.Factorial design indicated that the scaling-up of the NLC production is clearly feasible. Spray-drying was the method selected to obtain dry particles, not only because it consists of a single step procedure, but also because it facilitates the coating process of NLC with different polymers. Modified NLC formulations with the polymers allowed obtaining distinct release mechanisms, comprising immediate, delayed and prolonged release. Sureteric:Combo-NLC provided a low cytotoxicity profile, along with a ca. 12-fold OL/3-fold SV higher intestinal permeability, compared to those obtained with commercial tablets. Such findings can be ascribed to drug protection and control over release promoted by NLC, supporting them as a versatile platform able to be modified according to the intended needs.
Keywords: Co-encapsulation; Nanostructured lipid carriers; Polymer coating; Scale-up; Spray-drying; Intestinal permeability;

Display OmittedIn this study, we developed the quaternized chitosan microgels without chemical crosslinking as an adjuvant of H5N1 split vaccine. The microgels with pH-sensitivity, positive surface charge and good biocompatibility, have been demonstrated in favor of enhancing both humoral and cellular immune response. However, the detailed mechanism of the chitosan-based microgels to enhance antigen specific immune responses remains unclear. Therefore, we prepared the quaternized chitosan microgels with well defined quaternization degrees (QDs, 20–80%) and particle sizes (800 nm–5 μm) by the premix membrane emulsification technique, and investigated the effect of quaternization degree (QD) and size on the adjuvanticity of microgels. Results suggested that microgels with relatively smaller size (807 nm) and moderate quaternization degree (QD 41% and 60%) were favorable for a maximum immune response. The mechanism was studied and explained by examining the characteristics of microgels and investigating the stimulation of bone-marrow derived dendritic cells (BMDCs). Moreover, they induced significantly stronger immune responses at lower antigen doses (known as antigen sparing effect) compared to aluminum adjuvant. These data indicated that a maximum immune response can be achieved by controlling properties of chitosan microgels, which also could serve as a significant guidance for rational design of chitosan-based particle adjuvant.
Keywords: Quaternized chitosan microgels; Hydrogel; Dose sparing effect; Adjuvant; H5N1 split vaccine;

Influence of process parameters on the preparation of pharmaceutical films by electrostatic powder deposition by Leena Kumari Prasad; Justin S. LaFountaine; Justin M. Keen; Robert O. Williams; James W. McGinity (94-103).
Display OmittedElectrostatic powder deposition (ESPD) has been developed as a solvent-free method to prepare pharmaceutical films. The aim of this work was to investigate the influence of process parameters during (1) electrostatic powder deposition, (2) curing, and (3) removal of the film from the substrate on the properties of the film. Polyethylene oxide (PEO) was used as the model polymer and stainless steel 316 as the substrate. Deposition efficiency (i.e. deposited weight) was measured with varying charging voltage, gun tip to substrate distance, and environmental humidity. Scanning electron microscopy was utilized to assess film formation, and adhesive and mechanical strength of films were measured with varying cure temperature and time. Adhesive strength was measured for films prepared on substrates of varying surface roughness. When deposition was performed at low humidity conditions, 25%RH, process parameters did not significantly affect deposition behavior. At 40%RH, increasing deposition efficiency with decreasing gun tip to substrate distance and increasing voltage (up to 60 kV) was observed. Complete film formation was seen by 30 min at 80 °C, compared to lower curing temperatures and times. All films were readily removed from the substrates. The results show the ESPD process can be modified to produce films with good mechanical properties (e.g. tensile strength > 0.06 MPa), suggesting it is a promising dry powder process for preparing pharmaceutical films.
Keywords: Electrostatic powder coating; Dry powder process; Pharmaceutical films; Adhesion; Polyethylene oxide;

Half generations magnetic PAMAM dendrimers as an effective system for targeted gemcitabine delivery by Maryam Parsian; Pelin Mutlu; Serap Yalcin; Aysen Tezcaner; Ufuk Gunduz (104-113).
Display OmittedTumor-specific delivery of anticancer drugs by magnetic nanoparticles will maximize the efficacy of the drug and minimize side effects, and reduce systemic toxicity. The magnetic core of these nanoparticles provides an advantage for selective drug targeting as they can be targeted to the tumor site and accumulated in cancer cells by means of an external magnetic field. Magnetic nanoparticles can be coated with Polyamidoamine (PAMAM) dendrimer and loaded with drugs. However, biomedical applications of PAMAM dendrimers are limited due to their toxicity associated with their multiple cationic charges due to terminal ―NH2 groups. Modifying the positively charged end groups with negatively charged ―COOH groups, is a satisfactory strategy for obtaining less toxic PAMAM dendrimers. Gemcitabine being an analogue of deoxycytidine, is an effective anticancer drug. However, clinical benefits of Gemcitabine are limited due to its short biological half-life. The aim of this study was to obtain an effective, less toxic targeted delivery system for Gemcitabine. Half generations, between G4.5 and G7.5, of PAMAM dendrimer coated magnetic nanoparticles (DcMNPs) were synthesized and conjugated with Gemcitabine. TEM images showed nanoscale size (12–14 nm) of the nanoparticles. The zeta-potential analysis indicated a decreased negativity of surface charge in drug bound dendrimer compared to the empty nanoparticles. Gemcitabine was effectively conjugated successfully onto the surface of half-generations of PAMAM DcMNPs. It was observed Gemcitabine did not effectively bind to Generations G4 and G5. The highest drug loading was obtained for DcMNPs with Generation 5.5. Empty nanoparticles showed no significant cytotoxicity on SKBR-3 and MCF-7 cells. On the other hand, Gemcitabine loaded nanoparticles were 6.0 fold more toxic on SKBR-3 and 3.0 fold more toxic on MCF-7 cells compared to free Gemcitabine. Gemcitabine loaded on Generation 5.5 DcMNPs showed a higher stability than free Gemcitabine. About 94% of the drug was retained over 6 weeks period, at pH 7.2. Due to their targetability under magnetic field, stability, size distribution, cellular uptake and toxicity characteristics the dendrimeric nanoparticles obtained in this study can be useful a delivery system for Gemcitabine in cancer therapy.
Keywords: Half generation PAMAM dendrimer; Gemcitabine; Magnetic nanoparticles; Chemotherapy; MCF-7; SKBR-3;

Continuous production of itraconazole-based solid dispersions by hot melt extrusion: Preformulation, optimization and design space determination by Justine Thiry; Pierre Lebrun; Chloe Vinassa; Marine Adam; Lauranne Netchacovitch; Eric Ziemons; Philippe Hubert; Fabrice Krier; Brigitte Evrard (114-124).
Display OmittedThe purpose of this work was to increase the solubility and the dissolution rate of itraconazole, which was chosen as the model drug, by obtaining an amorphous solid dispersion by hot melt extrusion. Therefore, an initial preformulation study was conducted using differential scanning calorimetry, thermogravimetric analysis and Hansen's solubility parameters in order to find polymers which would have the ability to form amorphous solid dispersions with itraconazole. Afterwards, the four polymers namely Kollidon® VA64, Kollidon® 12PF, Affinisol® HPMC and Soluplus®, that met the set criteria were used in hot melt extrusion along with 25 wt.% of itraconazole. Differential scanning confirmed that all four polymers were able to amorphize itraconazole. A stability study was then conducted in order to see which polymer would keep itraconazole amorphous as long as possible. Soluplus® was chosen and, the formulation was fine-tuned by adding some excipients (AcDiSol®, sodium bicarbonate and poloxamer) during the hot melt extrusion process in order to increase the release rate of itraconazole. In parallel, the range limits of the hot melt extrusion process parameters were determined. A design of experiment was performed within the previously defined ranges in order to optimize simultaneously the formulation and the process parameters. The optimal formulation was the one containing 2.5 wt.% of AcDiSol® produced at 155 °C and 100 rpm. When tested with a biphasic dissolution test, more than 80% of itraconazole was released in the organic phase after 8 h. Moreover, this formulation showed the desired thermoformability value. From these results, the design space around the optimum was determined. It corresponds to the limits within which the process would give the optimized product. It was observed that a temperature between 155 and 170 °C allowed a high flexibility on the screw speed, from about 75 to 130 rpm.
Keywords: Amorphous solid dispersions; Itraconazole; Solubility enhancement; Design of experiments; Design space determination;

Buccal absorption of diazepam is improved when administered in bioadhesive tablets—An in vivo study in conscious Göttingen mini-pigs by Emil Meng-Lund; Jette Jacobsen; Anette Müllertz; Erling B. Jørgensen; René Holm (125-131).
Display OmittedBuccal delivery may be clinically beneficial for compounds with a high gastrointestinal and hepatic first pass metabolism or in situations where a fast systemic absorption is desired. The delivery of a crystalline low soluble compounds, e.g. diazepam, may be limited due to the low volume of saliva available to facilitate solvation in order to drive the permeation of drug through the buccal mucosa. Therefore, the present study investigated the potential benefits of administering diazepam either as an amorphous or as a crystalline form in mucoadhesive tablets to conscious Göttingen mini-pigs. Presentation of the compound in the amorphous form lead to a very fast absorption, however, the obtained bioavailability was at the same level observed following buccal administration of a commercially immediate release tablet. Addition of chitosan, as a mucoadhesive excipient, resulted in a higher absolute bioavailability compared to tablets without chitosan. The absorption rate for the chitosan-based tablets was significant slower, probably due to the slower diffusion of the compound out of the tablet. In vitro release data was able to predict the variations in tmax, but otherwise no correlation could be found between in vitro and in vivo data.
Keywords: Diazepam; Buccal drug delivery; Chitosan; Mucoadhesive; Mini-pigs; Amorphous;

Nanomedicines for advanced cancer treatments: Transitioning towards responsive systems by Merel van Elk; Bruce P. Murphy; Tatiane Eufrásio-da-Silva; Daniel P. O’Reilly; Tina Vermonden; Wim E. Hennink; Garry P. Duffy; Eduardo Ruiz-Hernández (132-164).
Display OmittedThe development of nanomedicines for the treatment of cancer focuses on the local targeted delivery of chemotherapeutic drugs to enhance drug efficacy and reduce adverse effects. The nanomedicines which are currently approved for clinical use are mainly successful in terms of improved bioavailability and tolerability but do not necessarily increase drug performance. Therefore, there is a need for improved drug carrier systems which are able to deliver high doses of anti-cancer drugs to the tumor. Stimuli responsive carriers are promising candidates since drug release can be triggered locally in the tumor via internal (i.e. pH, redox potential, metabolite or enzyme concentration) or external (i.e. heat, ultrasound, light, magnetic field) stimuli. This review summarizes the recent progress in the transition towards stimuli responsive nanomedicines (i.e. liposomes, polymeric micelles, nanogels and mesoporous silica nanoparticles) and other therapy modalities that are currently developed in the fight against cancer like the application of ultrasound, tumor normalization and phototherapy. Furthermore, the potential role of image guided drug delivery in the development of new nanomedicines and its clinical application is discussed.
Keywords: Drug delivery; Cancer; Nanomedicine; Stimuli-responsive; Image guided therapies;

Multifunctional nanogels with dual temperature and pH responsiveness by Loredana Elena Nita; Aurica P. Chiriac; Alina Diaconu; Nita Tudorachi; Liliana Mititelu-Tartau (165-175).
Display OmittedOver the last 10 years, the development of intelligent biomaterials for medical and pharmaceutical applications has attracted growing interest by combining interdisciplinary efforts. Between them nanogels represent one of the most attractive carriers for innovative drug delivery systems. In the present investigation new variants of multi-responsive nanogels have been synthesized by crosslinking poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro [5.5] undecane) copolymer (having different molar ratios between comonomers) with 1,12-dodecandiol. The new structures were obtained by using modification of itaconic anhydride moieties in the copolymer. This is a convenient method for the preparation of a network with increased functionality, which further may ensure new strategies for coupling various bioactive compounds, especially owing to the behavior of the used copolymers, which present dual pH and temperature sensitive characteristics. The chemical structure of the new compounds was confirmed by FTIR and 1H RMN spectra. Also, the evaluation of thermal stability by thermogravimetric analysis sustains the covalent bonds occurring between the copolymer and diol. The dual responsiveness of the nanogel structures to temperature and pH was put into evidence by DLS studies. This feature can be used for the development of drug delivery systems, which can mimic biological response behavior to a certain extent. The new synthesized nanogels were tested as drug delivery systems by using diclofenac as a model drug. The results obtained from in vitro and in vivo investigation confirm the bioactivity of the nanogel networks.
Keywords: Nanogel network; Poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane); 1,12-Dodecandiol; Dual responsiveness; In vitro and in vivo tests;

Display OmittedIn recent years, oral colon specific drug delivery system has been paid more attention in the treatment of inflammatory bowel disease (IBD). As the special pH condition in gastrointestinal tract, the challenge for treatment of IBD was that the colon drug delivery system should endure the low pH in stomach and release drugs quickly in high pH in colon. Icariin with the poor solubility and low bioavailability limited the treatment of many diseases in clinic. In this study, the protective mechanism of alginate-chitosan microspheres loaded with icariin were investigated with trinitrobenzene sulfonic acid (TNBS)/ethanol induced colonic mucosal injury in rats. The results of drug release showed that the icariin loaded into microspheres released only 10% in simulated gastric fluid and a high amount of 65.6% released in simulated colonic fluid. The fluorescence tracer indicated high retention of targeted microspheres more than 12 h in colon. The microspheres loaded with icariin could not only reduce the colonic injury by decreasing the colon mucosa damage index in rats, but also reduce the inflammatory response by reducing the production and gene expression of inflammatory mediators and cytokines in colonic mucosa. All the results indicate that targeted microspheres loaded with icariin could exert the colon-protective effects through reducing the inflammatory response, which would be developed as a potential drug controlled release system for treatment of ulcerative colitis.
Keywords: Icariin; Microspheres; Colon specific drug delivery system; Anti-inflammatory; Colonic mucosal injury;

Dipeptide-functionalized polyamidoamine dendrimer-mediated apoptin gene delivery facilitates apoptosis of human primary glioma cells by Yoonhee Bae; Eric S. Green; Goo-Young Kim; Su Jeong Song; Ji Young Mun; Sunray Lee; Jong-Il Park; Jong-sang Park; Kyung Soo Ko; Jin Han; Joon Sig Choi (186-200).
Display OmittedGlioblastoma multiform (GBM) is the most frequent and aggressive form of brain tumors in adults. However, the development of more efficient and safe nonviral vector gene therapy represents a promising therapeutic approach, using a tumor-specific killer gene, named apoptin. In this study, we describe the efficacy of non-viral gene delivery vectors, the amino acid-conjugated PAMAM derivatives (PAMAM-H-R and PAMAM-H-K) in delivering a therapeutic gene, displaying affinity toward human primary glioma cells (GBL-14 cells) and dermal fibroblasts. We analyzed transfection efficiency, using luciferase (Luci) and a pDNA encoding for enhanced fluorescent protein (EGFP), and cytotoxicity in both cells. The results show that transfection efficiency of PAMAM-H-R improved compared to native PAMAM dendrimer, but cytotoxicity of PAMAM-H-R and PAMAM-H-K were very low. We treated both cells with a polyplex formation of PAMAM-H-R or PAMAM-H-K/apoptin, and analyzed their cellular uptake and localization by flow cytometry and confocal microscopy. Furthermore, we analyzed the endosomal escape effect using TEM images, and found that PAMAM-H-R showed very fast escape from endosome to the cytosol. Caspase 3 activity assay, cell cycle distribution, and JC-1 analysis showed apoptosis induced by apoptin in GBL-14 cells. This indicates that PAMAM-H-R can be a potential nonviral vector gene delivery carrier for brain tumor therapy. The present study demonstrates that PAMAM-H-R/apoptin gene polyplex can be used as an effective therapeutic candidate for GBM due to its selective induction of apoptosis in primary glioma cells as a potential nonviral gene delivery carrier for brain tumor therapy.
Keywords: Apoptin; Gene therapy; PAMAM dendrimer; Human primary glioma cells; Apoptosis;

Segmental-dependent permeability throughout the small intestine following oral drug administration: Single-pass vs. Doluisio approach to in-situ rat perfusion by Isabel Lozoya-Agullo; Moran Zur; Avital Beig; Noa Fine; Yael Cohen; Marta González-Álvarez; Matilde Merino-Sanjuán; Isabel González-Álvarez; Marival Bermejo; Arik Dahan (201-208).
Display OmittedIntestinal drug permeability is position dependent and pertains to a specific point along the intestinal membrane, and the resulted segmental-dependent permeability phenomenon has been recognized as a critical factor in the overall absorption of drug following oral administration. The aim of this research was to compare segmental-dependent permeability data obtained from two different rat intestinal perfusion approaches: the single-pass intestinal perfusion (SPIP) model and the closed-loop (Doluisio) rat perfusion method. The rat intestinal permeability of 12 model drugs with different permeability characteristics (low, moderate, and high, as well as passively and actively absorbed) was assessed in three small intestinal regions: the upper jejunum, mid-small intestine, and the terminal ileum, using both the SPIP and the Doluisio experimental methods. Excellent correlation was evident between the two approaches, especially in the upper jejunum (R 2  = 0.95). Significant regional-dependent permeability was found in half of drugs studied, illustrating the importance and relevance of segmental-dependent intestinal permeability. Despite the differences between the two methods, highly comparable results were obtained by both methods, especially in the medium-high P eff range. In conclusion, the SPIP and the Doluisio method are both equally useful in obtaining crucial segmental-dependent intestinal permeability data.
Keywords: Biopharmaceutics classification system; Intestinal permeability; Segmental-dependent permeability; Oral drug absorption;

Development of controlled-release cisplatin dry powders for inhalation against lung cancers by Vincent Levet; Rémi Rosière; Romain Merlos; Luca Fusaro; Gilles Berger; Karim Amighi; Nathalie Wauthoz (209-220).
Display OmittedThe present study focuses on the development of dry powders for inhalation as adjuvant chemotherapy in lung cancer treatment. Cisplatin was chosen as a potential candidate for a local treatment as it remains the main platinum component used in conventional chemotherapies, despite its high and cumulative systemic toxicities. Bulk cisplatin was reduced to submicron sizes using high-pressure homogenization, mixed with a solubilized lipid and/or PEGylated component and then spray-dried to produce controlled-release dry powder formulations. The obtained formulations were characterized for their physicochemical properties (particle size and morphology), aerodynamic performance and release profiles. Cisplatin content and integrity were assessed by electrothermal atomic absorption spectrometry and 195Pt nuclear magnetic resonance spectroscopy. DPI formulations with cisplatin contents ranging from 48.5 to 101.0% w/w exhibited high fine particle fractions ranging from 37.3% to 51.5% of the nominal dose. Formulations containing cisplatin microcrystals dispersed in solid lipid microparticles based on acceptable triglycerides for inhalation and PEGylated excipients showed a controlled-release for more than 24 h and a limited burst effect. These new formulations could provide an interesting approach to increasing and prolonging drug exposure in the lung while minimizing systemic toxicities.
Keywords: Dry powder inhaler; Lung cancer chemotherapy; Pulmonary delivery; Controlled-release; Cisplatin; Solid lipid microparticles;

Novel morphology change of Au-Methotrexate conjugates: From nanochains to discrete nanoparticles by Wei-Yuan Wang; Xiu-Fen Zhao; Xiao-Han Ju; Yu Wang; Lin Wang; Shu-Ping Li; Xiao-Dong Li (221-232).
Display OmittedA novel morphology change of Au-methotrexate (Au-MTX) conjugates that could transform from nanochains to discrete nanoparticles was achieved by a simple, one-pot, and hydrothermal growth method. Herein, MTX was used efficiently as a complex-forming agent, reducing agent, capping agent, and importantly a targeting anticancer drug. The formation mechanism suggested a similarity with the molecular imprinting technology. The Au–MTX complex induced the MTX molecules to selectively adsorb on different crystal facets of gold nanoparticles (AuNPs) and then formed gold nanospheres. Moreover, the abundantly binding MTX molecules promoted directional alignment of these gold nanospheres to further form nanochains. More interestingly, the linear structures gradually changed into discrete nanoparticles by adding different amount of ethylene diamine tetra (methylene phosphonic acid) (EDTMPA) into the initial reaction solution, which likely arose from the strong electrostatic effect of the negatively charged phosphonic acid groups. Compared with the as-prepared nanochains, the resultant discrete nanoparticles showed almost equal drug loading capacity but with higher drug release control, colloidal stability, and in vitro anticancer activity.
Keywords: Gold; Methotrexate; Nanochains; Discrete nanoparticles; Anticancer drug;

Dual release and molecular mechanism of bilayered aceclofenac tablet using polymer mixture by Hien Van Nguyen; Van Hong Nguyen; Beom-Jin Lee (233-244).
Display OmittedThe objectives of the present study were to develop a controlled-release bilayered tablet of aceclofenac (AFN) 200 mg with dual release and to gain a mechanistic understanding of the enhanced sustained release capability achieved by utilizing a binary mixture of the sustained release materials. Different formulations of the sustained-release layer were formulated by employing hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) as the major retarding polymers. The in vitro dissolution studies of AFN bilayered tablets were carried out in intestinal fluid (pH 6.8 buffer). The mechanism of the synergistic rate-retarding effect of the polymer mixture containing HPC and carbomer was elucidated by the rate of swelling and erosion in intestinal fluid and the molecular interactions in the polymer network. The optimized bilayered tablets had similar in vitro dissolution profiles to the marketed tablet Clanza®CR based on the similarity factor (f2) in combination with their satisfactory micromeritic, physicochemical properties, and stability profiles. Drug release from HPMC-based matrix was controlled by non-Fickian transport, while drug release from HPC-based matrix was solely governed by drug diffusion. The swelling and erosion data exhibited a dramatic increase of water uptake and a reduction of weight loss in the polymer mixture-loaded tablet. Fourier transform infrared (FTIR) spectra revealed strong hydrogen bonding between HPC and carbomer in the polymer mixture. Regarding spatial distribution of polymers in the polymer mixture-loaded tablet, carbomer was found to be the main component of the gel layer during the first 2 h of the hydration process, which was responsible for retarding drug release at initial stage. This process was then followed by a gradual transition of HPC from the glassy core to the gel layer for further increasing gel strength.
Keywords: Aceclofenac; Dual release; Bilayered tablet; Swelling and erosion; Spatial distribution of polymers; Mechanistic understanding; Polymer mixture;

Display OmittedIn vivo fate of nanomaterials is influenced by the particle size among other parameters. Thus, Health Agencies have identified the size of nanomaterial as an essential physicochemical property to characterize. This parameter can be explored by dynamic light scattering (DLS) that is described in the ISO standard 22412:2008(E) and is one of the methods recognized by Health Agencies. However, no protocol of DLS size measurement has been validated over a large range of size so far. In this work, we propose an extension of validation of a protocol of size measurement by DLS previously validated with certified reference materials (CRM) at 60 and 203 nm. The present work reports robustness, precision and trueness of this protocol that were investigated using CRM at 100 and 400 nm. The protocol was robust, accurate and consistent with the ISO standard over the whole range of size that were considered. Expanded uncertainties were 4.4 and 3.6% for CRM at 100 and 400 nm respectively indicating the reliability of the protocol. The range of application of the protocol previously applied to the size measurement of liposomes and polymer nanoparticles was extended to inorganic nanomaterial including silica nanoparticles.
Keywords: Nanomaterials; Dynamic light scattering; Standardized protocol; Validation; Range of size; Analysis of variance;

Immobilization and delivery of biologically active Lipoxin A4 using electrospinning technology by Eleonora Cianci; Oriana Trubiani; Francesca Diomede; Ilaria Merciaro; Ida Meschini; Pantaleone Bruni; Fausto Croce; Mario Romano (254-261).
Display OmittedLipoxin (LX)A4 is a lipoxygenase-formed arachidonic acid metabolite with potent anti-inflammatory, pro-resolution properties. Its therapeutic efficacy has been largely demonstrated in a variety of cellular, preclinical and clinical models. Among these, periodontal disease, where LXA4 promotes tissue repair, also by modulating functions of human periodontal ligament stem cells (hPDLSCs). As medicated biomembranes may be particularly useful in clinical settings, where local stimulation of tissue repair is needed, we used electrospinning to embed LXA4 in membranes made of poly(ethylene oxide) (PEO) and poly(d,l-lactide) (PDLLA). These membranes were fully characterized by scanning electron microscopy, differential scanning calorimetry and biocompatibility with hPDLSCs. Here, we report that LXA4 is retained in these membranes and that ∼15–20% of the total LXA4 amount added to the reaction can be eluted from the membranes using an aqueous buffered medium. The eluted LXA4 fully retained its capability to stimulate hPDLSC proliferation. A similar effect was obtained by adding directly the LXA4-containing membranes to cells. These results demonstrate for the first time that LXA4 can be incorporated into biomembranes, which may be useful to combat local inflammation and promote tissue repair in selected clinical settings.
Keywords: Biomembranes; Electrospinning; Inflammation; Lipoxin; Stem cells;

Evaluation of mesoporous carbon aerogels as carriers of the non-steroidal anti-inflammatory drug ibuprofen by Georgios K. Eleftheriadis; Maria Filippousi; Vassiliki Tsachouridou; Maria-Anna Darda; Lamprini Sygellou; Ioanna Kontopoulou; Nikolaos Bouropoulos; Theodore Steriotis; Georgia Charalambopoulou; Ioannis S. Vizirianakis; Gustaaf Van Tendeloo; Dimitrios G. Fatouros (262-270).
Mesoporous carbon aerogels can modulate the release profiles of ibuprofen whereas in vitro toxicity profiles appear to be compatible as carriers for the oral delivery of poorly soluble drugs.Display OmittedTowards the development of novel drug carriers for oral delivery of poorly soluble drugs mesoporous aerogel carbons (CAs), namely CA10 and CA20 with different pore sizes (10 and 20 nm, respectively), were evaluated. The non-steroidal anti-inflammatory lipophilic compound ibuprofen was incorporated via passive loading. The drug loaded carbon aerogels were systemically investigated by means of High-Resolution Transmission Electron Microscopy (HR-TEM), Nitrogen physisorption studies, X-ray diffraction (XRD), Differential Scanning Calorimetry (DSC), X-ray photon electron spectroscopy (XPS) and ζ-potential studies. In vitro release studies were performed in simulated intestinal fluids reflecting both fasted (FaSSIF) and fed (FeSSIF) state conditions. Cytotoxicity studies were conducted with human intestinal cells (Caco-2). Drug was in an amorphous state in the pores of the carbon carrier as shown from the physicochemical characterization studies. The results showed marked differences in the release profiles for ibuprofen from the two aerogels in the media tested whereas in vitro toxicity profiles appear to be compatible with potential therapeutic applications at low concentrations.
Keywords: Carbon aerogels; Poorly soluble drugs; Simulated intestinal fluids; Oral delivery;

In vitro and in silico characterisation of Tacrolimus released under biorelevant conditions by A. Mercuri; S. Wu; S. Stranzinger; S. Mohr; S. Salar-Behzadi; M. Bresciani; E. Fröhlich (271-280).
Display OmittedThis work aims to better understand the in vivo behaviour of modified release (MR) formulations (Envarsus® tablets and Advagraf® capsules) using in vitro properties of tacrolimus and in silico simulations. The in silico concentration profiles of tacrolimus released from the MR formulations were predicted after building a three compartments PK model with GastroPlus™, and using the experimentally determined in vitro physico-chemical properties as input parameters. In vitro-in vivo correlations (IVIVC) were obtained after deconvolution of in vivo data from a clinical trial. The IVIVC showed that the in vitro dissolution was faster than the in vivo deconvoluted dissolution for Advagraf®, while the in vitro dissolution was slightly slower than the in vivo deconvoluted dissolution for Envarsus®. Population PK simulation showed that variability in the simulation was lower for Envarsus® compared to Advagraf®. The in silico predicted preferential absorption sites were the proximal and distal tract for Advagraf® and Envarsus®, respectively. The integration of experimental in vitro solubility, permeability and biorelevant dissolution data allowed to generate in silico tacrolimus concentrations for two different MR formulations. This permitted to compare the two formulations in a single PK profile, in a simulated population PK study and with respect to their absorption sites.
Keywords: Biorelevant dissolution; PK modelling; Tacrolimus; Permeability; Solubility;

Redox-sensitive mPEG-SS-PTX/TPGS mixed micelles: An efficient drug delivery system for overcoming multidrug resistance by Dujuan Zhao; Huiyuan Zhang; Shengfeng Yang; Wenxiu He; Yuxia Luan (281-292).
The constructed mPEG-SS-PTX/TPGS mixed micelles can obviously improve the chemotherapy effect of PTX by reversing MDR.Display OmittedThe main cause of multidrug resistance (MDR) is overexpression of active efflux transporters, such as P-glycoprotein (P-gp). To reverse MDR and improve the chemotherapy effect of paclitaxel (PTX), we propose a new drug delivery system based on mixed micelles constructed with d-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS) and the mPEG-SS-PTX conjugate with consideration that TPGS is a P-gp inhibitor that can block the cancer cell action of pumping drugs outside of cells and can enhance the anticancer effect. mPEG-SS-PTX is synthesized by conjugating hydrophilic mPEG with a hydrophobic drug, PTX, via a redox-sensitive disulfide bond. The mPEG-SS-PTX conjugate is amphiphilic and can self-assemble in water. Mixed micelles formed by the mPEG-SS-PTX conjugate and TPGS have a low critical micelle concentration (CMC, ∼1.05 × 10−3  mg/mL) and high drug loading content (∼19.6%). The disulfide bond in the mPEG-SS-PTX conjugate can be broken in cancer cells (a reductive environment) and release PTX to kill cancer cells. In vitro cytotoxicity and cell uptake suggest that mixed micelles can effectively improve the accumulation of PTX in multidrug-resistant MCF-7 cells. Therefore, the present as-prepared mixed micelles very effectively reverse the MDR and enhance the therapeutic effect.
Keywords: Redox-sensitive; Polymer-drug conjugate; TPGS; Multidrug resistance;

In-vitro investigation regarding the effects of Gelucire® 44/14 and Labrasol® ALF on the secretory intestinal transport of P-gp substrates by Océane Dubray; Vincent Jannin; Fréderic Demarne; Yann Pellequer; Alf Lamprecht; Arnaud Béduneau (293-299).
Display OmittedIn this present study, the secretory transport of P-gp substrates, rhodamine 123 and digoxin, was evaluated using a Caco-2/HT29-MTX co-culture characterized by an efflux mechanism and a paracellular permeability closer to the human intestinal barrier compared to the Caco-2 monolayer gold standard. The influence of simulated intestinal fluids termed FeSSIF and FaSSIF on the intestinal absorption was also assessed in comparison with a conventional saline buffer. Labrasol® ALF and Gelucire® 44/14 in saline buffer significantly decreased to 83% and 62%, the P-gp-mediated transport of rhodamine 123 across the co-culture, respectively. The effects of Gelucire® 44/14 were much more exacerbated with the Caco-2 monolayer model with a reduced permeability to 34% but they were partially reversed in the co-culture with FeSSIF. The modulation by the lipid excipients of digoxin secretory transport across the Caco-2 monolayer and the co-culture was reduced compared with the rhodamine 123. This work also emphasizes the numerous parameters that have to be considered for predicting accurately the effects of potential P-gp inhibitors including the in-vitro model, the incubation media and the intrinsic properties of P-gp substrates.
Keywords: Labrasol®; Gelucire®; P-glycoprotein; Permeability; Intestinal barrier; Caco-2/HT29-MTX co-culture; Digoxin;

Display OmittedReactive oxygen species (ROS) play an important role in the development of inflammatory bowel diseases. Superoxide dismutase (SOD) has a great therapeutic potential by scavenging superoxide that is one of ROS; however, in vivo application is limited especially when it is orally administered. SOD is easily degraded in vivo by the harsh conditions of gastrointestinal tract. Here, we design a zein-alginate based oral drug delivery system that protects SOD from the harsh conditions of gastrointestinal tract and releases it in the environment of the small intestine. SOD is encapsulated in zein-alginate nanoparticles (ZAN) via a phase separation method. We demonstrate that ZAN protect SOD from the harsh conditions of the stomach or small intestine condition. ZAN (200:40) at the weight ratio of 200 mg zein to 40 mg of alginate releases SOD in a pH dependent manner, and it releases 90.8 ± 1.2% of encapsulated SOD at pH 7.4 in 2 h, while only 11.4 ± 0.4% of SOD was released at pH 1.3. The encapsulation efficiency of SOD in ZAN (200:40) was 62.1 ± 2.0%. SOD in ZAN (200:40) reduced the intracellular ROS level and it saved 88.9 ± 7.5% of Caco-2 cells from the toxic superoxide in 4 hours. Based on the results, zein-alginate based oral drug delivery systems will have numerous applications to drugs that are easily degradable in the harsh conditions of gastrointestinal tract.
Keywords: Zein; Alginate; Oral drug delivery; Superoxide dismutase;

Promises of a biocompatible nanocarrier in improved brain delivery of quercetin: Biochemical, pharmacokinetic and biodistribution evidences by Pramod Kumar; Gajanand Sharma; Rajendra Kumar; Bhupinder Singh; Ruchi Malik; Om Prakash Katare; Kaisar Raza (307-314).
Display OmittedIn various neurological disorders, antioxidants are frequently prescribed along with the specific treatment modalities. One such promising natural flavonoid is quercetin, offering better outcomes than established vitamins E and C. Though with immense promises, various challenges like poor oral-bioavailability (<2%), extensive first-pass metabolism, poor brain permeability, hydrophobic nature and physiological pH instability hinder its proper usage. Hence, it was planned to prepare quercetin-loaded nano lipidic carriers (NLCs) employing biocompatible components like phospholipids and tocopherol acetate for enhanced brain delivery. The outcomes were also compared with solid lipid nanoparticles (SLNs) of comparable composition. Both the nanocolloids offered better drug loading and controlled drug release with appreciable stability. In vitro antioxidant performance was improved after encapsulation in nanoparticles and the nanoparticles were substantially uptaken by Caco-2 cells. The difference in outcomes was vivid in pharmacokinetic studies, where nanoparticles, esp. NLCs substantially enhanced the relative bioavailability (approx. 6 folds), biological residence (2.5 times) and appreciably retarded the drug clearance (approx. 6 folds). On the other hand, both nanoparticles were able to substantially deliver the drug to brain. NLCs were observed to enhance the brain permeability of drug in a noticeable manner. In Conclusion, SLNs/NLCs can offer a better-platform for brain-delivery of quercetin.
Keywords: Neuroprotection; Neurological disorders; Bioavailability; Nanoparticles; SLNs; NLCs;

Display OmittedThis study aimed to build an innovative system to deliver a systemic small interfering RNA (siRNA) treatment for rheumatoid arthritis. We combined arginine-histidine-cysteine based oligopeptide-modified polymer micelles with siRNA targeting the nuclear factor κB subunit, RelA (siRelA). This is a key molecule in the control of inflammation. We tested the cellular uptake of siRNA and its effects on inflammatory cytokine levels in vitro using synoviocytes, and siRNA distribution and therapeutic effects in vivo in mice with collagen-induced arthritis (CIA). These studies showed that arginine-histidine based oligopeptide modified micelles produced effective cellular siRNA uptake and suppressed inflammatory cytokine levels in synoviocytes. In vivo, these micelles produced marked accumulation of siRNAs in arthritic paws in CIA mice, with much less accumulation in healthy mice. The siRelA-polymer micelle complexes also produced more effective suppression of RelA mRNA expression and inflammatory cytokine levels in the arthritic paws of CIA mice and reduced their clinical symptom scores and paw thickness.
Keywords: Rheumatoid arthritis; Polymeric micelles; Systemic siRNA delivery; Nuclear factor κB; RelA; Functional peptides;

Near-infrared chemical imaging (NIR-CI) of 3D printed pharmaceuticals by Milad Khorasani; Magnus Edinger; Dhara Raijada; Johan Bøtker; Johanna Aho; Jukka Rantanen (324-330).
Display OmittedHot-melt extrusion and 3D printing are enabling manufacturing approaches for patient-centred medicinal products. Hot-melt extrusion is a flexible and continuously operating technique which is a crucial part of a typical processing cycle of printed medicines. In this work we use hot-melt extrusion for manufacturing of medicinal films containing indomethacin (IND) and polycaprolactone (PCL), extruded strands with nitrofurantoin monohydrate (NFMH) and poly (ethylene oxide) (PEO), and feedstocks for 3D printed dosage forms with nitrofurantoin anhydrate (NFAH), hydroxyapatite (HA) and poly (lactic acid) (PLA). These feedstocks were printed into a prototype solid dosage form using a desktop 3D printer. These model formulations were characterized using near-infrared chemical imaging (NIR-CI) and, more specifically, the image analytical data were analysed using multivariate curve resolution-alternating least squares (MCR-ALS). The MCR-ALS algorithm predicted the spatial distribution of IND and PCL in the films with reasonable accuracy. In the extruded strands both the chemical mapping of the components in the formulation as well as the solid form of the active compound could be visualized. Based on the image information the total nitrofurantoin and PEO contents could be estimated., The dehydration of NFMH to NFAH, a process-induced solid form change, could be visualized as well. It was observed that the level of dehydration increased with increasing processing time (recirculation during the mixing phase of molten PEO and nitrofurantoin). Similar results were achieved in the 3D printed solid dosage forms produced from the extruded feedstocks. The results presented in this work clearly demonstrate that NIR-CI in combination with MCR-ALS can be used for chemical mapping of both active compound and excipients, as well as for visualization of solid form variation in the final product. The suggested NIR-CI approach is a promising process control tool for characterization of innovative patient-centred medicinal products.
Keywords: Hot-melt extrusion; Near-infrared chemical imaging; Multivariate data analysis; MCR-ALS; Patient-centered medicinal products;

Nanomedicine as a non-invasive strategy for drug delivery across the blood brain barrier by Vivienne H. Tam; Chris Sosa; Rui Liu; Nan Yao; Rodney D. Priestley (331-342).
Display OmittedThe blood brain barrier (BBB) is a major obstacle to drug delivery for diseases of the central nervous system (CNS). This brief review highlights the current invasive and non-invasive technologies available to address this problem. In particular, nanomedicine has shown much promise as a non-invasive strategy due to its drug loading capabilities, ease of targeting to the BBB, and small size. The versatility of this technology in terms of type of drug and imaging agent, carrier material, and targeting mechanism is highlighted in this review. The recent inclusion of imaging agents in the nanocarriers has important consequences for the field of theranostics.
Keywords: Nanomedicine; Theranostics; Blood brain barrier; Drug delivery; Imaging;

Display OmittedDesign of Experiments (DoE) is a powerful tool for systematic evaluation of process parameters’ effect on nanoparticle (NP) quality with minimum number of experiments. DoE was employed for optimization of ciprofloxacin loaded PLGA NPs for pulmonary delivery against Pseudomonas aeruginosa infections in cystic fibrosis (CF) lungs. Since the biofilm produced by bacteria was shown to be a complicated 3D barrier with heterogeneous meshes ranging from 100 nm to 500 nm, nanoformulations small enough to travel through those channels were assigned as target quality. Nanoprecipitation was realized utilizing MicroJet Reactor (MJR) technology based on impinging jets principle. Effect of MJR parameters flow rate, temperature and gas pressure on particle size and PDI was investigated using Box-Behnken design. The relationship between process parameters and particle quality was demonstrated by constructed fit functions (R2  = 0.9934 p < 0.0001 and R2  = 0.9983 p < 0.0001, for particle size and PDI, respectively). Prepared nanoformulations varied between 145.2 and 979.8 nm with PDI ranging from 0.050 to 1.00 and showed encapsulation efficiencies >65%. Response surface plots provided experimental data-based understanding of MJR parameters’ effect, thus NP quality. Presented work enables ciprofloxacin loaded PLGA nanoparticle preparations with pre-defined quality to fulfill the requirements of local drug delivery under CF disease conditions.
Keywords: Antibiotic-loaded nanoparticles; Design-of-experiments; Process optimization; Quality-by-design;

In vitro evaluation of the impact of gastrointestinal transfer on luminal performance of commercially available products of posaconazole and itraconazole using BioGIT by Alexandros Kourentas; Maria Vertzoni; Mira Symillides; Bart Hens; Joachim Brouwers; Patrick Augustijns; Christos Reppas (352-358).
Display OmittedBiorelevant Gastrointestinal Transfer system (BioGIT) has been shown to be useful in reproducing concentrations of drugs in the fasted upper small intestine after their administration in the stomach. In the present investigation, we evaluated the impact of gastrointestinal transfer on luminal performance of commercially available products of two highly lipophilic weak bases, posaconazole (Noxafil® suspension) and itraconazole (Sporanox® hard gelatin capsules and Sporanox® oral solution) by comparing % solid fraction, concentrations and supersaturation in the duodenal compartment of BioGIT with recently reported data in the upper small intestine of healthy adults. BioGIT was useful for estimating the % solid fraction in the upper small intestine, in cases where dissolution during gastric residence was incomplete, i.e. after administration of Noxafil® and Sporanox® capsules, and the precipitated fraction of itraconazole in the upper small intestine after administration of Sporanox® solution; median values in vitro were similar to the luminal values. Based on the values for the area under the concentration vs. time data estimated up to 45 min post initiation of the experiment, concentrations in the duodenal compartment of BioGIT were similar to previously measured concentrations in the upper small intestine of healthy adults or they overestimated them by up to 2.5 times. In most cases, supersaturation of contents in the upper small intestine was overestimated, partly due to underestimation of luminal solubility.
Keywords: BioGIT; Dissolution; Precipitation; Supersaturation; Posaconazole; Itraconazole;

Juglone loaded poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer by Xin Jin; Youwen Zhang; Zhenhai Zhang; Danbiao Che; Huixia Lv (359-366).
Display OmittedInvestigating the effects of juglone loaded P188/phospholipid mixed micelles (J-MM) in breast cancer. In vitro cytotoxicity, apoptotic effects, in vivo therapeutic efficacy and toxicity were used to assess its antitumour effect. Uptake and imaging were used to evaluate the effect on the uptake and passive targeting.Mixed micelle carrier enhanced the targeting and uptake by MB-231 cells. The tumour inhibition rates in tumour xenograft models for paclitaxel, juglone, J-MM (10 mg/kg) and J-MM (40 mg/kg) were 46%, 27%, 39% and 53%, respectively. J-MM (10 mg/kg) exhibited lower toxicity compared with that by free juglone or high dose J-MM.J-MM exhibited low toxicity, improved cellular uptake, passive targeting and anti-cancer effects in breast cancer model.
Keywords: Juglone; Mixed micelles; Antitumor; Toxicity; Uptake;

Trospium chloride is absorbed from two intestinal “absorption windows” with different permeability in healthy subjects by Tobias Tadken; Michael Weiss; Christiane Modess; Danilo Wegner; Tarek Roustom; Claudia Neumeister; Ulrich Schwantes; Hans-Ulrich Schulz; Werner Weitschies; Werner Siegmund (367-373).
Display OmittedIntestinal P-glycoprotein is regio-selectively expressed and is a high affinity, low capacity efflux carrier for the cationic, poorly permeable trospium. Organic cation transporter 1 (OCT1) provides lower affinity but higher capacity for trospium uptake. To evaluate regional intestinal permeability, absorption profiles after gastric infusion of trospium chloride (30 mg/250 ml = [I]2) for 6 h and after swallowing 30 mg immediate-release tablets in fasted and fed healthy subjects, were evaluated using an inverse Gaussian density function to model input rate and mean absorption time (MAT). Trospium chloride was slowly absorbed (MAT ∼10 h) after gastric infusion involving two processes with different input rates, peaking at about 3 h and 7 h. Input rates and MAT were influenced by dosage form and meal. In conclusion, trospium is absorbed from two “windows” located in the jejunum and cecum/ascending colon, whose uptake capacity might result from local abundance and functional interplay of P-glycoprotein and OCT1.
Keywords: Trospium chloride; Pharmacokinetics; Gastric infusion; Food effects; P-glycoprotein; OCT1;

Multispectral UV imaging for surface analysis of MUPS tablets with special focus on the pellet distribution by Anna Novikova; Jens M. Carstensen; Thomas Rades; Prof. Dr. Claudia S. Leopold (374-383).
Display OmittedIn the present study the applicability of multispectral UV imaging in combination with multivariate image analysis for surface evaluation of MUPS tablets was investigated with respect to the differentiation of the API pellets from the excipients matrix, estimation of the drug content as well as pellet distribution, and influence of the coating material and tablet thickness on the predictive model. Different formulations consisting of coated drug pellets with two coating polymers (Aquacoat® ECD and Eudragit® NE 30 D) at three coating levels each were compressed to MUPS tablets with various amounts of coated pellets and different tablet thicknesses. The coated drug pellets were clearly distinguishable from the excipients matrix using a partial least squares approach regardless of the coating layer thickness and coating material used. Furthermore, the number of the detected drug pellets on the tablet surface allowed an estimation of the true drug content in the respective MUPS tablet. In addition, the pellet distribution in the MUPS formulations could be estimated by UV image analysis of the tablet surface. In conclusion, this study revealed that UV imaging in combination with multivariate image analysis is a promising approach for the automatic quality control of MUPS tablets during the manufacturing process.
Keywords: Multispectral UV imaging; MUPS tablets; Multivariate image analysis; PLS; Pellet distribution; Content uniformity;

In vivo skin penetration of macromolecules in irritant contact dermatitis by Mona M.A. Abdel-Mottaleb; Alf Lamprecht (384-389).
Although nanostructures could accumulate preferentially in the inflamed skin, macromolecules behaved differently and their penetration was not enhanced in case of dermatitis.Display OmittedRecently, a selective preferential accumulation of polymeric nanoparticles (in the size range around 100 nm) has been observed in the follicular system of dermatitis skin. The present investigation aimed at clearly investigating the effect of irritant contact dermatitis on the barrier permeability for colloidal systems below this size range, namely quantum dots and hydrophilic macromolecules. Irritant dermatitis was induced in mice and the penetrability of quantum dots (5 nm) and hydrophilic dextran molecules has been tracked in both healthy and inflamed skin using confocal laser scanning microscopy. The selective accumulation of the quantum dots was clearly observed in inflamed skin while hydrophilic dextran behaved similarly in both healthy and inflamed skin. The therapeutic potential for the transdermal delivery of peptide drugs through inflamed skin has been also tested in rats. Results revealed that the transdermal permeation of insulin and calcitonin was not significantly enhanced in dermatitis compared to healthy skin. On the other side, permeation through stripped skin was significantly higher. However, the effect was limited and shorter compared to the SC injection where tmin was 0.5 h and 2 h with a 70% and 46% reduction in blood glucose levels for the stripped skin and the SC injection respectively. Similarly, tmin was 4 h and 8 h with area under the curve of 161 ± 65% and 350 ± 97% for the stripped skin and the SC injection respectively. In conclusion, the changes in skin permeability accompanied with skin inflammation did not affect its permeability to peptide drugs. Our findings also underline that experiments with the tape stripped skin model as a surrogate for inflamed skin can risk misleading conclusions due to significant difference of skin permeability between the tape stripped skin and inflamed skin.
Keywords: Skin; Dermatitis; Protein; Peptide; Quantum dots; Tape stripping;

Advancing pharmaceutical quality: An overview of science and research in the U.S. FDA’s Office of Pharmaceutical Quality by Adam C. Fisher; Sau L. Lee; Daniel P. Harris; Lucinda Buhse; Steven Kozlowski; Lawrence Yu; Michael Kopcha; Janet Woodcock (390-402).
Display OmittedFailures surrounding pharmaceutical quality, particularly with respect to product manufacturing issues and facility remediation, account for the majority of drug shortages and product recalls in the United States. Major scientific advancements pressure established regulatory paradigms, especially in the areas of biosimilars, precision medicine, combination products, emerging manufacturing technologies, and the use of real-world data. Pharmaceutical manufacturing is increasingly globalized, prompting the need for more efficient surveillance systems for monitoring product quality. Furthermore, increasing scrutiny and accelerated approval pathways provide a driving force to be even more efficient with limited regulatory resources. To address these regulatory challenges, the Office of Pharmaceutical Quality (OPQ) in the Center for Drug Evaluation and Research (CDER) at the U.S. Food and Drug Administration (FDA) harbors a rigorous science and research program in core areas that support drug quality review, inspection, surveillance, standards, and policy development. Science and research is the foundation of risk-based quality assessment of new drugs, generic drugs, over-the-counter drugs, and biotechnology products including biosimilars. This is an overview of the science and research activities in OPQ that support the mission of ensuring that safe, effective, and high-quality drugs are available to the American public.
Keywords: Pharmaceutical quality; Regulatory science; Emerging technology; Quality standards; Policy;

Co amorphous systems: A product development perspective by Rahul B Chavan; Rajesh Thipparaboina; Dinesh Kumar; Nalini R Shastri (403-415).
Display OmittedSolubility is one of the major problems associated with most of the new chemical entities that can be reasonably addressed by drug amorphization. However, being a high-energy form, it usually tends to re-crystallize, necessitating new formulation strategies to stabilize amorphous drugs. Polymeric amorphous solid dispersion (PASD) is one of the widely investigated strategies to stabilize amorphous drug, with major limitations like limited polymer solubility and hygroscopicity. Co amorphous system (CAM), a new entrant in amorphous arena is a promising alternative to PASD. CAMs are multi component single phase amorphous solid systems made up of two or more small molecules that may be a combination of drugs or drug and excipients. Excipients explored for CAM preparation include amino acids, carboxylic acids, nicotinamide and saccharine. Advantages offered by CAM include improved aqueous solubility and physical stability of amorphous drug, with a potential to improve therapeutic efficacy. This review attempts to address different aspects in the development of CAM as drug products. Criterion for co-former selection, various methods involved in CAM preparation, characterization tools, stability, scale up and regulatory requirements for the CAM product development are discussed.
Keywords: Co amorphous system; Amorphous solid dispersion; Polymers; Miscibility; Phase separation; Stability;

The effect of structurally related impurities on crystallinity reduction of sulfamethazine by grinding by Yoshito Hamada; Makoto Ono; Motomu Ohara; Etsuo Yonemochi (416-421).
Display OmittedIn this study, the effect of structurally related impurities on crystallinity reduction of sulfamethazine by grinding was evaluated. The crystallinity of sulfamethazine was not decreased when it was ground alone. However, when structurally related impurities with sulfonamide derivatives were blended, the crystallinity of sulfamethazine was decreased by grinding. Other materials without a sulfonamide moiety showed no such effect. The Raman spectra of sulfamethazine demonstrated that there was a difference between its crystalline and amorphous states within its sulfonamide structure. It was suggested that the sulfonamide structure of the impurities was important in causing the inhibition of recrystallization of sulfamethazine during grinding.
Keywords: Sulfamethazine; Crystallinity reduction; Grinding; Effect of impurity; Raman spectroscopy;

Delivery system for berberine chloride based on the nanocarrier ZnAl-layered double hydroxide: Physicochemical characterization, release behavior and evaluation of anti-bacterial potential by Mohamed Amine Djebbi; Alae Elabed; Zaineb Bouaziz; Moulay Sadiki; Soumya Elabed; Philippe Namour; Nicole Jaffrezic-Renault; Abdesslem Ben Haj Amara (422-430).
Display OmittedLayered double hydroxide (LDH) has attracted major interest as one of the most versatile drug delivery systems especially for adsorption capacity and/or controlled delivery property of bioactive agents owing to their combining features of biohybrid. ZnAl synthesized layered double hydroxide can offer a platform to immobilize various types of bioactive compounds, particularly berberine chloride (BBC). However, the immobilization reaction of berberine chloride into ZnAl-LDH was performed by direct co-precipitation method at different ratios of BBC/LDH. BBC–ZnAl-LDH biohybrids were characterized in terms of structure, surface morphology, in vitro drug release profile and antibacterial assay against various bacterial cells. The BBC biomolecules were attached by coordinate bond. Structural and microstructural characterization confirms that interaction of BBC with ZnAl-LDH occurs by adsorption rather than intercalation of BBC within LDH layers. The BBC release profiles from BBC–ZnAl-LDH had a longer release duration compared to the physical mixture, and the drug release seemed faster with the low ratio of BBC/LDH. BBC−ZnAl-LDH can be internalized into bacterial cells. In vitro experiments in PBS medium showed that BBC−ZnAl-LDH biohybrid had higher cytotoxicity and inhibitory effects against three pathogenic bacteria; Staphylococcus aureus CIP 543154, Pseudomonas aeruginosa A22 and Bacillus subtilus ILP 1428B upon the drug release profiles and its destructive potential depends on the loading BBC on the LDH layers. Nonetheless these results prove that the prepared BBC–ZnAl-LDH biohybrids retain the anti-bacterial character of the BBC molecules and are therefore potential modified drug delivery system (DDS).
Keywords: Layered double hydroxide (LDH); Berberine chloride (BBC); Co-precipitation; Drug delivery system (DDS); Anti-bacterial activity;

CXCR4-targeted modular peptide carriers for efficient anti-VEGF siRNA delivery by Anna Egorova; Anastasia Shubina; Dmitriy Sokolov; Sergey Selkov; Vladislav Baranov; Anton Kiselev (431-440).
Display OmittedThe application of small interfering RNA (siRNA) for specific gene inhibition is a promising strategy in gene therapy treatments. The efficient cellular delivery of therapeutic siRNA is a critical step in RNA interference (RNAi) application. Highly efficient siRNA carriers should be developed for specific cellular uptake, stable RNA-complexes formation and intracellular RNA release. To study these features, we evaluated modular peptide carriers bearing CXCR4 targeting ligand for their ability to condense siRNA, facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. Peptide carriers were shown to condense and protect siRNA from RNAse degradation. Various N/P ratios were used for physicochemical characterization to optimize siRNA/peptide complexes for in vitro studies. On average, cytotoxicity of siRNA-polyplexes depended on cell type and was not higher than that of PEI/siRNA complexes. VEGFA gene knockdown was significantly improved with CXCR4-targeted carriers in contrast to nontargeted peptides. siRNA delivery by means of ligandconjugated carriers resulted in 2.5–3-fold decrease of VEGF expression in glioblastoma cells and in 1.5–2-fold decrease of VEGF expression in endothelial cells. Delivery of siRNA/peptide complexes resulted in 2–6- fold decrease in VEGF protein yield and in significant inhibition of endothelial cells migration. The study shows that implication of peptide carriers modified with CXCR4 ligand is a promising approach to develop targeted siRNA delivery system into CXCR4-expressing cancer and endothelial cells.
Keywords: CXCR4; VEGF; siRNA; Glioblastoma; Endothelium; Cross-linking peptides;

Impact of sodium lauryl sulfate in oral liquids on e-tongue measurements by Laura Isabell Immohr; Roy Turner; Miriam Pein-Hackelbusch (441-448).
Display OmittedDuring development of oral liquid medicines taste assessment is often required to evaluate taste and taste masking. Electronic tongue analysis can provide taste assessment of medicinal products but should only be conducted with medicines that interact with the instrument without damaging the sensor membranes or interfering with their electrical output so that robust data is generated. To explore the impact of a substance deemed unsuitable for electronic tongue analysis the influence of the anionic surfactant sodium lauryl sulfate (SLS), on the performance of the electronic tongue was conducted using electronic tongues equipped with self-developed PVC based sensors. The results showed a significant impact of SLS on all applied sensor types and an alteration of the sensor’s sensitivity. Nevertheless, concentration dependent sensor responses could still be obtained and the sensor performance was not impacted negatively. Assessment of unsuitable substances should therefore be evaluated prior to performing electronic tongue analysis so that their impact is understood fully.
Keywords: Electronic-tongue sensors; Sensitivity; Performance test;

Polysialic acid-modifying liposomes for efficient delivery of epirubicin, in-vitro characterization and in-vivo evaluation by Ting Zhang; Songlei Zhou; Ling Hu; Bo Peng; Yang Liu; Xiang Luo; Yanzhi Song; Xinrong Liu; Yihui Deng (449-459).
Display OmittedPolysialic acid (PSA) serves as a hydrophilic polymer and affords conjugated biologically active molecules a longer circulation time in vivo. Furthermore, PSA could potentially target tumor tissues and help achieve better curative effects. In this study, PSA was conjugated with octadecyl dimethyl betaine (BS18) to yield a PSA-BS18 conjugate. The PSA-BS18 modified liposomal epirubicin (EPI-SL), had a particle size of 133.63 ± 0.92 nm, a zeta potential of −26.23 ± 1.50 mV and an encapsulation efficiency (%EE) of 96.23 ± 1.16%. In vitro release studies showed that PSA-BS18 could delay EPI release from the modified liposomes. The MTT assay suggested that EPI-SL led to stronger cytotoxic activity than that exhibited by common and PEGylated liposomes. The pharmacokinetic study showed that EPI-SL prolonged the residence time of the EPI in the blood compared with that observed from common liposomes. Bio-distribution results obtained from tumor-bearing mice clearly demonstrated that PSA-BS18 increased the accumulation of modified liposomes in tumors compared with that of common liposomes. In the antitumor efficacy study, EPI-SL showed the best antitumor and life-prolonging effects among all of the tested formulations. These findings strongly indicate EPI-SL might have great potential as an effective approach for anticancer therapy.
Keywords: Polysialic acid; Long circulation time; Tumor shed; Antitumor efficacy;

Drug-eluting coating of ginsenoside Rg1 and Re incorporated poly(lactic-co-glycolic acid) on stainless steel 316L: Physicochemical and drug release analyses by Zulaika Miswan; Siti Khadijah Lukman; Fadzilah Adibah Abd Majid; Mun Fai Loke; Syafiqah Saidin; Hendra Hermawan (460-466).
Display OmittedActive ingredients of ginsenoside, Rg1 and Re, are able to inhibit the proliferation of vascular smooth muscle cells and promote the growth of vascular endothelial cells. These capabilities are of interest for developing a novel drug-eluting stent to potentially solve the current problem of late-stent thrombosis and poor endotheliazation. Therefore, this study was aimed to incorporate ginsenoside into degradable coating of poly(lactic-co-glycolic acid) (PLGA). Drug mixture composed of ginseng extract and 10% to 50% of PLGA (xPLGA/g) was coated on electropolished stainless steel 316L substrate by using a dip coating technique. The coating was characterized principally by using attenuated total reflectance-Fourier transform infrared spectroscopy, scanning electron microscopy and contact angle analysis, while the drug release profile of ginsenosides Rg1 and Re was determined by using mass spectrometry at a one month immersion period. Full and homogenous coating coverage with acceptable wettability was found on the 30PLGA/g specimen. All specimens underwent initial burst release dependent on their composition. The 30PLGA/g and 50PLGA/g specimens demonstrated a controlled drug release profile having a combination of diffusion- and swelling-controlled mechanisms of PLGA. The study suggests that the 30PLGA/g coated specimen expresses an optimum composition which is seen as practicable for developing a controlled release drug-eluting stent.
Keywords: Dip coating; Drug release; Ginsenoside; Poly(lactic-co-glycolic acid); Stent;

Controlled drug release from hydrogels for contact lenses: Drug partitioning and diffusion by A.F.R. Pimenta; J. Ascenso; J.C.S. Fernandes; R. Colaço; A.P. Serro; B. Saramago (467-475).
Display OmittedOptimization of drug delivery from drug loaded contact lenses assumes understanding the drug transport mechanisms through hydrogels which relies on the knowledge of drug partition and diffusion coefficients. We chose, as model systems, two materials used in contact lens, a poly-hydroxyethylmethacrylate (pHEMA) based hydrogel and a silicone based hydrogel, and three drugs with different sizes and charges: chlorhexidine, levofloxacin and diclofenac. Equilibrium partition coefficients were determined at different ionic strength and pH, using water (pH 5.6) and PBS (pH 7.4). The measured partition coefficients were related with the polymer volume fraction in the hydrogel, through the introduction of an enhancement factor following the approach developed by the group of C. J. Radke (). This factor may be decomposed in the product of three other factors EHS , Eel and Ead which account for, respectively, hard-sphere size exclusion, electrostatic interactions, and specific solute adsorption. While EHS and E el are close to 1, Ead  > > 1 in all cases suggesting strong specific interactions between the drugs and the hydrogels. Adsorption was maximal for chlorhexidine on the silicone based hydrogel, in water, due to strong hydrogen bonding. The effective diffusion coefficients, De , were determined from the drug release profiles. Estimations of diffusion coefficients of the non-adsorbed solutes D  =  De  ×  Ead allowed comparison with theories for solute diffusion in the absence of specific interaction with the polymeric membrane.
Keywords: Drug release; Contact lens; Hydrogel membrane; Partition coefficient; Diffusion coefficient;

Enhanced oral delivery of alendronate by sucrose fatty acids esters in rats and their absorption-enhancing mechanisms by Tammam Alama; Hiroko Katayama; Sachiko Hirai; Shizuka Ono; Ayumi Kajiyama; Kosuke Kusamori; Hidemasa Katsumi; Toshiyasu Sakane; Akira Yamamoto (476-489).
Display OmittedOral delivery is the most fascinating route for interminable drug remedy. However, the intestinal absorption of alendronate (ALN), a bisphosphonate drug after oral administration is very poor. Absorption enhancers, which help to achieve the efficiency-safety balance, are considered one of the most promising agents for the improvement the intestinal absorption of drugs. In the current study, we focused on using sucrose fatty acid esters (SEs) as promising absorption enhancers to enhance the intestinal absorption of alendronate using an in situ closed-loop method in rats. The intestinal absorption of alendronate was significantly enhanced in the presence of SEs, especially L-1695. In addition, no considerable increase was observed in the activity of lactate dehydrogenase (LDH) or in protein release from the intestinal epithelium in the presence of sugar esters at concentrations equivalent to or lower than 1.0% (w/v), suggesting that these compounds are safe. Furthermore, mechanistic studies revealed increased membrane fluidity and loosening of the tight junctions (TJs) might be the underlying mechanism by which SEs improve the intestinal intake of alendronate, via transcellular and paracellular routes, respectively. These findings suggest that SEs are effective absorption enhancers for improving the intestinal absorption of alendronate, without causing serious damage to the enteric epithelium.
Keywords: Intestinal absorption; Absorption enhancer; Sucrose fatty acid ester; Poorly absorbed drug; Alendronate; Bisphosphonate;

Development of a novel oral delivery system of edaravone for enhancing bioavailability by Ankit Parikh; Krishna Kathawala; Chun Chuan Tan; Sanjay Garg; Xin-Fu Zhou (490-500).
Display OmittedEdaravone (EDR), a strong free radical scavenger, is known for its promising therapeutic potential in oxidative stress (OS) associated diseases, however poor oral bioavailability is the major obstacle in its potential use. Oral liquid dosage form is the most preferred delivery method in paediatric, geriatric and specialised therapies. The present research discusses the development of a Novel Oral Delivery System (NODS) of EDR to enhance oral bioavailability. From preformulation study, solubility, and stability were identified as key challenges and the requirement of an acidic environment and protection against oxidation were found to be critical. The NODS made up of a mixture of Labrasol (LBS) and an acidic aqueous system, was optimized on the basis of solubility and stability study. It can be stored ≤40 °C for at least one month. Drug release from NODS was slow, sustained and significantly better as compared to suspension. The significant reduction in metabolism and improvement in permeability across the small intestine were observed with NODS compared to free EDR. The oral pharmacokinetic study showed 571% relative bioavailability with NODS compared to EDR suspension. From the results obtained, NODS is a promising candidate for use in OS associated diseases.
Keywords: Edaravone; Labrasol; Aqueous stability; Metabolism; Permeability; Oral bioavailability;

Display OmittedCubosomes were used to increase the aqueous solubility of the water insoluble anticancer drug SN38. The results showed that the use of a common cubosome formulation consisting of phytantriol (PHYT) as the matrix amphiphile (PHYT-cubosome) led to a 6-fold increase in the solubility of SN38. However, mean hydrodynamic diameter (D H) and polydispersity index (PDI) of these PHYT-cubosome particles were 345 ± 49 nm and 0.37 ± 0.05, respectively, making them unsuitable for intravenous applications. Several additives were investigated to increase the solubility of SN38 and reduce the DH and PDI values of the resulting particles. Charged additives such as didodecyldimethyl ammonium bromide (DDAB) and sodium dodecyl sulfate (SDS) led to improvements in the physiochemical properties of the cubosomes. Notably, the PHYT-DDAB and PHT-SDS cubosomes led to 15- and 14-fold increases in the aqueous solubility of SN38, respectively. Moreover, the SN38 loaded into the PHYT-DDAB and PHYT-SDS cubosomes was found to be highly stable, with very little hydrolysis to its inactive acid form. In summary, the addition of DDAB and SDS to PHYT-cubosome nanoparticle drug delivery systems not only led to considerable improvements in their physiochemical properties, but also enhanced the aqueous solubility of SN38 and increased its chemical stability.
Keywords: Cubosomes; Poorly water-soluble drug; SN38; Hydrolysis; Chemical stability;

Nanoemulsion enhances α-tocopherol succinate bioavailability in rats by Yanan Gao; Xuejing Qi; Yiping Zheng; Hongyu Ji; Linhua Wu; Nannan Zheng; Jingling Tang (506-514).
Display OmittedThe vitamin E analogue, α-tocopherol succinate (α-TOS), has a broad anti-tumor effect. α-TOS can induce cancer cells apoptosis and suppress tumor growth by targeting mitochondria. Low bioavailability of α-TOS is the major problem encountered with formulation development. In our study, α-TOS nanoemulsion (α-TOS-NE) was demonstrated as a new drug delivery system of α-TOS to increase the bioavailability. MTT-based cytotoxicity assay and mitochondrial membrane potential (ΔY) were performed on human breast cancer cell lines MCF-7 and human oral epithelial cancer cell lines KB to evaluate in vitro anticancer efficacy of α-TOS-NE. In comparison with free α-TOS, α-TOS-NE exhibited a stronger cytotoxicity and decreased ΔΨ. Pharmacokinetic profiles of I.V. α-TOS-NE group, I.P. α-TOS-NE group, and I.P. free α-TOS group (7% DMSO/93% PEG) were drawn. First of all, nanoemultion (NE) enables the I.V. injection of α-TOS, make it possible to be an I.V. preparation. Second, compare to the I.P. free α-TOS group, I.P. α-TOS-NE group had a higher bioavailability. Thus, NE improved the strong anti-cancer efficacy of α-TOS while increasing its in vivo bioavailability in rats. In conclusion, our laboratory-made NE was a safe drug delivery system for clinical trials and could be a promising formulation for α-TOS by I.V administration.
Keywords: α-tocopherol succinate; Nanoemulsion; Cancer; Mitochondria; Bioavailability;

Miktoarm star copolymers from D-(−)-salicin core aggregated into dandelion-like structures as anticancer drug delivery systems: synthesis, self-assembly and drug release by Anna Mielańczyk; Justyna Odrobińska; Sebastian Grządka; Łukasz Mielańczyk; Dorota Neugebauer (515-526).
Display OmittedThe β-glucoside-based heterofunctional initiator was used in the synthesis of well-defined eight-armed miktopolymers by sequential ring opening polymerization (ROP) of ε-caprolactone (CL) and atom transfer radical (co)polymerization (ATRP) of methyl methacrylate (MMA) and/or tert-butyl methacrylate (tBMA). Consequently, methacrylic acid (MAA) repeating units were introduced via selective cleavage of pendant tert-butyl protecting groups. Both the amphiphilic copolymers and miktoarm copolymers were self-assembled at 37 °C and pH 7.4. The aggregates of miktoarm polymers were larger than that formed by polymethacrylate homoarm stars (≥250 nm vs ≤200 nm). The critical aggregation concentrations (CAC) of (mikto)stars were relatively low (0.006–0.411 mg/mL) and decreased with the increase in MAA fraction content. Both MAA-based mikto- and homoarmed (co)polymers with shorter arms exhibited lower doxorubicin (DOX) loading capacity, whereas camptothecin (CPT) was encapsulated preferably by miktostars. The kinetic profiles of drug release showed that the rate of release was higher at acidic environment (pH 5.0) than in neutral pH. In the most cases the studied miktopolymer systems demonstrated the well-controlled delivery of the model anticancer drugs, which can be adjusted by structural parameters of polymeric carriers.
Keywords: Miktoarm star polymers; Drug-loading; Drug delivery; Doxorubicin; Camptothecin;

Delivery of poorly soluble GNF-5837 via albumin hybrid nanoparticles (GNF-Alb-HNPs) shows significantly superior anticancer efficacy in a panel of breast cancer cell lines.Display OmittedNovel albumin hybrid nanoparticles (Alb-HNPs) loaded with tyrosine kinase A (TrkA) inhibitor GNF-5837 were prepared and evaluated for antineoplastic efficacy in a panel of breast cancer cell lines. The nanomedicines (GNF-Alb-HNPs, hydrodynamic diameter ∼150 nm) were formed through a unique polyelectrolyte complexation process where albumin and GNF-5837 were encapsulated by a stabilizing layer of oppositely charged chitosan and dextran sulfate polysaccharides. GNF-Alb-HNPs showed an excellent colloidal stability and a sustained drug release over more than 24 h. We found that these nanomedicines inhibited TrkA phosphorylation and downstream mitogen-activated protein kinase (MAPK) signaling in breast cancer cells specifically, resulting in anti-proliferative and pro-apoptotic effects. Moreover, the migration and invasion activities of cancer cells were dramatically suppressed and the inhibitory effects were much more prominent with GNF-Alb-HNPS than the drug alone. These results show that the GNF-Alb-HNPs may represent a novel approach for targeted breast cancer therapy.
Keywords: Albumin hybrid nanoparticles; TrkA inhibitor; Breast cancer; Targeted therapy;

Gellan gum fluid gels for topical administration of diclofenac by Mohammed H. Mahdi; Barbara R. Conway; Thomas Mills; Alan M. Smith (535-542).
Display OmittedDiclofenac topical formulations are often preferred for drug administration to patients who experience serious GIT problems. Absorption of the drug through the skin, however, can be challenging due to the natural protective feature of the stratum corneum (SC). In this article, fluid gels prepared from gellan gum were explored as a topical drug delivery vehicle. Rheological analysis of the formulations showed that it was possible to produce a topical gel with a viscosity and the mechanical strength similar to that of the commercially available Voltaren® gel using 1% w/w of a 50:50 low acyl/high acyl (LA/HA) gellan blend. Soft-tribology was used to assess the lubrication properties of gellan fluid gels. The lubrication of the gellan gum fluid gel formulations at high rubbing speeds was similar to the lubrication of the Voltaren® gel. The use of gellan gum dramatically increased skin permeation of diclofenac when compared with the commercially available formulation and could be controlled by changing the gellan gum concentration and/or sodium ion concentration in the formulation. This study highlights the potential use of fluid gels that can be easily tuned to have physical properties suitable for topical formulations with the added advantage of increasing drug permeation.
Keywords: Gellan gum; Rheology; Tribology; Topical; Diclofenac; Skin permeation; Fluid gel;

Microwave-assisted formulation of solid lipid nanoparticles loaded with non-steroidal anti-inflammatory drugs by Rohan M. Shah; Daniel S. Eldridge; Enzo A. Palombo; Ian H. Harding (543-554).
Display OmittedStearic acid-based solid lipid nanoparticles (SLNs) were prepared using the microwave assisted one-pot microemulsions procedure pioneered by our group. In this study, non-steroidal anti-inflammatory drugs (NSAIDs) including indomethacin, ketoprofen and nimesulide were selected as ideal “test” drugs, based on their poor water solubility. The model drugs were incorporated within the SLNs by the microwave-assisted procedure at the time of SLN production. The microwave-produced drug-loaded SLNs were evaluated in terms of their physicochemical characteristics, drug release behavior and their uptake into against A549 cell line (human lung epithelial cells). The microwave-produced drug-loaded SLNs had a small particle size distribution, negative zeta potential and high encapsulation efficiency. The drug release studies were consistent with a core-shell structure of SLNs (probably a drug-loaded shell) which results in biphasic drug release from the SLNs. The drug release kinetics suggested a good fit of the release data to the Makoid-Banakar model and was governed by Fickian diffusion. The drug-loaded SLNs showed concentration-dependent cytotoxicity and reduced IL-6 and IL-8 secretion in lipopolysaccharide-induced cells. All of the above findings suggest that the microwave-produced SLNs could be promising drug carriers of NSAIDs and will further facilitate their development for topical, oral and/or nasal administration.
Keywords: Microwave-assisted microemulsion; Solid lipid nanoparticles; Non-steroidal anti-inflammatory drugs; Drug release; Release kinetic models; LPS-induced inflammation;

Porous microscaffolds for 3D culture of dental pulp mesenchymal stem cells by Ronak S. Bhuptani; Vandana B. Patravale (555-564).
Display OmittedThe collective power of stem cells due to their evident advantages is incessantly investigated in regenerative medicine to be the next generation exceptional remedy for tissue regeneration and treatment of diseases. Stem cells are highly sensitive and a 3D culture environment is a requisite for its successful transplantation and integration with tissues. Porous microscaffolds can create a 3D microenvironment for growing stems cells, controlling their fate both in vitro and in vivo. In the present study, interconnected porous PLGA microscaffolds were fabricated, characterized and employed to propagate human dental pulp mesenchymal stem cells (DPMSCs) in vitro. The porous topography was investigated by scanning electron microscopy and the pore size was controlled by fabrication conditions such as the concentration of porogen. DPMSCs were cultured on microscaffolds and were evaluated for their morphology, attachment, proliferation, cell viability via MTT and molecular expression (RT-PCR). DPMSCs were adequately proliferated and adhered over the microscaffolds forming a 3D cell-microscaffold construct. The average number of DPMSCs grown on PLGA microscaffolds was significantly higher than monolayer 2D culture during 5th and 7th day. Moreover, cell viability and gene expression results together corroborated that microscaffolds maintained the viability, stemness and plasticity of the cultured dental pulp mesenchymal stem cells. The novel porous microscaffold developed acts as promising scaffold for 3D culture and survival and transplantation of stem cells for tissue engineering.
Keywords: Dental pulp stem cell; Stem cells; Microscaffolds; 3D culture; PLGA; Scaffolds; Microcarriers;

Buccal delivery of low molecular weight heparin by cationic polymethacrylate nanoparticles by Samiha Mouftah; Mona M.A. Abdel-Mottaleb; Alf Lamprecht (565-574).
Display OmittedBuccal delivery seems to be a very promising administration route for macromolecular drugs. Here, we explored the potential of cationic polymethacrylate nanoparticles (NPs) as a carrier system for the buccal delivery of low molecular weight heparin (LMWH). LMWH-loaded NPs were prepared by emulsification solvent diffusion method and the NPs were analyzed for their physiochemical properties, rheological evaluations and ex vivo transport studies across buccal mucosa. The prepared LMWH-loaded NPs showed a mean diameter between 400 and 500 nm with unimodal size distribution with negative surface charge. Viscosity measurements revealed a positive rheological synergism between the prepared NPs and mucin when mixed under physiological conditions. After 4 h, about 6.3 ± 0.9% of LMWH was released in case of using Eudragit® RS (ERS); while Eudragit® RL (ERL) NPs released only 3.0 ± 0.3 % of its LMWH content and this incomplete release was slightly ameliorated in the presence of mucin reaching to 7.2 ± 0.3 % and 4.8 ± 0.3 % for ERS and ERL, respectively. The ex-vivo permeability of heparin through the buccal mucosa was significantly increased after using polymetharylate NPs while no heparin permeation was detected from free heparin solution. Confocal laser scanning microscopy (CLSM) imaging indicated the mucoadhesive properties of the polymetharylate NPs where the drug-free NPs were detected in the superficial layers of buccal mucosa. LMWH-loaded NPs had less mucoadhesive properties showing significant deeper penetration of the mucosa. The results indicated that mucoadhesive cationic polymethacrylate NPs offer a possible approach for the buccal delivery of heparin.
Keywords: Buccal mucosa; Polymethacrylate; Nanoparticles; LMWH (Low molecular weight heparin); Mucoadhesion;

Preformulation of cysteamine gels for treatment of the ophthalmic complications in cystinosis by Barbara McKenzie; Graeme Kay; Kerr H. Matthews; Rachel Knott; Donald Cairns (575-582).
Display OmittedNephropathic cystinosis is a rare autosomal recessive disease characterised by raised lysosomal levels of cystine in the cells of all organs. It is treated by regular administration of the aminothiol, cysteamine. Corneal crystal deposition is one of the most troublesome complications affecting patients and requires the hourly administration of cysteamine eye drops. In an attempt to reduce this frequency and improve the treatment, the preformulation and evaluation of cysteamine containing gels is reported. Suitability for ophthalmic delivery was determined by analysis of rheology, bioadhesion, dissolution and stability. The results demonstrated that three polymers were suitable for ophthalmic delivery of cysteamine; namely sodium hyaluronate, hydroxyethyl cellulose and carbomer 934. Sodium hyaluronate displayed optimum performance in the preformulation tests, being pseudoplastic (reduction in apparent viscosity under increasing shear rate), bioadhesive, releasing cysteamine over 40 min and displaying stability over time. In conclusion these results offer the possibility to formulate cysteamine in an ocular applicable gel formulation.
Keywords: Cystinosis; Ophthalmic delivery; Gel; Cysteamine; Preformulation;

Rapid and cost-effective xenograft hepatocellular carcinoma model in Zebrafish for drug testing by Federica Tonon; Cristina Zennaro; Barbara Dapas; Michele Carraro; Massimo Mariotti; Gabriele Grassi (583-591).
Display OmittedWe developed a novel, rapid and cost-effective Zebrafish xenograft model of hepatocellular carcinoma (HCC) for drug screening in the disease.Following injection into the yolk sack of Zebrafish larvae of the human HCC cell line JHH6 stained by a vital dye, tumor mass growth was followed by fluorescence microscopy and by human Ki67 quantification. Tumor induced neo-angiogenesis was evaluated by alkaline phosphatase staining of the vessels, by using the Tg(fli1:EGFP)y1 strain of Zebrafish and by the quantification of the zebrafish vascular endothelial growth factor and of its receptor.We show that it is feasible to micro-inject JHH6 in Zebrafish larvae, that injected cells can grow for different days and that this induces a marked neo-angiogenesis. Finally, we show that our model allows testing the effects of anti-HCC drugs such as Bortezomib. Compared to more complex HCC mouse models, our model is far less expensive, faster to set up and does not need immunosuppressant treatment. Finally, the model makes use of JHH6, an aggressive form of HCC cell line never tested before in Zebrafish.In conclusion, the possibility to test anti HCC/neo-angiogenesis drugs makes our JHH6 model useful to select therapeutic molecules for a highly vascularized tumor such as HCC.
Keywords: Hepatocellular carcinoma; Zebrafish larvae xenograft; JHH6 cell line; Neo-angiogenesis; Bortezomib;

Fabrication of antimicrobial silver-doped carbon structures by combinatorial pulsed laser deposition by Ion N. Mihailescu; Dorota Bociaga; Gabriel Socol; George E. Stan; Mariana-Carmen Chifiriuc; Coralia Bleotu; Marius A. Husanu; Gianina Popescu-Pelin; Liviu Duta; Catalin R. Luculescu; Irina Negut; Claudiu Hapenciuc; Cristina Besleaga; Irina Zgura; Florin Miculescu (592-606).
Display OmittedWe report on the selection by combinatorial pulsed laser deposition of Silver-doped Carbon structures with reliable physical-chemical characteristics and high efficiency against microbial biofilms. The investigation of the films was performed by scanning electron microscopy, high resolution atomic force microscopy, energy dispersive X-Ray Spectroscopy, X-ray diffraction, Raman spectroscopy, bonding strength “pull-out” tests, and surface energy measurements. In vitro biological assays were carried out using a large spectrum of bacterial and fungal strains, i.e., Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Enterococcus faecalis and Candida albicans. The biocompatibility of the films obtained was evaluated on MG63 mammalian cell cultures. The optimal combination with reasonable physical-chemical properties, efficient protection against microbial colonization and beneficial effects on human cells was found for Silver-doped Carbon films containing 2 to 7 at.% silver. These mixtures can be used to fabricate safe and efficient coatings of metallic implants, with the goal to decrease the risk of implant associated biofilm infections which are difficult to treat and often responsible for implant failure.
Keywords: C:Ag coatings; Microbial biofilm; Combinatorial pulsed laser deposition; Mammalian cells; Biocompatibility;

Specific targeting delivery to MUC1 overexpressing tumors by albumin-chitosan nanoparticles conjugated to DNA aptamer by Mehdi Esfandyari-Manesh; Ali Mohammadi; Fatemeh Atyabi; Seyedeh Maryam Nabavi; Seyedeh Masoumeh Ebrahimi; Elnaz Shahmoradi; Behrang Shiri Varnamkhasti; Mohammad Hossein Ghahremani; Rassoul Dinarvand (607-615).
Display OmittedChitosan-coated human serum albumin nanoparticles were functionalized by MUC1 aptamer to obtain a selective drug carrier toward cancers overexpressing MUC1. The negative charges of albumin nanoparticles were shifted to positive charges by surface modification with chitosan, and MUC1 was conjugated through an acrylate spacer. The cytotoxicity of targeted nanoparticles was significantly more than non-aptamer nanoparticles, and also the chitosan-coated nanoparticles had more cytotoxic effects than the negatively charged albumin nanoparticles. The IC50 of targeted nanoparticles was 28 and 26% of free paclitaxel in MCF7 and T47D cells at 48 h, respectively. Confocal laser scanning electron microscopy showed that aptamer conjugation and positive charge increase the cellular uptake. 66% of paclitaxel was released within 32 h, but 100% of drug was released at pH = 5.5 (similar cancer cells). The paclitaxel plasma amount was at a good level of 17.6% at 2 h for increasing the chance of cellular uptake.
Keywords: Nanoparticles; MUC1 aptamer; Specific targeting; Chitosan-coated albumin; Surface modification;

Development of aqueous ternary nanomatrix films: a novel ‘green’ strategy for the delivery of poorly soluble drugs Adeola Tawakalitu Kola-Mustapha; David Armitage; *Amos Olusegun Abioye Display OmittedAqueous polymeric films have potentially great values in drug development, particularly in controlled drug release and taste masking strategies. However the progressive polymer-particle coalescence that occurs randomly during film formation, curing and storage may render the film less permeable leading to erratic and unpredictable drug release profile. The focus of this study was to investigate the impacts of the in situ formation of polymer-drug nanoconjugate, at the interfacial nano-domains of two oppositely charged polymers, on the mechanism of film formation and to prepare aqueous ternary polymer-drug-polymer nanomatrix films as a novel green strategy for the delivery of ibuprofen, a model poorly soluble drug. Composite and Layer-by-Layer films were prepared by aqueous casting technique using the concept of combined polymer-drug self-assembly and polyelectrolyte complexation. The plain and drug-loaded nanomatrix films were characterized using SEM, AFM, FTIR, DSC and TGA. Ibuprofen formed spherical core-shell microstructures (4.55–9.73 μm) in gellan film. However in the presence of cationic dextran (Ddex), nanoconjugates (61.49 ± 5.97–447.52 ± 37.51 nm) were formed within the core of the film matrix. The composite films exhibited reduced tensile strength and lower elastic modulus with optimal conjugation efficiency of 98.14 ± 1.19%, which correlates with higher dissolution efficiency (99.76%) compared to 47.37% in layer-by-layer (LbL) films, dictated by Ddex concentration. Generally, the mechanism of drug release was by Fickian diffusion, however anomalous transport or polymer relaxation was also observed at higher concentration of Ddex. This study demonstrated the potential application of aqueous drug-loaded nanomatrix films as controlled drug delivery strategy for ibuprofen, a model poorly soluble drug.
Keywords: Aqueous nanomatrix film; Controlled polymeric self-assembly; Tuneable polymeric nanodomain; Ternary nanoconjugate; Green strategy for drug delivery; Mechanism of drug release;

Development of a Targeted anti-HER2 scFv Chimeric Peptide for Gene Delivery into HER2-Positive Breast Cancer Cells by Roya Cheraghi; Mahboobeh Nazari; Mohsen Alipour; Asia Majidi; Saman Hosseinkhani (632-643).
Display OmittedChimeric polymers are known as suitable carriers for gene delivery. Certain properties are critical for a polymer to be used as a gene delivery vector. A new polymer was designed for the targeted delivery of genes into breast cancer cell lines, based on MPG peptide. It is composed of different functional domains, including HIV gp41, nuclear localization sequence of SV40 T-antigen, two C-terminus repeats of histone H1, and the scFv of anti-HER2 antibody. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with an average size of 250 nm. Moreover, fusion of the scFv portion to the carrier brought about the specific recognition of HER2. Overall, the transfection efficiency of the vector demonstrated that it could deliver the desired gene into BT-474 HER2-positive breast cancer cells.

Chitosan nanoparticles and their Tween 80 modified counterparts disrupt the developmental profile of zebrafish embryos by Zhongyue Yuan; Ying Li; Yulan Hu; Jian You; Kazuma Higashisaka; Kazuya Nagano; Yasuo Tsutsumi; Jianqing Gao (644-656).
Display OmittedChitosan nanoparticles (CS-NPs) and their Tween 80 modified counterparts (TmCS-NPs) are among the most commonly used brain-targeted vehicles. However, their potential developmental toxicity is poorly understood. In this study, zebrafish embryos are introduced as an in vivo platform. Both NPs showed a dose-dependent increase in developmental toxicity (decreased hatching rate, increased mortality and incidences of malformation). Neurobehavioral changes included decreased spontaneous movement in TmCS-NP treated embryos and hyperactive effect in CS-NP treated larvae. Both NPs remarkably inhibited axonal development of primary and secondary motor neurons, and affected the muscle structure. Overall, this study demonstrated that CS-NPs and TmCS-NPs could affect embryonic development, disrupt neurobehavior of zebrafish larvae and affect muscle and neuron development, suggesting more attention on biodegradable chitosan nanoparticles.
Keywords: Chitosan nanoparticles; Tween 80; Developmental; Developmental neurotoxicity; Zebrafish;

Display OmittedOsteoarthritis is a propagated debilitating condition affecting patients' quality of life. Intra-articular injection approach was investigated as a localized treatment strategy providing: site-specific delivery, decreased side effects and, increased patient compliance. A 32 full factorial experimental design was employed to prepare the indomethacin-loaded self-assembling nanosystems (SANS). The surfactant (Poloxamer 407/Tetronic 90R4) ratio and the poly(lactic-co-glycolic acid) (PLGA) concentration significantly affected encapsulation efficiency and drug release (p < 0.05). The optimized formula was subjected to modification by addition of different proteoglycans, as a compensatory treatment, to improve its pharmacological properties. The modified SANS, containing glucosamine (150 mg), was selected for in-vivo studies as it had a sustained drug release profile and a small particle size (173.90 nm). The effect of the optimized SANS, with or without PLGA, was compared with the modified formula containing glucosamine and, with the drug suspension on the arthritic knee joints of rats. It was found that the formulation containing PLGA and glucosamine showed significantly higher reduction in both, knee diameter and TNF-α levels, compared to other groups. Furthermore, all SANS showed histological improvement in the cellularity of the synovial membranes and joints. Our results indicate that SANS containing PLGA and glucosamine is capable of treating arthritic joints.
Keywords: Self-assembly; Micelles; Indomethacin; Intra-articular; PLGA; Osteoarthritis;

Complexation of curcumin with 2-aminoethyl diphenyl borate and implications for spatiotemporal fluorescence monitoring by Yoon Young Kang; Heesun Jung; Gyeonghui Yu; Youhoon Chong; Hyejung Mok (669-676).
In this study, we successfully determined spatiotemporal distribution of curcumin in mice, without complicated extraction procedures, via simple and fast fluorescence detection of native curcumin and stabilized curcumin with 2-aminoethyl diphenyl borate (DPBA) as a stabilizer. Direct fluorescent monitoring could allow evaluation of in vivo distribution and fate of curcumin, which could be also applied to diverse natural polyphenols with fluorescent signals.Display OmittedIn this study, we successfully determined spatiotemporal distribution of curcumin in mice via simple and fast fluorescence detection of native curcumin and stabilized curcumin. We used 2-aminoethyl diphenyl borate (DPBA) as a stabilizer of curcumin, which binds to curcumin and enhances its aqueous stability. After intravenous injection, curcumin and DPBA–curcumin complexes showed similar fluorescence intensities in the brain, pancreas, lungs, and kidneys at 15 min. However, stabilized DPBA–curcumin complexes exhibited much stronger fluorescent signals at metabolically active sites such as liver tissues than native curcumin. After incubation for 1–3 h, native curcumin showed significantly rapid reduction of fluorescent signals, compared to DPBA–curcumin complexes, probably due to degradation and reduction. In addition, complicate extraction procedures inhibited precise fluorescent monitoring of unstable curcumin, which result in different biodistribution of curcumin before and after extraction. Direct fluorescent monitoring could allow evaluation of in vivo distribution and fate of curcumin, which could be also applied to diverse natural polyphenols with fluorescent signals.
Keywords: Curcumin; 2-aminoethyl diphenyl borate; Spatiotemporal imaging; Stability; Biodistribution;

Oral sustained-release suspension based on a lauryl sulfate salt/complex by Yuuki Kasashima; Shinya Uchida; Keiichi Yoshihara; Takehiko Yasuji; Kazuhiro Sako; Noriyuki Namiki (677-683).
Display OmittedThe objective of this study was to evaluate the feasibility of lauryl sulfate (LS) salt/complex as a novel carrier in oral sustained-release suspensions. Mirabegron, which has a pH-dependent solubility, was selected as the model drug. Sodium lauryl sulfate (SLS) was bound to mirabegron in a stoichiometric manner to form an LS salt/complex. LS salt/complex formulation significantly reduced the solubility of mirabegron and helped mirabegron achieve sustained-release over a wide range of pH conditions. Microparticles containing the LS salt/complex were prepared by spray drying with the aqueous dispersion of ethylcellulose (Aquacoat® ECD). The diameter of the microparticles was less than 200 μm, which will help avoid a gritty taste. In vitro results indicated the microparticles had slower dissolution profiles than the LS salt/complex. The dissolution rate could be controlled flexibly by changing the amount of Aquacoat® ECD. The microparticle suspension retained the desired sustained-release property and dissolution profile after being stored for 30 days at 40 °C. In addition, the suspension displayed sustained-release behavior in dogs without a pronounced Cmax peak, which will help prevent side effects. These results suggest that microparticles containing LS salt/complex may be useful as a novel sustained-release suspension for oral delivery.
Keywords: Sustained-release; Suspension; Lauryl sulfate; Salt; Complex; Spray drying;

Display OmittedThe combined strategy of drug-cyclodextrin (CD) complexation and complex loading into nanocarriers (deformable liposomes or nanostructured lipid carriers (NLC)), was exploited to develop effective topical formulations for oxaprozin transdermal administration. Oxaprozin was loaded as ternary complex with randomly-methylated-ßCD and arginine, selected as the best system in improving drug solubility. The colloidal dispersions, characterized for particle size, zeta-potential and entrapment efficiency, were investigated for drug permeation properties in comparison with a plain drug aqueous suspension, a ternary complex aqueous solution and a plain drug liposomal or NLC dispersion. Experiments with artificial membranes showed that the joined use of CD and both liposomes or NLC enabled a marked increase of the drug permeability (16 and 8 times, respectively) and was significantly more effective (P < 0.05) than the drug as ternary complex (3.2 times increase), and the corresponding liposomal or NLC dispersion of plain drug (5.6 and 4.3 times increase, respectively). Experiments with excised human skin confirmed the significantly (P < 0.05) better performance of deformable liposomes than NLC in promoting drug permeation; moreover, they evidenced a more marked permeability increase compared to the plain drug (24 and 12 fold, respectively), attributed to a possible enhancer effect of the nanocarriers components and/or of the randomly-methylated-ßCD.
Keywords: Deformable liposomes; Nanostructured lipid carriers (NLC); Oxaprozin; Randomly methylated ß-cyclodextrin; Arginine; In vitro skin permeation; Excised human skin;

Radiolabeled block copolymer micelles for image-guided drug delivery by Elisabete Ribeiro; Irina Alho; Fernanda Marques; Lurdes Gano; Isabel Correia; João D.G. Correia; Sandra Casimiro; Luís Costa; Isabel Santos; Célia Fernandes (692-701).
Display OmittedWe aimed at exploring block copolymer micelles (BCMs) for the simultaneous delivery of radiation/chemotherapy to cancer cells. To achieve that goal, we have prepared and characterized a novel type of docetaxel (DTX) loaded and non-loaded BCMs. The micelles were decorated with pyrazolyl-diamine chelating units to stabilize the matched pair 99mTc/Re for image-guided delivery of therapeutic drugs. The in vitro studies have shown that DTX release is pH-dependent increasing at lower pH values. Anti-proliferative studies in different cancer cell lines showed that DTX-loaded BCMs present relevant anti-proliferative activity. In comparison to free DTX, the loaded-micelles exhibited higher anti-proliferative activity for the same DTX concentration, which mean that a similar therapeutic outcome may be achieved with reduced side effects. The pyrazolyl-diamine-functionalized micelles were labeled with fac-[99mTc(CO)3(H2O)3]+ in high radiochemical yield and purity. The radiolabeled micelles are stable in phosphate buffer and in cell culture media. Cellular uptake studies in different cancer cell lines indicate a cell type and time-dependent uptake, in agreement with the anti-proliferative activity. Early biodistribution studies in healthy BALB/c mice has shown prolonged circulation lifetime in the bloodstream and relevant in vivo stability, important features when considering an effective DTX delivery system and image-guided delivery agent for cancer theranostics.
Keywords: PEG-b-PCL; Block copolymer micelle; Drug delivery system; Radiolabeled micelles; 99mTc;

Impact of sodium chloride on the expansion of a liquid-liquid miscibility gap in an API/water system. Case study of Brivaracetam by Nicolas Couvrat; Julien Mahieux; Baptiste Fours; Yohann Cartigny; Eric Schenkel; Luc Aerts; Luc Quéré; Gérard Coquerel (702-707).
The intravenous formulations of brivaracetam, a new antiepileptic drug, marketed under the name Briviact® are explored by means of a thermodynamic investigation and the impact of sodium chloride in isotonic and hypertonic solutions is established with respect to the formation of liquid-liquid miscibility gaps in the respective phase diagrams.Display OmittedBrivaracetam, or (2S)-2-[(4R)-2-oxo-4-propyl-pyrrolidin-1-yl] butanamide, is an active pharmaceutical ingredient designed for the treatment of epilepsy. During the development of the IV administration mode, a liquid-liquid miscibility gap has been observed with pure water, isotonic and hypertonic solutions (vehicle at 0.9% w/w and 5%w/w NaCl respectively). The study reveals that the NaCl concentration has a direct impact on the extent of the demixing domain; from a sub-micronic demixing in pure water towards a macroscopic miscibility gap in hypertonic aqueous solutions. The thorough exploration of these heterogeneous equilibria led to define experimental parameters for safe IV injections without risk of liquid – liquid miscibility gap at 37 °C.
Keywords: Brivaracetam; Liquid-liquid miscibility gap; Phase diagrams; IV administration; Monotectic invariant; Tyndall effect;

Induction of a balanced Th1/Th2 immune responses by co-delivery of PLGA/ovalbumin nanospheres and CpG ODNs/PEI-SWCNT nanoparticles as TLR9 agonist in BALB/c mice by Mahboubeh Ebrahimian; Maryam Hashemi; Mohsen Maleki; Khalil Abnous; Gholamreza Hashemitabar; Mohammad Ramezani; Alireza Haghparast (708-720).
Display OmittedTo develop effective and safe vaccines with reduced dose of antigen and adjuvant, intelligent delivery systems are required. Many delivery systems have been developed to enhance the biological activity of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) as both immunotherapeutic agents and vaccine adjuvants. In this study we designed a novel CpG ODN delivery system based on single-walled carbon nanotube (SWCNT) functionalized with polyethylenimine (PEI) and alkylcarboxylated PEI (AL-PEI). The physicochemical characteristics, cytotoxicity and cellular uptake studies of these carriers were performed.All carriers were conjugated with CpG ODN followed by co-delivery with ovalbumin (OVA) encapsulated into poly (lactic-co-glycolic acid) nanospheres (PLGA NSs) to enhance the induction of immune responses. The effect of these formulations on antibody (IgG1, IgG2a) and cytokine (IL-1β, IFN-γ, IL-4) production was evaluated in an in vivo experiment. The results showed that all nano-adjuvant formulations had a strong influence in up-regulation of IFN-γ and IL-4 in parallel with high IgG1-IgG2a isotype antibody titers in mice. In particular, SWCNT-AL-PEI nano-adjuvant formulation generated a balanced Th1 and Th2 immune response with more biased toward Th1 response without exhibiting any inflammatory and toxic effects. Therefore this nano-adjuvant formulation could be used as an efficient prophylactic immune responses agent.
Keywords: Single-walled carbon nanotubes; Polyethylenimine; CpG ODN; Adjuvant delivery; PLGA; Ovalbumin;

Where does hydrolysis of nandrolone decanoate occur in the human body after release from an oil depot? by R.W. Kalicharan; M.R. Bout; C. Oussoren; H. Vromans (721-728).
Display OmittedLong-term therapy of nandrolone (N) is recommended to increase mineral density and muscle strength. Using a parenteral sustained release drug formulation with nandrolone decanoate (ND), therapeutic N levels can be achieved and maintained. Until now, it is unknown if hydrolysis of ND into N occurs in tissue at the injection site or after systemic absorption. Therefore, hydrolysis studies were conducted to investigate the location and rate of ND hydrolysis after its release from the oil depot.ND hydrolysis was studied in porcine tissues, to mimic the human muscular and subcutaneous tissues. Additionally, the ND hydrolysis was studied in human whole blood, plasma and serum at a concentration range of 23.3–233.3 μM.ND hydrolysis only occurred in human whole blood. The hydrolysis did not start immediately, but after a lag time. The mean lag time for all studied concentrations was 34.9 ± 2.5 min. Because of a slow penetration into tissue, hydrolysis of ND is found to be very low in surrounding tissue. Therefore the local generation of the active compound is clinically irrelevant.It is argued that after injection of the oil depot, ND molecules will be transported via the lymphatic system towards lymph nodes. From here, it will enter the central circulation and within half an hour it will hydrolyse to the active N compound.

Display OmittedWe report the design of a nanoparticle depot (NPD) system for local delivery of gold nanoparticles (AuNP) that facilitates their controlled release and is implantable into tumors by permanent seed implantation (PSI) brachytherapy techniques. Various sizes (5, 15, 30, and 50 nm) of polyethylene glycol (PEG) coated AuNP and concentrations (6%, 8%, and 10% w/v) of calcium alginate used to form the NPD were studied. AuNP release rate, diffusion characteristics and spatial distribution were characterized in a tissue equivalent phantom model, and in a breast cancer tumor xenograft model and compared to a Fickian diffusion computational model, to identify the optimal NPD composition. In phantoms, 5 nm and 15 nm AuNP were released more rapidly than 30 nm or 50 nm AuNP but when implanted into tumor xenografts, AuNP exhibited slower release from NPD. Controlled prolonged release of AuNP was observed in tumor tissue over durations which were dependent on AuNP size. Maximum release and distribution in tumors were achieved using 5 nm AuNP incorporated into the NPD. These results demonstrate the potential for the NPD as an effective local delivery system for AuNP-based therapies.
Keywords: Nanoparticle depot; Gold nanoparticles; Calcium alginate; Tissue-equivalent phantom; Tumor xenograft; Diffusion;

Efficient approach to enhance drug solubility by particle engineering of bovine serum albumin by Mouhamad Khoder; Hamdy Abdelkader; Amr ElShaer; Ayman Karam; Mohammad Najlah; Raid G. Alany (740-748).
Display OmittedThe aim of this study was to investigate the use of bovine serum albumin (BSA) as a solubility enhancer for indometacin (IND) as a model drug. IND-BSA solid dispersions were prepared by both spray drying and freeze drying techniques using IND:BSA solution (20:1 Molar Ratio (MR)) and IND:BSA suspension (100:1 MR). The solid state of IND in solid dispersions was characterised by SEM, DSC and XRD. The aqueous solubility of IND in the presence of increased amounts of BSA was evaluated. Additionally, IND dissolution and release profiles were evaluated. IND in solid dispersions with BSA showed significantly higher solubility in water than that of the physical mixture of both. Enhancement factors of 24,000 and 100,000 were obtained for the solid dispersion formulated in 20:1 MR and 100:1 MR, respectively. Dissolution studies in-vitro indicated a significant increase in the dissolution rate of IND from solid dispersions compared to that of the free drug, with almost 95% of the drug dissolved in the first 5 min. Furthermore, an immediate release of IND from BSA solid dispersions was shown. The potential use of albumin as solubility enhancer for poorly soluble drugs, particularly, for immediate release volume-limited dosage forms is reported.
Keywords: Bovine serum albumin; Indometacin; Solid dispersion; Solubility enhancer; Dissolution rate;

Oil-in-water nanoemulsions are suitable for carrying hydrophobic compounds: Indomethacin as a model of anti-inflammatory drug by Björn Kwasigroch; Elvira Escribano; María del Carmen Morán; Josep Queralt; Maria Antònia Busquets; Joan Estelrich (749-756).
Display OmittedOil-in-water nanoemulsions are increasingly being used as delivery systems for encapsulating lipophilic components in functional food, personal care and pharmaceutical products. In the current study, we developed a multimodal platform to carry hydrophobic indomethacin or magnetic nanoparticles, or both. As a consequence, this platform has great potential for therapeutic or imaging purposes. By optimizing the system composition and homogenization conditions, a nanoemulsion with a mean droplet diameter of about 200 nm and a low polydispersity index (<0.2) was formed. The plain nanoemulsion was shown to be innocuous in cellular studies and did not present acute toxicity (observed in a rat model). More interesting was the finding that nanoemulsions loaded with indomethacin presented a significantly different anti-inflammatory than the free drug.
Keywords: Nanoemulsion; Indomethacin; Anti-inflammation; Cell viability; Acute toxicity;

Encapsulation of NSAIDs for inflammation management: Overview, progress, challenges and prospects by Waisudin Badri; Karim Miladi; Qand Agha Nazari; Hélène Greige-Gerges; Hatem Fessi; Abdelhamid Elaissari (757-773).
Display OmittedNon-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed drugs. Debilitating diseases such as rheumatoid arthritis and osteoarthritis are commonly managed by NSAIDs. However, NSAIDs pharmacological mechanism is often associated with the presence of gastrointestinal side effects. NSAIDs encapsulation is performed in order to overcome some of the drawbacks linked to their clinical use. To fulfill this purpose, various vectors like polymer-based nanoparticles, liposomes and solid lipid nanoparticles have been proposed. Such vehicles could have advantages but some limitations as well. This manuscript highlights current NSAIDs encapsulation approaches based on either preformed polymers or lipids. Moreover, properties of the prepared carriers and their applications are also discussed. Many factors are taken into account for selecting carrier type and encapsulation method. It was concluded that different vehicles and preparation methods have been employed for NSAIDs encapsulation. Mostly, vehicles sizes ranged within the nanoscale. Main advantages that have been confirmed by in vitro and in vivo studies include promoted stability, sustained release and bioavailability enhancement.
Keywords: Non-steroidal anti-inflammatory drugs; Encapsulation; Carriers; Liposomes; Particles; Solid lipid nanoparticles; In vivo;

Display OmittedThe objective of this study was to evaluate five commercial ready-to-use transdermal vehicles (Phytobase®, Lipovan®, Pentravan®, Pentravan® Plus and Pluronic Lecithin Organogel (PLO)), for the compounding of three antiemetic drugs (ondansetron, dexamethasone and aprepitant) and their administration in combination to treat chemotherapy-induced nausea and vomiting (CINV) at the hospital. Drugs were individually formulated in these vehicles and in mixture in Pentravan® Plus using different penetration enhancers. Quality control of the forms has demonstrated that formulation process was mastered and convenient for the hospital (time required: 20 min). Diffusion experiments through synthetic membranes and pig ear epidermis performed using Franz-type diffusion cells, have shown that the release and permeation process were greater for ondansetron than for dexamethasone and aprepitant, with a release step not limiting. As permeation of aprepitant was too low, it was discarded of the study. When ondansetron and dexamethasone were compounded in combination in Pentravan® Plus, the most efficient vehicle, a permeation decrease was observed. Finally, the use of tween 20 instead of EtOH as chemical enhancer has led to 2-fold factor increase in the flux of dexamethasone, resulting in fluxes convenient for transdermal administration of ondansetron to a child, but insufficient for an adult and for dexamethasone.
Keywords: Ondansetron; Dexamethasone; Aprepitant; Transdermal vehicle; Franz-type diffusion cell; Kinetic;

Display OmittedThe intrinsic osmolality of aqueous solutions of sodium salt antisense oligonucleotides (ASOs) has been studied to inform formulation practices, understand the molecular basis underlying the difference between theoretical and empirical results, and determine suitable measurement methods. It was found that regardless of nucleotide sequence, ASO concentration of ∼140 mg/mL has isotonic osmolality of ∼290 mOsm/kg water (SI unit: mmol osmotically-active particles/kg water), such that lower concentration formulations require excipients for tonicity adjustment. The range of osmolality values at a given active ingredient concentration can be ascribed to drug substance lot-to-lot purity differences impacting total oligonucleotide content (i.e., including oligonucleotide-related impurities). Empirical osmolality measurements were found to be ∼70% of theoretical values, which corresponds to an osmotic coefficient value of ∼0.7, thus inferring incomplete counterion dissociation. When comparing theoretical (ideal) osmolality of multiple sequences with various nucleotide compositions and chemistries at the same w/v concentration, the “average osmolar mass” (molar mass of the oligonucleotide, including the sodium counterions, divided by the ideal Van’t Hoff factor, i i d ) appears to be the strongest factor governing theoretical osmolality values. Other factors examined were the sequence length, backbone chemistry, 2′ sugar chemistry, and nucleotide composition. A head-to-head comparison between two osmolality techniques showed that vapor pressure osmometry is generally more suitable than freezing point osmometry for oligonucleotide solutions greater than ∼150 mg/mL due to viscosity effects, but the two techniques are comparable otherwise.
Keywords: Osmolality; Osmometry; Counterion dissociation; Antisense oligonucleotide;