International Journal of Pharmaceutics (v.513, #1-2)

Near infrared spectroscopy to monitor drug release in-situ during dissolution tests by Mafalda Cruz Sarraguça; Rita Matias; Raquel Figueiredo; Paulo Roberto S. Ribeiro; Ana Teixeira Martins; João Almeida Lopes (1-7).
Display OmittedDissolution tests can be used to demonstrate suitable in vivo drug release through in vivo/in vitro correlations. This work explores the possibility of using near infrared spectroscopy (NIRS) to monitor in-situ dissolution tests. It aims at expanding surrogate methods in quality control of drug products. Laboratory designed tablets of an immediate-release formulation containing folic acid and four excipients were used as case study. The dissolution tests were performed on a 1 L vessel filled with 500 ml of Milli-Q water with a rotating paddle apparatus (apparatus 2, Ph. Eur.) at 50 rpm and 37 ± 0.5 °C. Near infrared (NIR) spectra were acquired in-situ with a transflectance probe connected to a Fourier-transform near infrared spectrometer. NIR spectra were regressed against folic acid concentration by partial least squares (PLS) regression. Folic acid concentrations during dissolution tests were obtained by periodically sampling the dissolution vessel and resourcing to an UV method. The proposed real-time NIR method was tested on a validation run yielding a root mean squared error of 0.25 μg ml−1 (0.16 μg ml−1 for the calibration runs) and a R2 of 0.93 (0.95 for the calibration runs). The results suggest that NIRS is a suitable analytical technique for monitoring in-situ dissolution tests.
Keywords: Folic acid; Dissolution tests; near infrared spectroscopy; In-situ measurements;

Scheme 1. Schematic illustration of co-delivery of PTX and BA via nanoemulsions (PTX/BA NE) to overcome MDR in MCF-7/Tax cells. Nanoemulsions can efficiently deliver the therapeutic agents to the tumor cells through typical endocytosis pathway. PTX/BA NE significantly increase the cellular reactive oxygen species (ROS), reduce the cellular glutathione (GSH), and enhance caspase-3 activity in MCF-7/Tax cells, eventually resulting in apoptosis.Display OmittedMultidrug resistance (MDR) is a major obstacle for clinical application of paclitaxel (PTX). Recent studies have suggested that baicalein (BA) might be a potent MDR reversal agent with the ability of P-glycoprotein inhibition and oxidative stress augmentation. Herein, we co-encapsulated PTX and BA in nanoemulsions (PTX/BA NE) for overcoming MDR in breast cancer. Paclitaxel-cholesterol complex and baicalein-phospholipid complex were prepared to improve the liposolubility of PTX and BA. The cytotoxicity of the combination of PTX and BA with different weight ratios were evaluated and the combination with a weight ratio of 1/1 exhibited the strongest synergistic effect. In vitro cytotoxicity study indicated that PTX/BA NE had a better antitumor efficacy in MCF-7/Tax cells than other PTX formulations. Studies on cellular uptake demonstrated that the PTX/BA NE could effectively accumulate in cancer cells. Mechanism research showed that PTX/BA NE could significantly increase the cellular reactive oxygen species (ROS), decrease cellular glutathione (GSH), and enhance caspase-3 activity in MCF-7/Tax cells. More importantly, in vivo antitumor study demonstrated that PTX/BA NE exhibited a much higher antitumor efficacy than other PTX formulations. These findings suggest that co-delivery of PTX and BA in nanoemulsions might provide us a potential combined therapeutic strategy for overcoming MDR.
Keywords: Multidrug resistance; Paclitaxel; Baicalein; Nanoemulsions; Oxidative stress; P-glycoprotein;

Layered nanohydroxyapatite as a novel nanocarrier for controlled delivery of 5-fluorouracil by Honglin Luo; Dehui Ji; Chunzhi Li; Yong Zhu; Guangyao Xiong; Yizao Wan (17-25).
Display OmittedLayered materials intercalated by drug molecules have attracted much attention since they exhibit improved safety and effectiveness in drug delivery. In this work, layered nanohydroxyapatite (L-nHAp) was synthesized by template method and a model anticancer drug 5-fluorouracil (5FU) was loaded by intercalation technique. The morphology and structure of L-nHAp/5FU hybrids were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). XRD and FTIR revealed the successful drug intercalation into the gallery of L-nHAp. TGA evidenced enhanced thermal stability of the drug molecules in the gallery of L-nHAp. The in vitro release experiments demonstrated that the release of 5FU from L-nHAp/5FU was slower than that from rodlike nanohydroxyapatite (R-nHAp)/5FU. Furthermore, the release of 5FU from L-nHAp/5FU hybrid could be fitted by first-order, Higuchi, and Rigter-Pappas models. It is believed that L-nHAp may be a promising carrier for anticancer drugs.
Keywords: Layered hydroxyapatite; 5-Fluorouracil; Intercalation; Controlled release;

Arginine as a protein stabilizer and destabilizer in liquid formulations by Nam Ah Kim; Sharvron Hada; Ritu Thapa; Seong Hoon Jeong (26-37).
Display OmittedEven though arginine monohydrochloride (ArgHCl) is a useful additive for protein stabilization, its mechanism is not yet fully elucidated. Moreover, there is a concern that ArgHCl may be a protein denaturant since it decreases transition melting temperature (Tm ) of certain proteins. It contains a guanidinium group, a critical structure for denaturating activity of guanidine hydrochloride (GndHCl). Effects of ArgHCl, GndHCl, and creatinine on a model protein, etanercept, were examined by biophysical analyses including DLS, DSC, FT-IR, microviscometer, and SEC. Accelerated storage stability of the protein was examined in the absence and presence of H2O2 at different incubation temperatures with pH monitoring. ArgHCl reduced protein aggregation and retained monomer, but increased fragmentation at high temperature. Tm 1 and Tm 2 of the protein increased with ArgHCl, but slight decrease (>1 °C) in Tm 3 was observed. GndHCl and creatinine decreased all three Tm s. In the presence of heat and H2O2, the effect of ArgHCl was significantly decreased compared to GndHCl and creatinine. In addition, it accelerated the loss of monomer and increased fragmentation with decreasing pH. ArgHCl differed from GndHCl in the mode of physical interaction with the protein, due to its unique balance of three steric functional groups (guanidinium, carboxylic acid, and carbon aliphatic straight chain). In contrast, ArgHCl acted as a protein denaturant at high temperature since NOx generated from the amine group at the 3-carbon aliphatic straight chain and it is supported by GndHCl which did not change the pH nor accelerated the monomer loss after oxidation by H2O2 at high temperature.
Keywords: Arginine oxidation; Protein denaturant; Protein aggregation; DSC; Etanercept; NOx;

Investigating mixing and segregation using discrete element modelling (DEM) in the Freeman FT4 rheometer by Zilin Yan; Sam K. Wilkinson; Edmund H. Stitt; Michele Marigo (38-48).
Display OmittedMixing and segregation in a Freeman FT4 powder rheometer, using binary mixtures with varied particle size ratio and volume fraction, were studied using the Discrete Element Method (DEM). As the blade moves within the particle bed, size induced segregations can occur via a sifting mechanism. A larger particle size ratio and/or a larger volume fraction of large particles lead to a quicker segregation process. A higher particle velocity magnitude can promote the segregation process and the rate for the segregation index increases in the radial direction: from the centre towards the outer layer. In the current DEM simulations, it is shown that the change in flow energy associated with segregation and mixing depends on the choice of frictional input parameters. FT4 is proposed as a potential tool to compare and rank the segregation tendency for particulate materials with distinct differences in flow energy of each component. This is achieved by measuring the flow energy gradient after a number of test cycles for mixing powders with different flow properties. Employing the FT4 dynamic powder characterisation can be advantageous to establish blending performances in an industrial context.
Keywords: Discrete element method; Dem; Input parameters; Powder segregation; Powder mixing; Freeman FT4 rheometer;

Preparation and characterization of biodegradable polyhydroxybutyrate-co-hydroxyvalerate/polyethylene glycol-based microspheres by Alexandre Monnier; Charlotte Rombouts; Dania Kouider; Imad About; Hatem Fessi; Nida Sheibat-Othman (49-61).
Display OmittedThe in vivo effectiveness of biomolecules may be limited by their rapid diffusion in the body and short half-life time. Encapsulation of these biomolecules allows protecting them against degradation and ensuring a controlled release over time. In this work, the production of polyhydroxybutyrate-co-hydroxyvalerate/polyethylene glycol-based microspheres loaded with heparin by double emulsion-solvent evaporation is investigated. Significant improvements are achieved after blending PHB-HV microspheres with PEG. First of all, an important decrease of the initial burst effect is ensured. Moreover, lower degradation of the microspheres is observed after 30 days in the release medium. Finally, the release rate could be controlled using different PEG molecular weights and concentrations. A toxic effect of PHB-HV 30% PEG 1100 g mol−1 microspheres is observed whereas PHB-HV and PHB-HV 30% PEG 10,000 g mol−1 microspheres are not toxic. These microspheres seem to be most suited for further tissue engineering applications. The effectiveness of direct PEG blending to PHB-HV is proved, limiting the use of chemical reagents for PHB-HV/PEG copolymer synthesis and steps for chemical reagents removal from the copolymer.
Keywords: Polyhydroxybutyrate-co-hydroxyvalerate; Polyethylene glycol; Microencapsulation; Toxicity; Double-emulsion; Drug release;

Display OmittedIn this research, a novel method is reported for the surface grafting of n-vinylcaprolactam as a thermosensitive agent and allylimidazole with affinity toward celecoxib onto magnetic nano-particles. The grafted nano-particles were characterized by Fourier transform infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The surface morphology was studied using Scanning Electron Microscopy. The resulting grafted nano-particles were used for the determination of trace celecoxib in biological human fluids and pharmaceutical samples. The profile of celecoxib uptake by the modified magnetic nano-particles indicated good accessibility of the active sites in the grafted copolymer. It was found that the adsorption behavior could be fitted by the Langmuir adsorption isotherm model. Solid phase extraction for biological fluids such as urine and serum were investigated. In this study, urine extraction recovery of more than 95% was obtained.
Keywords: Celecoxib; Magnetic nano-particles; Drug delivery; Human biological fluid;

Tissue distribution and enhanced in vivo anti-hyperlipidemic-antioxidant effects of perillaldehyde-loaded liposomal nanoformulation against Poloxamer 407-induced hyperlipidemia by Emmanuel Omari-Siaw; Qilong Wang; Congyong Sun; Zengquan Gu; Yuan Zhu; Xia Cao; Caleb Kesse Firempong; Rita Agyare; Ximing Xu; Jiangnan Yu (68-77).
Display OmittedAn optimized perillaldehyde-loaded liposomal nanoformulation (PAH-LNF) was successfully applied to improve the pharmacological effect of perillaldehyde (PAH) in poloxamer 407-induced hyperlipidemia. Oral administration of PAH-LNF (240 mg/kg per body weight) in rats significantly enhanced solubility and relative bioavailability (270.7%) compared to the free PAH with about 2.7-, 1.5-, 1.3-, 1.3- and 1.5-fold increase in AUC, T1/2, MRT, Cmax and Tmax, respectively. Tissue distribution study also revealed the accumulation of PAH in the liver, lungs, spleen, kidney, brain and heart in order of decreasing affinity. Moreover, a significant decrease in serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) with simultaneous increase in high-density lipoprotein cholesterol (HDL-C) level was observed in the chemically-induced hyperlipidemic mice which further confirmed PAH’s anti-hyperlipidemic properties. Additionally, PAH-LNF also significantly increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) with a concurrent decrease in malondialdehyde (MDA) to affirm the antioxidant and hepatoprotective effects of PAH. Thus, liposomal nanoformulation promises to be a useful drug delivery system for the development of PAH.
Keywords: Perillaldehyde; Anti-hyperlipidemic activity; Antioxidant; Liposomal nanoformulations; Poloxamer 407;

Display OmittedThe aim was to investigate the ability of polysorbate 20 to alter oral digoxin absorption in vitro and drug exposure in vivo via modulation of transporter mediated efflux. Transport studies were performed in MDCKII-MDR1 and Caco-2 cells using 3H-digoxin. Pharmacokinetic studies were performed in wild type and mdr1a deficient Sprague Dawley rats. 3H-digoxin was quantified using liquid scintillation counting. The results showed an increased absorptive transport and a reduced secretory transport in MDCKII-MDR and Caco-2 cells as a function of polysorbate 20 concentrations. The secretory transport (B–A) of digoxin was reduced by 50% at lower polysorbate 20 concentrations than required to increase the absorptive transport (A-B). In vivo, the oral bioavailability of digoxin in wild type animal was increased by 10–25% (w/v) polysorbate 20. In mdr1a deficient Sprague Dawley rats 25% (w/v) polysorbate 20 did not alter the absorption of digoxin after oral administration, but digoxin exposure was significantly different between wild type and mdr1a deficient rats. In conclusion, polysorbate 20 increased absorptive transport across Caco-2 cell monolayers and in vivo in rats in a concentration dependent manner, most likely via inhibition of P-gp rather than through solubilization of digoxin.
Keywords: Digoxin; P-gp; Polysorbate; mdr1a(-/-) Sprague Dawley rats; MDCKII-MDR1 cells; Caco-2 cells;

Display OmittedConvective flow is one of the main mechanisms of mass transfer employed in drug delivery (e.g. osmotic pumps) and working in material transport in the body (e.g. blood circulation). Although convective flow has been investigated extensively, not much data is available on convective behavior of nanoparticles, the subject of the present investigation.Here, liposomes with different sizes, charges, bilayer flexibilities and medium viscosities were encountered convective flow and changes in their properties were monitored over 48 h. For large particles (>350 nm), neutral liposomes (NL) showed significant phase separation and decreased lipid content over time, while positively or negatively charged liposomes remained homogenous. Reduction of size in NLs to about 100 nm resolved phase separation problem, but their lipid content still showed reduction; no such a problem was observed in charged small liposomes. When bilayer flexibility of large NLs was increased, neither phase separation nor decreased lipid content was observed. Increasing medium viscosity for large NLs from 3.4 cP to 45.2 cP again solved the problem and a uniform liposomal delivery was observed. These results indicate that size, charge, bilayer flexibility and viscosity affect convective liposomal delivery and that uniform delivery is possible even in large liposomes by optimizing such factors.
Keywords: Liposomes; Nanoparticles; Convection; Convective flow; Drug delivery; Mass transport;

An experimental investigation of temperature rise during compaction of pharmaceutical powders by Alexander Krok; Andreja Mirtic; Gavin K. Reynolds; Serena Schiano; Ron Roberts; Chuan-Yu Wu (97-108).
Display OmittedDuring pharmaceutical powder compaction, temperature rise in the compressed powder can affect physiochemical properties of the powder, such as thermal degradation and change in crystallinity. Thus, it is of practical importance to understand the effect of process conditions and material properties on the thermal response of pharmaceutical formulations during compaction. The aim of this study was to examine the temperature rise of pharmaceutical powders during tableting, in particular, to explore how the temperature rise depends on material properties, compression speed and tablet shape. Three grades of microcrystalline cellulose (MCC) were considered: MCC Avicel pH 101, MCC Avicel pH 102 and MCC DG. These powders were compressed using a compaction simulator at various compaction speeds (10–500 mm/s). Flat faced, shallow convex and normal convex tablets were produced and temperature distributions on the surface of theses tablets upon ejection were examined using an infrared thermoviewer. It was found that an increase in the compaction speed led to an increase in the average surface temperature. A higher surface temperature was induced when the powder was compressed into a tablet with larger surface curvature. This was primarily due to the increasing degree of powder deformation (i.e. the volume reduction) and the effect of interparticule/wall friction.
Keywords: Temperature rise; Powder compaction; Tabletting; Microcrystalline cellulose;

An in vitro and in silico study of the impact of engineered surface modifications on drug detachment from model carriers by Shengqian Wu; Sarah Zellnitz; Annalisa Mercuri; Sharareh Salar-Behzadi; Massimo Bresciani; Eleonore Fröhlich (109-117).
Display Omitted In silico modeling was used to predict the impact of carrier surface modifications on the in vivo plasma concentration of an active pharmaceutical ingredient (API) and as a tool to support formulation development. In vitro fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) of salbutamol sulphate delivered from Cyclocaps®, detached from unmodified and surface engineered glass beads were measured using a Next Generation Impactor (NGI). Surface roughness was chosen to classify surface modification/engineering and it was evaluated via scanning electron microscopy (SEM) and image analysis. An in silico pharmacokinetic (PK) model was built and the quality confirmed with available literature data. Plasma profiles were generated combining the PK model with in silico deposition models for salbutamol sulphate released from Cyclocaps®, unmodified and surface engineered glass beads.The increased roughness of the surface of engineered beads resulted in a FPF 1.36 times higher than that of untreated beads. Cmax from the in silico plasma profile of salbutamol released from the surface engineered beads was 1.20 fold higher than that from untreated beads.Increasing the surface roughness was found to augment the amount of drug loading and detaching from the carrier both in vitro and in silico.
Keywords: Salbutamol sulphate; Carrier surface engeenering; NGI; In silico pharmacokinetic modeling; Lung deposition;

Preparation and evaluation of teniposide-loaded polymeric micelles for breast cancer therapy by Bingyang Chu; Shuai Shi; Xingyi Li; Lufeng Hu; Lu Shi; Haina Zhang; Qiaoqiao Xu; Lei Ye; Guanyang Lin; Nansheng Zhang; Xiuhua Zhang (118-129).
Display OmittedSelf-assembled polymeric micelles have been widely applied in anticancer drug delivery systems. Teniposide is a broad spectrum and effective anticancer drug, but its poor water-solubility and adverse effects of commercial formulation (VM-26) restrict its clinical application. In this work, teniposide-loaded polymeric micelles were prepared based on monomethoxy-poly(ethylene glycol)-poly(ε-caprolactone-co-d,l- lactide) (MPEG-PCLA) copolymers through a thin-film hydration method to improve the hydrophilic and reduce the systemic toxicity. The prepared teniposide micelles were without any surfactants or additives and monodisperse with a mean particle size of 29.6 ± 0.3 nm. The drug loading and encapsulation efficiency were 18.53 ± 0.41% and 92.63 ± 2.05%, respectively. The encapsulation of teniposide in MPEG-PCLA micelles showed a slow and sustained release behavior of teniposide in vitro and improved the terminal half-life (t1/2), the area under the plasma concentration-time curve (AUC) and retention time of teniposide in vivo compared with VM-26. In addition, teniposide micelles also enhanced the cellular uptake by MCF-7 breast cancer cells in vitro and increased the distribution in tumors in vivo. Teniposide micelles showed an excellent safety with a maximum tolerated dose (MTD) of approximately 50 mg teniposide/kg body weight, which was 2.5-fold higher than that of VM-26 (about 20 mg teniposide/kg body weight). Furthermore, the intravenous application of teniposide micelles effectively suppressed the growth of subcutaneous MCF-7 tumor in vivo and exhibited a stronger anticancer effect than that of VM-26. These results suggested that we have successfully prepared teniposide-loaded MPEG-PCLA micelles with improved safety, hydrophilic and therapeutic efficiency, which are efficient for teniposide delivery. The prepared teniposide micelles may be promising in breast cancer therapy.
Keywords: Teniposide; Polymeric micelles; MPEG-PCLA; Breast cancer therapy;

Protein encapsulation and release from PEO-b-polyphosphoester templated calcium carbonate particles by Zeynep Ergul Yilmaz; Thomas Cordonnier; Antoine Debuigne; Brice Calvignac; Christine Jerome; Frank Boury (130-137).
Display OmittedCalcium carbonate particles are promising candidates as proteins carriers for their controlled delivery in the body. The present paper aims at investigating the protein encapsulation by in situ precipitation of calcium carbonate particles prepared by a process based on supercritical CO2 and using a new type of degradable well-defined double hydrophilic block copolymers composed of poly(ethylene oxide) and polyphosphoester blocks acting as templating agent for the calcium carbonate. For this study, lysozyme was chosen as a model for therapeutic protein for its availability and ease of detection. It was found that by this green process, loading into the CaCO3 microparticles with a diameter about 2 μm can be obtained as determined by scanning electron microscopy. A protein loading up to 6.5% active lysozyme was measured by a specific bioassay (Micrococcus lysodeikticus). By encapsulating fluorescent-labelled lysozyme (lysozyme-FITC), the confocal microscopy images confirmed its encapsulation and suggested a core–shell distribution of lysozyme into CaCO3, leading to a release profile reaching a steady state at 59% of release after 90 min.
Keywords: Polyphosphoester; CaCO3 particles; Protein loading; Release test; Supercritical CO2;

Discovery of PSMA-specific peptide ligands for targeted drug delivery by Wei Jin; Bin Qin; Zhijin Chen; Hao Liu; Ashutosh Barve; Kun Cheng (138-147).
Display OmittedProstate-specific membrane antigen (PSMA) has been widely used as a biomarker and targeting receptor for prostate cancer therapy because of its overexpression in most prostate cancer cells. In this study, a novel combinatorial phage biopanning procedure was developed to discover PSMA-specific peptides that can be potentially used as ligands for targeted drug delivery to prostate cancer cells. Five rounds of biopanning against recombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice were conducted. Various affinity assays were conducted to identify high-affinity peptides for PSMA ECD and PSMA-positive prostate cancer cells. Among them, the GTI peptide shows the highest affinity as well as specificity to PSMA in prostate cancer cells. The apparent Kd values of the GTI peptide to PSMA-positive LNCaP and C4-2 cells are 8.22 μM and 8.91 μM, respectively. The GTI peptide can specifically deliver the proapoptotic peptide D(KLAKLAK)2 to LNCaP cells to induce cell death. In the biodistribution study, the GTI peptide shows the highest uptake in C4-2 xenografts, while its uptake in other major organs, such as the liver and spleen, are either low or negligible. Compared to its scrambled control (random permutation of the GTI peptide), the GTI peptide exhibits higher and more specific uptake in C4-2 xenografts. All the results suggest that the GTI peptide is a potentially promising ligand for PSMA-targeted drug delivery for prostate cancer therapy.
Keywords: Prostate cancer; PSMA; Peptide ligand; Phage display; Combinatorial biopanning;

Hydrophilic-hydrophobic polymer blend for modulation of crystalline changes and molecular interactions in solid dispersion by Hai Van Ngo; Phuc Kien Nguyen; Toi Van Vo; Wei Duan; Van-Thanh Tran; Phuong Ha-Lien Tran; Thao Truong-Dinh Tran (148-152).
Display OmittedThis research study aimed to develop a new strategy for using a polymer blend in solid dispersion (SD) for dissolution enhancement of poorly water-soluble drugs. SDs with different blends of hydrophilic-hydrophobic polymers (zein/hydroxypropyl methylcellulose – zein/HPMC) were prepared using spray drying to modulate the drug crystal and polymer-drug interactions in SDs. Physicochemical characterizations, including power X-ray diffraction and Fourier transform infrared spectroscopy, were performed to elucidate the roles of the blends in SDs. Although hydrophobic polymers played a key role in changing the model drug from a crystal to an amorphous state, the dissolution rate was limited due to the wetting property. Fortunately, the hydrophilic-hydrophobic blend not only reduced the drug crystallinity but also resulted in a hydrogen bonding interaction between the drugs and the polymer for a dissolution rate improvement. This work may contribute to a new generation of solid dispersion using a blend of hydrophilic-hydrophobic polymers for an effective dissolution enhancement of poorly water-soluble drugs.
Keywords: Hydrophilic-hydrophobic polymer; Drug crystal; Molecular interaction; Solid dispersion;

Effect of quercetin and resveratrol co-incorporated in liposomes against inflammatory/oxidative response associated with skin cancer by Carla Caddeo; Amparo Nacher; Antonio Vassallo; Maria Francesca Armentano; Ramon Pons; Xavier Fernàndez-Busquets; Claudia Carbone; Donatella Valenti; Anna Maria Fadda; Maria Manconi (153-163).
Display OmittedThe present investigation reports the development of liposomes for the co-delivery of naturally occurring polyphenols, namely quercetin and resveratrol. Small, spherical, uni/bilamellar vesicles were produced, as demonstrated by light scattering, cryo-TEM, SAXS. The incorporation of quercetin and resveratrol in liposomes did not affect their intrinsic antioxidant activity, as DPPH radical was almost completely inhibited. The cellular uptake of the polyphenols was higher when they were formulated in liposomes, and especially when co-loaded rather than as single agents, which resulted in a superior ability to scavenge ROS in fibroblasts. The in vivo efficacy of the polyphenols in liposomes was assessed in a mouse model of skin lesion. The topical administration of liposomes led to a remarkable amelioration of the tissue damage, with a significant reduction of oedema and leukocyte infiltration. Therefore, the proposed approach based on polyphenol vesicular formulation may be of value in the treatment of inflammation/oxidative stress associated with pre-cancerous/cancerous skin lesions.
Keywords: Quercetin; Resveratrol; Liposome; Antioxidant; Fibroblast; Skin lesion;

Rapid determination of drug solubilization versus supersaturation in natural and digested lipids by Nicolas Gautschi; Christel A.S. Bergström; Martin Kuentz (164-174).
Display OmittedLipid-based formulations (LBFs) represent one of the successful formulation approaches that enable oral delivery of poorly water-soluble drugs. This work presents a simple equilibrium approach based on solubility in lipids and their corresponding digestion media to estimate a maximum drug supersaturation ratio (SRmax ). This value of drug concentration normalized by the solubility in the aqueous digestion phase indicates the propensity for drug precipitation. A set of 16 structurally diverse drugs was first measured for their solubility in tricaprin and tricaprylin and results were compared to an empirical model based on molecular predictors. In the next step, digestion media were either prepared by in vitro lipolysis or by assembling a composition to mimic the endpoint of digestion. It was found that drug solubility in the pure lipids mainly was related to the melting point in that increased values resulted in reduced solubility. The solubility values measured in the lipolysis media correlated well with those obtained from assembled digestion media. Interestingly, the solubilization upon digestion was typically higher when using tricaprin than tricaprylin in spite of that the latter oil (as pure excipient) generally was a more potent solvent. This work suggests that a simplified digestion screen can be used to facilitate evaluation of formulations during early development. Estimation of SRmax provides an early risk assessment of drug precipitation for LBFs. The method is easily scaled down to the microtiter plate format and can be used for selecting candidate formulations that merit further evaluation in more complex and dynamic in vitro tests.
Keywords: Poorly soluble drug; Solubilization; Lipid-based formulation; Digestion; Drug precipitation; Formulation screening;

Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme by Shuying Ji; Peter Waaben Thulstrup; Huiling Mu; Steen Honoré Hansen; Marco van de Weert; Jukka Rantanen; Mingshi Yang (175-182).
Display OmittedIn the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant impact of ethanol; however, the lysozyme enzymatic activity was ca. 25% lower compared to the starting material. In conclusion, the addition of ethanol as a co-solvent in the spray drying feed for lysozyme did not compromise the conformation of the protein after drying, while it improved the inhaled aerosol performance.
Keywords: Spray drying; Lysozyme; Ethanol; Aerosol performance; Protein stability;

PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model by Pan Li; Catpagavalli Asokanathan; Fang Liu; Kyi Kyi Khaing; Dorota Kmiec; Xiaoqing Wei; Bing Song; Dorothy Xing; Deling Kong (183-190).
The pertussis toxoid (PTd) loaded nano/micro poly(lactic-glycolic acid) (PLGA) particles elicited a more balanced Th1/Th2 immune responseDisplay OmittedPoly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response.
Keywords: Pertussis; PLGA nano/micro particles; Phagocytosis; Adjuvant;

Design, fabrication and biomedical applications of zein-based nano/micro-carrier systems by Yong Zhang; Lili Cui; Feng Li; Nianqiu Shi; Chunlei Li; Xianghui Yu; Yan Chen; Wei Kong (191-210).
Classes, size range of zein-based particles/capsules fabricated using various methods and their biomedical applications.Display OmittedNano/micro-carrier systems have shown promising application in the biomedical field as various delivery carriers. The composite material and fabrication method determine their microstructures, properties and thus their potential applications. Since approved as tablet coating material by the U.S. Food and Drug Administration (US-FDA), zein has been widely investigated as one of protein-based materials in the past few decades. Zein is renewable, biodegradable and relatively inexpensive in comparison with animal proteins (e.g., gelatin and albumin). This paper reviews the current landscape of zein-based nano/micro-carrier systems, with particular emphasis on nano/microparticles, nano/microcapsules and their design, fabrication, assembly mechanisms and biomedical applications especially for controlled drug delivery. The benefits, challenges and related solutions of zein-based colloidal carrier systems are also discussed. In addition, investigations on the molecular structure, biocompatibility and immunogenicity of zein are summarized and discussed.
Keywords: Biopolymer; Nano/microparticle; Nanocapsule; Microcapsule; Delivery carrier;

In vivo evaluation of lipid-based formulations for oral delivery of apomorphine and its diester prodrugs by Nrupa Borkar; René Holm; Mingshi Yang; Anette Müllertz; Huiling Mu (211-217).
Display OmittedIn the present study, the differences in oral absorption of apomorphine and its diester prodrugs and the effect of lipid-based formulations on the absorption of apomorphine or its prodrugs were investigated. Apomorphine, dilauroyl apomorphine (DLA) and dipalmitoyl apomorphine (DPA) were orally administered (0.24 mmol/kg) to rats as: DLA-o/w emulsion, DPA-o/w emulsion, apomorphine-o/w emulsion, apomorphine aqueous suspension, DLA-Maisine, DLA-soybean oil, DLA-self-emulsifying drug delivery systems (SEDDS), and DLA-w/o emulsion. The o/w and w/o emulsion consisted of Maisine 35-1 emulsified with water in 1:3 and 4:1 ratio, respectively. Tmax of diesters was significantly increased (p ≤ 0.05) compared to apomorphine in o/w emulsion, suggesting that esterification yielded prolonged drug absorption. Cmax, AUC and the relative bioavailability of apomorphine after DLA-SEDDS administration was higher (p ≤ 0.05) than after DLA-w/o administration, indicating that triglycerides and surfactants improved the oral absorption of DLA. Similarly, Cmax and AUC after dosing apomorphine-o/w were significantly higher (p ≤ 0.05) than that of aqueous suspension. This suggested that lipids and lipolysis products possibly aided apomorphine micellar solubilization in intestinal fluids. A combination of prodrug strategy and lipid-based formulations facilitated a higher and prolonged absorption of apomorphine from its diester prodrugs.
Keywords: Apomorphine; Lipophilic diester prodrug; SEDDS; Lipid-based formulation; In vivo absorption;

Novel nanostructured enoxaparin sodium-PLGA hybrid carriers overcome tumor multidrug resistance of doxorubicin hydrochloride by Jia Wang; Lei Wu; Longfa Kou; Meng Xu; Jin Sun; Yongjun Wang; Qiang Fu; Peng Zhang; Zhonggui He (218-226).
Graphic abstract of the proposed mechanism by which DOX-EPNs overcome MDR.Display OmittedNovel enoxaparin sodium-PLGA hybrid nanocarries (EPNs) were successfully designed for sustained delivery of hydrophilic cationic doxorubicin hydrochloride (DOX) and to overcome multidrug resistance (MDR). By incorporation of the negative polymer of enoxaparin sodium (ES), DOX was highly encapsulated into EPNs with an encapsulation efficiency of 92.49%, and ES effectively inhibited the proliferation of HUVEC cell lines. The in vivo pharmacokinetics study after intravenous injection indicated that DOX-loaded EPNs (DOX-EPNs) exhibited a higher area under the curve (AUC) and a longer half-life (t1/2) in comparison with DOX solution (DOX-Sol). The biodistribution study demonstrated that DOX-EPNs increased the DOX level in plasma and decreased the accumulation of DOX in liver and spleen. Compared with DOX-Sol, DOX-EPNs increased the cytotoxicity in P-gp over-expressing MCF-7/Adr cells, attributed to the higher intracellular efficiency of DOX produced by the EPNs. DOX-EPNs entered into resistant tumor cells by multiple endocytosis pathways, which resulted in overcoming the multidrug resistance of MCF-7/Adr cells by escaping the efflux induced by P-gp transporters.
Keywords: Doxorubicin hydrochloride; Enoxaparin sodium-PLGA hybrid nanocarriers; Pharmacokinetics; Anti-proliferation; Cellular uptake; Multidrug resistance;

Drug-eluting biodegradable ureteral stent: New approach for urothelial tumors of upper urinary tract cancer by Alexandre A. Barros; Shane Browne; Carlos Oliveira; Estevão Lima; Ana Rita C. Duarte; Kevin E. Healy; Rui L. Reis (227-237).
Display OmittedUpper urinary tract urothelial carcinoma (UTUC) accounts for 5–10% of urothelial carcinomas and is a disease that has not been widely studied as carcinoma of the bladder. To avoid the problems of conventional therapies, such as the need for frequent drug instillation due to poor drug retention, we developed a biodegradable ureteral stent (BUS) impregnated by supercritical fluid CO2 (scCO2) with the most commonly used anti-cancer drugs, namely paclitaxel, epirubicin, doxorubicin, and gemcitabine. The release kinetics of anti-cancer therapeutics from drug-eluting stents was measured in artificial urine solution (AUS). The in vitro release showed a faster release in the first 72 h for the four anti-cancer drugs, after this time a plateau was achieved and finally the stent degraded after 9 days. Regarding the amount of impregnated drugs by scCO2, gemcitabine showed the highest amount of loading (19.57 μg drug/mg polymer: 2% loaded), while the lowest amount was obtained for paclitaxel (0.067 μg drug/mg polymer: 0.01% loaded). A cancer cell line (T24) was exposed to graded concentrations (0.01–2000 ng/ml) of each drugs for 4 and 72 h to determine the sensitivities of the cells to each drug (IC50). The direct and indirect contact study of the anti-cancer biodegradable ureteral stents with the T24 and HUVEC cell lines confirmed the anti-tumoral effect of the BUS impregnated with the four anti-cancer drugs tested, reducing around 75% of the viability of the T24 cell line after 72 h and demonstrating minimal cytotoxic effect on HUVECs.
Keywords: Ureteral stents; Upper tract urothelial carcinoma; Anti-cancer drugs; T24 cell line; Biodegradable polymers; Supercritical fluid technology;

Intravitreal injection of rapamycin-loaded polymeric micelles for inhibition of ocular inflammation in rat model by Wei Wu; Zhifen He; Zhaoliang Zhang; Xinxin Yu; Zongming Song; Xingyi Li (238-246).
Display OmittedThe therapeutic efficacy of rapamycin conjugated monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles (rapamycin micelles) was evaluated in a rat experimental autoimmune uveitis (EAU) model. Rapamycin micelles exhibited spherical morphology and had a mean particle size of 40 nm and a zeta-potential of −0.89 mv. The water solubility of rapamycin improved by more than 1000-fold in a micellar formulation. Intravitreal injection of MPEG-PCL micelles did not result in vitreous hemorrhage or retinal detachment. Fluorescence microscopy demonstrated that labeled micelles localized to the retinal pigment epithelium for at least 14 days following injection and the drug concentration of rapamycin micelles in the retinal tissue was significantly higher than unconjugated rapamycin over this period. At the optimal concentration of rapamycin micelles (9 μg/eye), clinical signs of EAU were abolished via the downregulation of the Th1 and Th17 response. There were no significant difference in T cell proliferation and delayed-type hypersensitivity between the treatment and control groups, suggesting that the therapeutic effect of rapamycin manifested locally in the eye and not systemically. These results indicate that intravitreal injection of rapamycin micelles is a promising therapy for controlling sterile intraocular inflammation.
Keywords: Rapamycin micelles; Ocular inflammation; In vivo; Intravitreal injection;

Comparison of different in vitro release methods used to investigate nanocarriers intended for dermal application by Benjamin Balzus; Miriam Colombo; Fitsum Feleke Sahle; Gaith Zoubari; Sven Staufenbiel; Roland Bodmeier (247-254).
Display Omitted In vitro drug release measurement is one of the most important methods used to assess the quality of a nanocarrier and estimate it́s in vivo performance. Different in vitro drug release methods have been used to investigate the drug release from nanocarriers, however, little information is available with regard to a comparison of these methods (e.g. discriminative power, reproducibility). Thus, drug release from four nanocarriers (nanocrystals, lipid nanoparticles, Eudragit® RS and ethyl cellulose nanoparticles) was investigated under sink and non-sink conditions with three drug release methods: an in situ method using Sirius® inForm and two in vitro methods using dialysis bags and Franz diffusion cells. Dexamethasone was used as the model drug. The in situ measurement was a simple and fast method but not adequately discriminating because of a too rapid drug dissolution/release. Franz diffusion cells and dialysis bags were in most cases discriminative for the different nanocarriers with the drug dissolution/release being in the order of nanocrystals > Eudragit® RS nanoparticles > lipid nanoparticles > ethyl cellulose nanoparticles. Drug release experiments with Franz diffusion cells had the highest reproducibility. The Franz diffusion cells could also be easily used with semisolid dosage forms.
Keywords: Dialysis bag; Discriminative dissolution testing; Franz diffusion cell; in situ drug release; in vitro drug release; Skin nanocarriers;

Development of solidified self-microemulsifying delivery systems with enhanced stability of sirolimus and extended release by Chun Tao; Juming Chen; Aiwen Huang; Jing Zhang; Bing Lin; Zhihong Liu; Minxin Zhang; Xu Chen; Lingjun Zeng; Lingna Zhang; Hongtao Song (255-261).
Display OmittedThe application of sirolimus (SRL) as immunosuppressive agent is hampered by its poor water solubility and narrow therapeutic range. The self-microemulsifying drug delivery system (SMEDDS) succeeded in improving the solubility of SRL in our previous work. In this study, the formulation of the SMEDDS was further optimized by investigating the influence of the excipients including the media, antioxidant and organic acid. It was demonstrated that addition of 0.20% of citric acid in SMEDDS most efficiently promoted the stability of SRL under high temperature (40 ± 2 °C), high humidity (relative humidity 90 ± 5%) or strong light irradiation (4500 ± 500 lx). SMEDDS absorbed by microcrystalline cellulose (MCC) was mixed with hydroxypropyl methylcellulose (HPMC) to prepare tablets. The optimal formulation composed of 15% of HPMC 100 LV with hardness of 120 N, which had a sustained release of 12 h. Results of X-ray powder diffraction and differential scanning calorimetry demonstrated that SRL in the tablets was in amorphous or molecularly dispersed state. The SMEDDS-tablets presented as promising substrates for water insoluble drugs with enhanced stability and extended release.
Keywords: Sirolimus; Self-microemulsifying drug delivery system; Tablet; Stability; Extended release;

Display OmittedIn this work, the sizing of microemulsion droplets of a lipid-based pharmaceutical excipient (Labrasol® ALF) is performed by Taylor dispersion analysis (TDA) using fluorescent detection. An hydrophobic fluorescent marker is used to tag the microemulsion droplet and to increase the sensitivity of detection (compared to UV detection). Combined with the frontal TDA mode, fluorescent detection was mandatory for an accurate sizing of microemulsions containing large coacervates. Microemulsion sizing of Labrasol was performed at various concentrations from 1 to 70 g.L−1 and at two different temperature (25 °C and 37 °C). Results obtained by TDA are compared to those derived from DLS measurements. The combination of both techniques allows estimating the size and proportion of coacervates in the microemulsion, as well as the polydispersity in size of the sample.
Keywords: Taylor dispersion analysis; Microemulsions; Polyoxylglycerides; Coacervation; Diffusion coefficient; Hydrodynamic radius; Fluorescent detection;

Targeting prostate cancer cells with genetically engineered polypeptide-based micelles displaying gastrin-releasing peptide by Wei Zhang; Sanjay Garg; Preethi Eldi; Fiona Huan-huan Zhou; Ian R.D. Johnson; Doug A. Brooks; Frankie Lam; Grigori Rychkov; John Hayball; Hugo Albrecht (270-279).
Display OmittedIn recent years G protein-coupled receptors (GPCRs) have emerged as crucial tumorigenic factors that drive aberrant cancer growth, metastasis and angiogenesis. Consequently, a number of GPCRs are strongly expressed in cancer derived cell lines and tissue samples. Therefore a rational anti-cancer strategy is the design of nano-medicines that specifically target GPCRs to bind and internalise cytotoxic drugs into cancer cells. Herein, we report the genetic engineering of a self-assembling nanoparticle based on elastin-like polypeptide (ELP), which has been fused with gastrin releasing peptide (GRP). These nanoparticles increased intracellular calcium concentrations when added to GRP receptor positive PC-3 prostate cancer cells, demonstrating specific receptor activation. Moreover, GRP-displaying fluorescent labelled nanoparticles showed specific cell-surface interaction with PC-3 prostate cancer cells and increased endocytic uptake. These nanoparticles therefore provide a targeted molecular carrier system for evaluating the delivery of cytotoxic drugs into cancer cells.
Keywords: G protein-coupled receptors (GPCRs); Elastin-like polypeptide (ELP); Gastrin releasing peptide (GRP); Micelle; Prostate cancer;

Display OmittedCentral nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220 nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11–14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2% ± 2.2% over 24 h and 9.4 ± 0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100 μM) demonstrating a decrease in cell viability to 58.2 ± 8.5% and curcumin an IC50 of 33 ± 0.18 μM. Furthermore confocal microscopy demonstrated nanoparticles of <500 nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2 h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.
Keywords: Mesoporous silica nanoparticle; Olfactory; Nose-to-brain; Flavonoid; Phytochemical;

The effect of device resistance and inhalation flow rate on the lung deposition of orally inhaled mannitol dry powder by Michael Y. Yang; Jordan Verschuer; Yuyu Shi; Yang Song; Andrew Katsifis; Stefan Eberl; Keith Wong; John D. Brannan; Weidong Cai; Warren H. Finlay; Hak-Kim Chan (294-301).
Display OmittedThe present study investigates the effect of DPI resistance and inhalation flow rates on the lung deposition of orally inhaled mannitol dry powder. Mannitol powder radiolabeled with 99mTc-DTPA was inhaled from an Osmohaler™ by healthy human volunteers at 50–70 L/min peak inhalation flow rate (PIFR) using both a low and high resistance Osmohaler™, and 110–130 L/min PIFR using the low resistance Osmohaler™ (n = 9). At 50–70 L/min PIFR, the resistance of the Osmohaler™ did not significantly affect the total and peripheral lung deposition of inhaled mannitol [for low resistance Osmohaler™, 20% total lung deposition (TLD), 0.3 penetration index (PI); for high resistance Osmohaler™, 17% TLD, 0.23 PI]. Increasing the PIFR 50–70 L/min to 110–130 L/min (low resistance Osmohaler™) significantly reduced the total lung deposition (10% TLD) and the peripheral lung deposition (PI 0.21). The total lung deposition showed dependency on the in vitro FPF (R2  = 1.0). On the other hand, the PI had a stronger association with the MMAD (R2  = 1.0) than the FPF (R2  = 0.7). In conclusion the resistance of Osmohaler™ did not significantly affect the total and regional lung deposition at 50–70 L/min PIFR. Instead, the total and regional lung depositions are dependent on the particle size of the aerosol and inhalation flow rate, the latter itself affecting the particle size distribution.
Keywords: Dry powder inhaler; Resistance; Flow rate; Lung deposition; Mannitol;

Display OmittedNanoparticle-loaded topical formulations can disrupt drug aggregation through controlled drug-nanoparticle interactions to enhance topical drug delivery. However, the complex relationship between the drug, nanoparticle and formulation vehicle requires further understanding. The aim of this study was to use nanoparticle-loaded hydroxypropyl methylcellulose (HPMC) and xanthan gum gels to probe how the drug, nanoparticle and formulation vehicle interactions influenced the delivery of an aggregated drug into the skin. Tetracaine was chosen as a model drug. It was loaded into HPMC and xanthan gum gels, and it was presented to porcine skin using infinite and finite dosing protocols. Gel infinite doses showed no important differences in tetracaine skin permeation rate, but HPMC gel finite doses delivered the drug more efficiently (46.99 ± 7.96 μg/cm2/h) compared to the xanthan gum (1.16 ± 0.14 μg/cm2/h). Finite doses of the nanoparticle-loaded HPMC gel generated a 10-fold increase in drug flux (109.95 ± 28.63 μg/cm2/h) compared to the equivalent xanthan gum system (14.19 ± 2.27 μg/cm2/h). Rheology measurements suggested that the differences in the gels ability to administer the drug into the skin were not a consequence of gel-nanoparticle interactions rather, they were a consequence of the dehydration mediated diffusional restriction imparted on the drug by xanthan gum compared to the viscosity independent interactions of HPMC with the drug.
Keywords: Tetracaine; Nanoparticles; Skin; Aggregation; Gel; Skin; Permeation; HPMC; Xanthan gum;

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles by Carlos Urey; Victor U. Weiss; Andreas Gondikas; Frank von der Kammer; Thilo Hofmann; Martina Marchetti-Deschmann; Günter Allmaier; György Marko-Varga; Roland Andersson (309-318).
Display OmittedFor drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability.
Keywords: Liposome; Gas-phase electrophoretic mobility molecular analysis; Transmission electron microscopy; Dynamic light scattering; Cytotoxicity; Flow field flow fractionation;

Fast dissolution of poorly water soluble drugs from fluidized bed coated nanocomposites: Impact of carrier size by Mohammad Azad; Jacqueline Moreno; Ecevit Bilgili; Rajesh Davé (319-331).
Display OmittedFormation of core-shell nanocomposites of Fenofibrate and Itraconazole, model poorly water soluble drugs, via fluidized bed (FB) coating of their well-stabilized high drug loaded nanosuspensions is investigated. Specifically, the extent of dissolution enhancement, when fine carrier particles (sub–50 μm) as opposed to the traditional large carrier particles (>300 μm) are used, is examined. This allows testing the hypothesis that greatly increased carrier surface area and more importantly, thinner shell for finer carriers at the same drug loading can significantly increase the dissolution rate when spray-coated nanosuspensions are well-stabilized. Fine sub–50 μm lactose (GranuLac® 200) carrier particles were made fluidizable via dry coating with nano-silica, enabling decreased cohesion, fluidization and subsequent nanosuspension coating. For both drugs, 30% drug loaded suspensions were prepared via wet-stirred media milling using hydroxypropyl methyl cellulose and sodium dodecyl sulfate as stabilizers. The stabilizer concentrations were varied to affect the milled particle size and prepare a stable nanosuspension. The suspensions were FB coated onto hydrophilic nano-silica (M-5P) dry coated sub-50 μm lactose (GranuLac® 200) carrier particles or larger carrier particles of median size >300 μm (PrismaLac®40). The resulting finer composite powders (sub-100 μm) based on GranuLac® 200 were freely flowing, had high bulk density, and had much faster, immediate dissolution of the poorly water-soluble drugs, in particular for Itraconazole. This is attributed to a much higher specific surface area of the carrier and corresponding thinner coating layer for fine carriers as opposed to those for large carrier particles.
Keywords: Poorly water soluble drug; Wet-stirred media milling; Fluidized bed coating; Fine vs large carrier particles; Thick vs thin polymer coating; Fast dissolving nanocomposite powders;

Long term performance evaluation of small-diameter vascular grafts based on polyvinyl alcohol hydrogel and dextran and MSCs-based therapies using the ovine pre-clinical animal model by Nuno Alexandre; Irina Amorim; Ana Rita Caseiro; Tiago Pereira; Rui Alvites; Alexandra Rêma; Ana Gonçalves; Guilherme Valadares; Elísio Costa; Alice Santos-Silva; Miguel Rodrigues; Maria Ascensão Lopes; André Almeida; José Domingos Santos; Ana Colette Maurício; Ana Lúcia Luís (332-346).
Display OmittedThe functional and structural performance of a 5 cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic/diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values.
Keywords: PVA; Dextran; Synthetic small diameter vascular graft; Mesenchymal stem cells; Ovine model;

Comparison in toxicity and solubilizing capacity of hydroxypropyl-β-cyclodextrin with different degree of substitution by Pengyu Li; Jia Song; Xiaomin Ni; Qin Guo; Hui Wen; Qiuyun Zhou; Yuanna Shen; Yijun Huang; Pengxin Qiu; Suizhen Lin; Haiyan Hu (347-356).
Display OmittedHydroxypropyl-β-cyclodextrin (HP-β-CD) has been widely used as an effective solubilizing agent in pharmaceutical industry for many years. However, the effect of degree of substitution (D.S.) of HP-β-CD on solubilizing capacity and toxicity has not been concerned. In this study, solubilizing capacity of HP-β-CDs with three different D.S. (4.55, 6.16 and 7.76) for 16 drugs were measured and their toxicities were compared by a 7-day i.v. administration (q.d.) study in rats. Generally, HP-β-CD with high D.S. (7.76) showed weaker solubilizing capacity for steroids and BCS class II drugs, but lower hemolytic activity, compared with that of HP-β-CD with low (4.55) or medium (6.16) D.S. HP-β-CD with low D.S. resulted in more changes in hematological and biochemical parameters, but the effects were reversible after a 7-day recovery. Moreover, HP-β-CD with medium D.S. may have slightly greater nephrotoxicity than the other two HP-β-CDs. HP-β-CDs with different D.S. had similar urine excretion percentage after i.v. administration and none of them was found to affect glomerular filtration function of rats. The results suggest that HP-β-CD with low D.S. would be a better choice considering both the solubilizing capacity and toxicity. However, comparison in toxicity of HP-β-CDs with different D.S. should be carried out in human in view of its species-dependence property.
Keywords: Hydroxypropyl-β-cyclodextrin; Degree of substitution; Solubilizing capacity; Toxicity;

Statistical analysis and comparison of a continuous high shear granulator with a twin screw granulator: Effect of process parameters on critical granule attributes and granulation mechanisms by Wei Meng; Lalith Kotamarthy; Savitha Panikar; Maitraye Sen; Shankali Pradhan; Michaelis Marc; James D. Litster; Fernando J. Muzzio; Rohit Ramachandran (357-375).
Display OmittedThis study is concerned with identifying the design space of two different continuous granulators and their respective granulation mechanisms. Performance of a continuous high shear granulator and a twin screw granulator with paracetamol formulations were examined by face-centered cubic design, which focused on investigating key performance metrics, namely, granule size, porosity, flowability and particle morphology of granules as a function of essential input process parameters (liquid content, throughput and rotation speed). Liquid and residence time distribution tests were also performed to gain insights into the liquid-powder mixing and flow behavior. The results indicated that continuous high shear granulation was more sensitive to process variation and produced spherical granules with monomodal size distribution and distinct internal structure and strength variation. Twin screw granulation with such a particular screw configuration showed narrower design space and granules were featured with multimodal size distribution, irregular shape, less detectible porosity difference and tighter range of strength. Granulation mechanisms explored on the basis of nucleation and growth regime maps revealed that for most cases liquid binder was uniformly distributed with fast droplet penetration into the powder bed and that granule consolidation and coalescence mainly took place in the nucleation, steady growth and rapid growth regimes.
Keywords: Continuous manufacturing; High shear granulation; Twin screw granulation; Design of experiments; Design space; Granulation mechanism;

Low density lipoprotein receptor targeted doxorubicin/DNA-Gold Nanorods as a chemo- and thermo-dual therapy for prostate cancer by Nan Zhang; Shasha Li; Haiying Hua; Dan Liu; Lili Song; Pengchao Sun; Weiwei Huang; Yafang Tang; Yongxing Zhao (376-386).
Display OmittedAs drug vehicles and therapeutics, Gold Nanorods (GNRs) have various merits such as easy preparation and modification, passive accumulation to tumor tissues, effective intracellular delivery of therapeutics, and thermal responses to laser radiation. Doxorubicin (DOX) has been the standard chemotherapy for cancer. To enhance the anti-cancer efficacy, chemotherapy and thermotherapy were combined in the present study. To load sufficient DOX, DOX was first intercalated into DNA double strands and then absorbed to GNRs. PEG (polyethylene glycol) was used to modify DOX/DNA-GNRs to prolong circulation in vivo and to enhance its stability. Low density lipoprotein receptor (LDLR) targeted peptide-RLT (R) was also bound to DOX/DNA-GNRs to increase their specificity to LDLR over-expressed cancer cells. DNA-GNRs-PEG/R was successfully prepared with high in vitro stability in this study and DOX was loaded sufficiently to obtain DOX/DNA-GNRs-PEG/R. DOX/DNA-GNRs-PEG/R with near infrared (NIR) laser treatment showed higher inhibition to MCF-7 cells and PC-3 cells and both DOX/DNA-GNRs-PEG/R with/without NIR laser treatment were more potent than free DOX. Cell uptake experiment indicated that DOX loaded in DNA-GNRs-PEG/R was taken by PC-3 cells much faster than free DOX. With DOX/DNA-GNRs-PEG/R, the apoptosis rate and necrosis rate of PC-3 cells increased 1.7 and 6.4 folds respectively compared to free DOX. Additional NIR laser treatment caused significantly increases in PC-3 cell necrosis. DOX/DNA-GNRs-PEG/R + laser also enhanced the inhibition of S phase of PC-3 cells by DOX. ROS (reactive oxygen species) assay showed that DOX/DNA-GNRs-PEG/R produced much more ROS than free DOX. With additional laser treatment, further increase in ROS was detected. Prostate cancer model was achieved by injecting PC-3 cells into nude mice and the results showed that more DNA-GNRs-PEG/R was observed in tumor cells and higher tumor inhibition rate was achieved in vivo with R modification. Conclusively, all results consistently indicated that DNA-GNRs-PEG/R was able to increase in vitro and in vivo anti-cancer efficacy of DOX. With additional NIR laser treatment, GNRs produced heat and further enhanced the anti-cancer effect achieved by DOX/DNA-GNRs-PEG/R. Therefore, the chemo- and thermo-dual therapy could be a potential combined therapy for more efficient anti-cancer treatment in clinical applications.
Keywords: Gold nanorods; Doxorubicin; Prostate cancer; Low density lipoprotein peptide; Chemotherapy; Thermotherapy;

Display OmittedA drug-cyclodextrin complex in liposome system was prepared in order to make a comparison with conventional risperidone-loaded liposome. Thin film hydration, reverse phase evaporation and ethanol injection methods were taken as preparation means to obtain the two types of liposome. Differential thermal analysis (DTA) and transmission electron microscopy (TEM) were used to investigate the thermal characters of inclusion complexes and morphology of liposome, respectively. Particle size, zeta potential and encapsulation efficiency were studied by light scattering analysis and ultrafiltration. In vitro release was carried out in the pH 7.4 phosphate buffer solution and samples were collected at the certain time. As a result, drug-cyclodextrin complex in liposome prepared by various methods displayed lower encapsulation efficiency than conventional liposome. However, size was larger and its stability was better than the latter. The second release phase of novel delivery system was slightly slower after initial burst release at the first phase, while the conventional liposome displayed a more regular trait. Thus, the novel liposome have potential to be developed as co-administration formulation with long-acting injection.
Keywords: Risperidone; Liposome; Cyclodextrin; Comparison;

Display OmittedThe aim of present investigation is to evaluate the feasibility of transdermal iontophoretic delivery of tacrine hydrochloride in Sprague Dawley (SD) rats using anodal iontophoretic patches and to correlate plasma tacrine concentration profiles to in vitro tacrine permeation flux. In vitro skin permeation studies were carried out across artificial membrane CELGRAD® 2400, freshly excised SD rat abdominal skin, freshly excised hairless rat abdominal skin, and frozen pig skin to examine the role of permeation membranes. Furthermore, plasma profiles with an application of 0.1–0.3 mA current strength and tacrine concentration loading of 5–20 mg/ml were obtained in SD rats. The tacrine plasma profiles were fitted to one-compartmental model using WinNonlin and in vivo transdermal absorption rates were then correlated to in vitro permeation profiles using various approaches. Tacrine permeation across membranes revealed current dependent interspecies differences at lower current strength application which diminished at higher current strength application, whereas, no significant difference in tacrine permeation was observed across fresh and frozen SD rat skin under 0.2 mA current application. In vivo studies confirmed current and concentration dependent tacrine plasma profiles with possible tacrine depot formation under the skin in-line with earlier in vitro results. Correlation of in vivo transdermal absorption rates to in vitro permeation profiles revealed higher in vitro permeation fluxes compare to in vivo transdermal absorption rates at varied combination of current strength and concentrations. Present in vivo studies support the earlier published in vitro findings and tacrine plasma profiles show a potential to reach therapeutic effective concentration of tacrine hydrochloride to provide a platform for pre-programmed tacrine delivery.
Keywords: Transdermal iontophoresis; Alzheimer’s disease; Tacrine hydrochloride; In vitro-in vivo correlation; WinNonlin;

Alginate nanoparticles protect ferrous from oxidation: Potential iron delivery system by Nuwanthi P. Katuwavila; A.D.L.C. Perera; Damayanthi Dahanayake; V. Karunaratne; Gehan A.J. Amaratunga; D. Nedra Karunaratne (404-409).
Display OmittedA novel, efficient delivery system for iron (Fe2+) was developed using the alginate biopolymer. Iron loaded alginate nanoparticles were synthesized by a controlled ionic gelation method and was characterized with respect to particle size, zeta potential, morphology and encapsulation efficiency. Successful loading was confirmed with Fourier Transform Infrared spectroscopy and Thermogravimetric Analysis. Electron energy loss spectroscopy study corroborated the loading of ferrous into the alginate nanoparticles. Iron encapsulation (70%) was optimized at 0.06% Fe (w/v) leading to the formation of iron loaded alginate nanoparticles with a size range of 15–30 nm and with a negative zeta potential (−38 mV). The in vitro release studies showed a prolonged release profile for 96 h. Release of iron was around 65–70% at pH of 6 and 7.4 whereas it was less than 20% at pH 2.The initial burst release upto 8 h followed zero order kinetics at all three pH values. All the release profiles beyond 8 h best fitted the Korsmeyer-Peppas model of diffusion. Non Fickian diffusion was observed at pH 6 and 7.4 while at pH 2 Fickian diffusion was observed.
Keywords: Anemia; Ferrous sulfate; Alginate nanoparticles; Iron loaded nanocomposite; Bioavailability;

Lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against Group A streptococcus by Nirmal Marasini; Zeinab G. Khalil; Ashwini Kumar Giddam; Khairunnisa Abdul Ghaffar; Waleed M. Hussein; Robert J. Capon; Michael R. Batzloff; Michael F. Good; Mariusz Skwarczynski; Istvan Toth (410-420).
Display OmittedRheumatic heart disease represents a leading cause of mortality caused by Group A Streptococcus (GAS) infections transmitted through the respiratory route. Although GAS infections can be treated with antibiotics these are often inadequate. An efficacious GAS vaccine holds more promise, with intranasal vaccination especially attractive, as it mimics the natural route of infections and should be able to induce mucosal IgA and systemic IgG immunity. Nanoparticles were prepared by either encapsulating or coating lipopeptide-based vaccine candidate (LCP-1) on the surface of poly(lactic-co-glycolic acid) (PLGA). In vitro study showed that encapsulation of LCP-1 vaccine into nanoparticles improved uptake and maturations of antigen-presenting cells. The immunogenicity of lipopeptide incorporated PLGA-based nanoparticles was compared with peptides co-administered with mucosal adjuvant cholera toxin B in mice upon intranasal administration. Higher levels of J14-specific salivary mucosal IgA and systemic antibody IgG titres were observed for groups immunized with encapsulated LCP-1 compared to LCP-1 coated nanoparticles or free LCP-1. Systemic antibodies obtained from LCP-1 encapsulated PLGA NPs inhibited the growth of bacteria in six different GAS strains. Our results show that PLGA-based lipopeptide delivery is a promising approach for rational design of a simple, effective and patient friendly intranasal GAS vaccine resulting in mucosal IgA response.
Keywords: Nanoparticles; Lipopeptides; PLGA; Vaccine; Mucosal immunology;

Biophysical evaluation of hybrid Fc fusion protein of hGH to achieve basal buffer system by Nam Ah Kim; In bok An; Hye Seong Lim; Sang In Yang; Seong Hoon Jeong (421-430).
Display OmittedA newly developed hybrid Fc (hyFc) is a non-immunogenic and non-cytolytic Fc with intact Ig structure derived from human IgD and IgG4. It is fused with the human growth hormone (GXD-9) and was evaluated by various biophysical techniques. Two thermal transitions were evident by DSC, reflecting the unfolding of IgG4 and the conjugated protein. The highest Tm of the initial GXD-9 was 68.17° C and the Tm of the two domains were around 66° C and 70° C. Although Tm increased with decreasing concentration, which reflects increasing conformational stability, aggregation issues were still observed by DLS. This might be caused by decreasing or low zeta potential due to a highly complex structure. The protein was dialyzed to various pH (6.2–8.2) values to enhance conformational stability and to overcome aggregation issues. The results of CD spectroscopy were correlated with DSC measurements to evaluate its conformational stability. Changes in secondary structural contents were similar as determined by DSC and DLS. In conclusion, GXD-9 was found to be most stable at pH 7.0. The investigation of the biophysical stability of a hyFc-fusion protein has demonstrated a positive feasibility of developing more stable formulations to facilitate the initial drug development process for further clinical trials.
Keywords: Hybrid fc; Fusion protein; hGH; pH; DSC; CD spectroscopy;

Cytotoxic and cytostatic side effects of chitosan nanoparticles as a non-viral gene carrier by Gizem Bor; Jennifer Mytych; Jacek Zebrowski; Maciej Wnuk; Gülşah Şanlı-Mohamed (431-437).
Display OmittedAlthough chitosan nanoparticles (CNs) became a promising tool for several biological and medical applications owing to their inherent biocompatibility and biodegradability features, studies regarding their effects on cytotoxic and cytostatic properties still remain insufficient. Therefore, in the present study, we decided to perform comprehensive analysis of the interactions between CNs–pKindling-Red-Mito (pDNA) and different cell line models derived from blood system and human solid tissues cancers. The resulting CNs-pDNA was investigated in terms of their cellular uptake, transfection efficiency, and physico-chemical, cytotoxic and cytostatic properties. The nanoparticles showed high encapsulation efficiency and physical stability for various formulations even after two days time period. Moreover, high gene expression levels were observed after 96 h of transfection. CNs-pDNA treatment, despite the absence of oxidative stress induction, caused cell cycle arrest in G0/G1 phase and as a consequence led to premature senescence which turned out to be both p21-dependent and p21-independent. Also, observed DNMT2 upregulation may suggest the activation of different pathways protecting from the results of CNs-mediated stress. In conclusion, treatment of different cell lines with CNs-pDNA showed that their biocompatibility was limited and the effects were cell type-dependent.
Keywords: Gene delivery; Chitosan; Nanoparticles; pDNA; Cytotoxicity; Monocytes; Cancer cells;

Opsonisation of nanoparticles prepared from poly(β-hydroxybutyrate) and poly(trimethylene carbonate)-b-poly(malic acid) amphiphilic diblock copolymers: Impact on the in vitro cell uptake by primary human macrophages and HepaRG hepatoma cells by Elise Vene; Ghislaine Barouti; Kathleen Jarnouen; Thomas Gicquel; Claudine Rauch; Catherine Ribault; Sophie M. Guillaume; Sandrine Cammas-Marion; Pascal Loyer (438-452).
Display OmittedThe present work reports the investigation of the biocompatibility, opsonisation and cell uptake by human primary macrophages and HepaRG cells of nanoparticles (NPs) formulated from poly(β-malic acid)-b-poly(β-hydroxybutyrate) (PMLA-b-PHB) and poly(β-malic acid)-b-poly(trimethylene carbonate) (PMLA-b-PTMC) diblock copolymers, namely PMLA800-b-PHB7300, PMLA4500-b-PHB4400, PMLA2500-b-PTMC2800 and PMLA4300-b-PTMC1400. NPs derived from PMLA-b-PHB and PMLA-b-PTMC do not trigger lactate dehydrogenase release and do not activate the secretion of pro-inflammatory cytokines demonstrating the excellent biocompatibility of these copolymers derived nano-objects. Using a protein adsorption assay, we demonstrate that the binding of plasma proteins is very low for PMLA-b-PHB-based nano-objects, and higher for those prepared from PMLA-b-PTMC copolymers. Moreover, a more efficient uptake by macrophages and HepaRG cells is observed for NPs formulated from PMLA-b-PHB copolymers compared to that of PMLA-b-PTMC-based NPs. Interestingly, the uptake in HepaRG cells of NPs formulated from PMLA800-b-PHB7300 is much higher than that of NPs based on PMLA4500-b-PHB4400. In addition, the cell internalization of PMLA800-b-PHB7300 based-NPs, probably through endocytosis, is strongly increased by serum pre-coating in HepaRG cells but not in macrophages. Together, these data strongly suggest that the binding of a specific subset of plasmatic proteins onto the PMLA800-b-PHB7300-based NPs favors the HepaRG cell uptake while reducing that of macrophages.
Keywords: Poly(trimethylene carbonate); Poly(hydroxy alkanoate); Poly(malic acid); Nanoparticle; Macrophages; HepaRG cells;

Statistical analysis of industrial-scale roller compactor ‘Freund TF-MINI model’ by M. Sajjia; Ahmad B. Albadarin; Gavin Walker (453-463).
Display OmittedThe aim of this study is to perform a statistical analysis on a pharmaceutical roller compaction process using an industrial-scale roller compactor “Freund TF-MINI model”. The process was modelled using response surface methodology (RSM) to better understand and control the process in order to produce ribbons and granules with optimised quality. The significant process parameters were determined to be (i) the screw speed to roll speed ratio and (ii) the roll pressure. The roll speed was kept constant and the roll gap was uncontrolled. The quality attributes of interest are: ribbon density, granule size (D10, D50, D90), amount of fines (granule size <157 μm), and tablet hardness. Microcrystalline cellulose (MCC) PH 102 was used as a model material. Design-Expert V9 was utilised to establish the design matrix and to analyse the experimental data. The relationships between the process parameters and the resultant ribbon/granule/tablet characteristics were established. This was followed by an optimisation of the process parameters to obtain the target responses. The results confirmed the attainment of significant models with satisfactory accurate measures. The optimisation allowed for the determination of the process parameters required to produce the best quality tablets.
Keywords: Design-Expert; Microcrystalline cellulose; Response surface methodology; Roller compaction; Statistical analysis;

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms by Shahrouz Taranejoo; Mohsen Janmaleki; Mohammad Pachenari; Seyed Morteza Seyedpour; Ramya Chandrasekaran; Wenlong Cheng; Kerry Hourigan (464-472).
Display OmittedA recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K1 and K2) and the coefficient of viscosity (μ). This difference was more pronounced for cancer cells after 48 h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach.
Keywords: Cytoskeleton; Actin microfilaments; Microtubules; Cancer; Microtubule-targeting agent; Albendazole;

Gelatin-based membrane containing usnic acid-loaded liposome improves dermal burn healing in a porcine model by Paula Santos Nunes; Alessandra Silva Rabelo; Jamille Cristina Campos de Souza; Bruno Vasconcelos Santana; Thailson Monteiro Menezes da Silva; Mairim Russo Serafini; Paula dos Passos Menezes; Bruno dos Santos Lima; Juliana Cordeiro Cardoso; Júlio César Santana Alves; Luiza Abrahão Frank; Sílvia Stanisçuaski Guterres; Adriana Raffin Pohlmann; Malone Santos Pinheiro; Ricardo Luiz Cavalcanti de Albuquerque; Adriano Antunes de Souza Araújo (473-482).
Display OmittedThere are a range of products available which claim to accelerate the healing of burns; these include topical agents, interactive dressings and biomembranes. The aim of this study was to assess the effect of a gelatin-based membrane containing usnic acid/liposomes on the healing of burns in comparison to silver sulfadiazine ointment and duoDerme® dressing, as well as examining its quantification by high performance liquid chromatography. The quantification of the usnic acid/liposomes was examined using high performance liquid chromatography (HPLC) by performing separate in vitro studies of the efficiency of the biomembranes in terms of encapsulation, drug release and transdermal absorption. Then, second-degree 5 cm2 burn wounds were created on the dorsum of nine male pigs, assigned into three groups (n = 3): SDZ – animals treated with silver sulfadiazine ointment; GDU – animals treated with duoDerme®; UAL – animals treated with a gelatin-based membrane containing usnic acid/liposomes. These groups were treated for 8, 18 and 30 days. In the average rate of contraction, there was no difference among the groups (p> 0.05). The results of the quantification showed that biomembranes containing usnic acid/liposomes were controlled released systems capable of transdermal absorption by skin layers. A macroscopic assay did not observe any clinical signs of secondary infections. Microscopy after 8 days showed hydropic degeneration of the epithelium, with intense neutrophilic infiltration in all three groups. At 18 days, although epidermal neo-formation was only partial in all three groups, it was most incipient in the SDZ group. Granulation tissue was more exuberant and cellularized in the UAL and GDU groups. At 30 days, observed restricted granulation tissue in the region below the epithelium in the GDU and UAL groups was observed. In the analysis of collagen though picrosirius, the UAL group showed greater collagen density. Therefore, the UAL group displayed development and maturation of granulation tissue and scar repair that was comparable to that produced by duoDerme ®, and better than that produced by treatment with sulfadiazine silver ointment In addition, the UAL group showed increased collagen deposition compared to the other two groups.
Keywords: Burn; Porcine; Gelatin membrane; Liposomes; Usnic acid;

Triclosan loaded electrospun nanofibers based on a cyclodextrin polymer and chitosan polyelectrolyte complex by Safa Ouerghemmi; Stéphanie Degoutin; Nicolas Tabary; Frédéric Cazaux; Mickaël Maton; Valérie Gaucher; Ludovic Janus; Christel Neut; Feng Chai; Nicolas Blanchemain; Bernard Martel (483-495).
Display OmittedThis work focuses on the relevance of antibacterial nanofibers based on a polyelectrolyte complex formed between positively charged chitosan (CHT) and an anionic hydroxypropyl betacyclodextrin (CD)-citric acid polymer (PCD) complexing triclosan (TCL). The study of PCD/TCL inclusion complex and its release in dynamic conditions, a cytocompatibility study, and finally the antibacterial activity assessment were studied. The fibers were obtained by electrospinning a solution containing chitosan mixed with PCD/TCL inclusion complex. CHT/TCL and CHT-CD/TCL were also prepared as control samples. The TCL loaded nanofibers were analyzed by Scanning Electron Microscopy (SEM), Fourier Transformed Infrared spectroscopy (FTIR) and X-Ray Diffraction (XRD). Nanofibers stability and swelling behavior in aqueous medium were pH and CHT:PCD weight ratio dependent. Such results confirmed that CHT and PCD interacted through ionic interactions, forming a polyelectrolyte complex. A high PCD content in addition to a thermal post treatment at 90 °C were necessary to reach a nanofibers stability during 15 days in soft acidic conditions, at pH = 5.5. In dynamic conditions (USP IV system), a prolonged release of TCL with a reduced burst effect was observed on CHT-PCD polyelectrolyte complex based fibers compared to CHT-CD nanofibers. These results were confirmed by a microbiology study showing prolonged antibacterial activity of the nanofibers against Escherichia coli and Staphylococcus aureus. Such results could be explained by the fact that the stability of the polyelectrolyte CHT-PCD complex in the nanofibers matrix prevented the diffusion of the PCD/triclosan inclusion complex in the supernatant, on the contrary of the similar system including cyclodextrin in its monomeric form.
Keywords: Electrospinning; Cyclodextrin polymer; Chitosan; Polyelectrolyte complex; Nanofibers; Triclosan; Antibacterial activity; Controlled release;

Permeation of topically applied caffeine from a food by—product in cosmetic formulations: Is nanoscale in vitro approach an option? by Francisca Rodrigues; Ana Catarina Alves; Claudia Nunes; Bruno Sarmento; M. Helena Amaral; Salette Reis; M. Beatriz P.P. Oliveira (496-503).
Display OmittedThe aim of the present work was to develop and evaluate the potential of nanostructured lipid carriers associated with caffeine extracted from Coffee Silverskin (NLC-CS), a food by-product, as a new possible topical therapy of cellulitis. Caffeine gain increasing research interest due to their cosmetic potential, particularly in gynoid lipodystrophy, commonly known as cellulite. NLC-CS were prepared via double emulsion technique using polysorbate 60 as surfactant and characterized for their morphology, particle size, zeta potential, association encapsulation and stability. The in vitro skin permeation studies were performed on Franz diffusion cells using pig skin ear as permeation membrane and the optimized formulation was compared with a hydroalcoholic solution of Coffee silverskin (CS) extract.NLC-CS were within the nanosized range (≈200 nm), with a low polydispersity index (<0.25) and zeta potential values around −30 Mv, presenting storage stability up to 180 days at 25 °C/65% relative humidity (RH) and 40 °C/75% RH. The association efficiency (AE) of caffeine was about 30% at production time and after storage period. Cryo-SEM images confirmed the spherical shape of NLC-CS.The in vitro skin permeation study demonstrated that NLC-CS had a similar skin permeation profile when compared to caffeine in CS extract.
Keywords: Caffeine; Nanostructured lipid carriers; Cellulitis; Coffee silverskin; Dermal delivery;

Nanostructured lipid carriers of artemether–lumefantrine combination for intravenous therapy of cerebral malaria by Priyanka Prabhu; Shital Suryavanshi; Sulabha Pathak; Aditya Patra; Shobhona Sharma; Vandana Patravale (504-517).
Display OmittedPatients with cerebral malaria (CM) are unable to take oral medication due to impaired consciousness and vomiting thus necessitating parenteral therapy. Quinine, artemether, and artesunate which are currently used for parenteral malaria therapy have their own drawbacks. The World Health Organization (WHO) has now banned monotherapy and recommends artemisinin-based combination therapy for malaria treatment. However, presently there is no intravenous formulation available for combination therapy of malaria. Artemether–Lumefantrine (ARM–LFN) is a WHO approved combination for oral malaria therapy. However, the low aqueous solubility of ARM and LFN hinders their intravenous delivery. The objective of this study was to formulate ARM–LFN nanostructured lipid carriers (NLC) for intravenous therapy of CM. ARM–LFN NLC were prepared by microemulsion template technique and characterized for size, drug content, entrapment efficiency, drug release, crystallinity, morphology, amenability to autoclaving, compatibility with infusion fluids, stability, antimalarial efficacy in mice, and toxicity in rats. The ARM–LFN NLC showed sustained drug release, amenability to autoclaving, compatibility with infusion fluids, good stability, complete parasite clearance and reversal of CM symptoms with 100% survival in Plasmodium berghei-infected mice, and safety in rats. The biocompatible ARM–LFN NLC fabricated by an industrially feasible technique offer a promising solution for intravenous therapy of CM.
Keywords: Artemether; Lumefantrine; Nanostructured lipid carriers; Intravenous; Cerebral malaria; Plasmodium;

HMDSO-plasma coated electrospun fibers of poly(cyclodextrin)s for antifungal dressings by Alejandro Costoya; Florencia Montini Ballarin; Jose Llovo; Angel Concheiro; Gustavo A. Abraham; Carmen Alvarez-Lorenzo (518-527).
Display OmittedElectrospun mats containing cyclodextrin polymers (poly-αCD or poly-βCD) were developed to act as wound dressings showing tunable release rate of the antifungal agent fluconazole incorporated forming inclusion complexes. Poly-αCD and poly-βCD were prepared via cross-linking with epichlorohydrin (EPI) as water-soluble large molecular weight polymers. Then, polyCDs forming complexes with fluconazole were mixed with poly-(ε-caprolactone) (PCL) or poly(N-vinylpyrrolidone) (PVP) for electrospinning. Obtained bead-free fibers showed a random distribution, diameters in the 350–850 nm range, and a variety of physical stability behaviors in aqueous environment. Mats were coated by hexamethyldisiloxane (HMDSO) plasma polymerization to create a hydrophobic layer that prevented rapid drug diffusion. HMDSO coating was evidenced by the Si content of mat surface (EDX analysis) and by the increase in the water contact angle (up to 130°). In physiological-mimicking medium, non-treated mats showed burst release of fluconazole, whereas HMDSO-coated mats sustained the release and delayed disintegration of PVP-based mats. Antifungal tests evidenced that both coated and non-coated mats efficiently inhibited the growth of Candida albicans.
Keywords: Electrospinning; Cyclodextrin polymers; Plasma polymerization; HMDSO coating; Fluconazole; Antifungal activity;

Display OmittedNovel fibrous materials with antioxidant and antibacterial properties from poly(3-hydroxybutyrate) (PHB), quaternized chitosan (QCh), κ-carrageenan (Car) and caffeic acid (CA) were obtained. These materials were prepared by applying electrospinning or electrospinning in conjunction with dip-coating and polyelectrolyte complex (PEC) formation. It was found that the CA release depended on the fiber composition. X-ray diffraction analysis (XRD) and differential scanning calorimetry (DSC) revealed that CA incorporated in the fibers was in the amorphous state, whereas CA included in the coating was in the crystalline state. In contrast to the neat PHB mats, the CA-containing mats and the PEC QCh/Car-coated mats were found to kill the Gram-positive bacteria S. aureus and the Gram-negative bacteria E. coli and were effective in suppressing the adhesion of pathogenic bacteria S. aureus. Enhancement of the antioxidant activity of the fibrous materials containing both CA and QCh/Car coating was observed.
Keywords: Quaternized chitosan; κ-Carrageenan; Caffeic acid; Electrospinning; Antibacterial fibers; Antioxidant activity;

Application of 13C NMR cross-polarization inversion recovery experiments for the analysis of solid dosage forms by Dariusz Maciej Pisklak; Monika Zielińska-Pisklak; Łukasz Szeleszczuk (538-542).
Display OmittedSolid-state nuclear magnetic resonance (ssNMR) is a powerful and unique method for analyzing solid forms of the active pharmaceutical ingredients (APIs) directly in their original formulations. Unfortunately, despite their wide range of application, the ssNMR experiments often suffer from low sensitivity and peaks overlapping between API and excipients. To overcome these limitations, the crosspolarization inversion recovery method was successfully used. The differences in the spin-lattice relaxation time constants for hydrogen atoms T1(H) between API and excipients were employed in order to separate and discriminate their peaks in ssNMR spectra as well as to increase the intensity of API signals in low-dose formulations. The versatility of this method was demonstrated by different examples, including the excipients mixture and commercial solid dosage forms (e.g. granules and tablets).
Keywords: Solid-state NMR; Cross-polarization; Inversion recovery; Solid dosage forms;

Herceptin-functionalized pure paclitaxel nanocrystals for enhanced delivery to HER2-postive breast cancer cells by Jin-Ki Noh; Muhammad Naeem; Jiafu Cao; Eun Hee Lee; Min-Soo Kim; Yunjin Jung; Jin-Wook Yoo (543-553).
Display OmittedThe objective of this study was to prepare Herceptin (HCT)-functionalized paclitaxel nanocrystals and evaluated their cell-specific interactions, cellular accumulation, and growth inhibition in HER2-positve breast cancer cells as a tumor-targeted delivery module. Paclitaxel (PTX) was fabricated in the form of nanocrystals (PNCs) by a sono-precipitation method, and HCT were coated using a facile non-covalent method (PNCs-HCT). Our results showed that the PNCs-HCT were stable for at least 1 month at 4 °C with no noticeable desorption of HCT. The release test showed that PNCs-HCT exhibited sustained drug release similar to only PNCs but with a higher release rate than only PTX powder. Cellular uptake, cytotoxicity, and cell cycle arrest studies revealed that PNCs-HCT exhibit greater binding affinity and higher cell-specific internalization to HER2-positive breast cancer cell lines as compared to PNCs, followed by enhanced cell growth inhibition. HCT-functionalized PNCs presented in this study offer a promising strategy for targeted pure drug nanocrystal delivery and enhancing the efficiency of anticancer therapy.
Keywords: Paclitaxel; Nanocrystals; Herceptin; HER2-positve breast cancer; Tumor-targeting;

Paclitaxel loaded magnetic nanocomposites with folate modified chitosan/carboxymethyl surface; a vehicle for imaging and targeted drug delivery by Shazia Bano; Muhammad Afzal; Mustansar Mahmood Waraich; Khalid Alamgir; Samina Nazir (554-563).
Display OmittedIn this study, Paclitaxel (PTX) containing, bovine serum albumin (BSA) nanoparticles were fabricated via a simple approach. Folic acid (FA) was conjugated to chitosan (CS)/carboxymethyl cellulose (CMC) through an esterification reaction to produce BSA–CS–FA or BSA-CMC-FA conjugates. NiFe2O4 noncore (NFs) and PTX were loaded through a heat treatment and by a diffusion process. NFs-BSA–CS and NFs-BSA–CMC–FA with size of about 80 nm, showed superior transversal R2 relaxation rate of 349 (mM)−1  s−1 along with folate receptor-targeted and magnetically directed functions. NFs-BSA–CS–FA or NFs-BSA–CS–FA were found stable and biocompatible. Application of an external magnetic field effectively enhanced the PTX release from PTX-NFs-BSA–CS–FA or PTX-NFs-BSA–CS–FA and hence tumor inhibition rate. This study validate that NFs-BSA–CS–FA or NFs-BSA–CMC–FA and PTX-NFs-BSA–CS–FA or PTX-NFs-BSA–CS–FA are suitable systems for tumor diagnosis and therapy.
Keywords: Biopolymers; Folate conjugation; Magnetic drug release; Magnetic resonance imaging; Drug delivery system;

Gemcitabine-based polymer-drug conjugate for enhanced anticancer effect in colon cancer by Tie-Jun Liang; Zhong-Mei Zhou; Ying-Qing Cao; Ming-Ze Ma; Xiao-Jun Wang; Kai Jing (564-571).
Display OmittedIn this study, we have demonstrated gemcitabine (GEM)-conjugated amphiphilic biodegradable polymeric drug carriers. Our aim was to increase the chemotherapeutic potential of GEM in colon cancer by forming a unique polymer-drug conjugates. The polymer-drug conjugate micelles were nanosized with a typical spherical shape. The GEM-conjugated methoxy poly(ethylene glycol)-poly(lactic acid) (GEM-PL) exhibited a controlled release of drug in both the pH conditions. The developed GEM-PL efficiently killed the HT29 cancers cells in a typical time dependent manner. The clonogenic assay further confirmed the superior anticancer effect of GEM-PL which showed least number of colonies. GEM-PL formulation exhibited a significantly higher apoptosis of cancer cells (∼25%) when stained using Annexin-V/PI kit. Conjugation of GEM to the mPEG-PLA significantly enhanced the blood circulation potential in animal model compared to that of free GEM. GEM-PL could prevent quick elimination of the drug and can provide sufficient time for the greater accumulation of GEM at the tumor sites. GEM-PL showed a remarkable tumor regression effect as evident from the lowest tumor volume in HT-29 containing tumor model. Overall, mPEG-PLA/GEM conjugates showed the potential of polymer-based drug targeting and might hold significant clinical potential in the treatment of colon cancers.
Keywords: Gemcitabine; Polylactide; Polymer-drug conjugates; Anticancer efficacy; Apoptosis;

Sugar-modified poly(propylene imine) dendrimers as drug delivery agents for cytarabine to overcome drug resistance by Aleksandra Szulc; Lukasz Pulaski; Dietmar Appelhans; Brigitte Voit; Barbara Klajnert-Maculewicz (572-583).
Display OmittedMaltose-modified poly(propylene imine) glycodendrimers (PPI-m OS) of the 4th generation provide a promising strategy for leukemia treatment. Anticancer therapy with nucleoside analog drugs such as cytarabine (Ara-C) frequently has limited efficacy due to drug resistance, inefficient uptake and accumulation of the drug inside cancer cells where it has to be transformed into the active triphosphate congener. The cationic nature of PPI dendrimers makes it possible to form complexes with nucleotide Ara-C triphosphate forms (Ara-CTP). The aim of this work was to test the concept of applying PPI glycodendrimers as drug delivery devices in order to facilitate the delivery of activated cytarabine to cancer cells to overcome metabolic limitations of the drug. The study has been carried out using 1301 and HL-60 leukemic cell lines as well as peripheral blood mononuclear cells. The results of cytotoxicity and apoptosis assays showed enhanced activity of Ara-C triphosphate form (Ara-CTP) complexed with PPI-m dendrimers in relation to free Ara-C and Ara-CTP against 1301 leukemic cells. Secondly, enhanced uptake and cytotoxicity of Ara-CTP-dendrimers complexes toward 1301 cells with blocked human equilibrative nucleoside transporter – hENT1 suggested that this combination might be a versatile candidate for chemotherapy against resistant acute lymphoblastic leukemia cells with lower expression of hENT1.
Keywords: Cytarabine; PPI dendrimers; Drug carriers; Leukemia; Drug resistance;

Photosensitizer-conjugated tryptophan-containing peptide ligands as new dual-targeted theranostics for cancers by Jisu Kim; Jihyun Chae; Jun Soo Kim; Sung-Ho Goh; Yongdoo Choi (584-590).
Display OmittedHere we report that new dual-targeted theranostic anti-cancer agents can be produced by simple conjugation of photosensitizers with tryptophan-containing peptide ligands via cyclic disulfide linkages. In the proof-of-concept study, photosensitizers conjugated with EGFR-targeting peptide GE11 (C-EGFR) were in close proximity with tryptophan residues in the conjugate, resulting in quenching of its fluorescence and singlet oxygen generation. C-EGFR specifically binds to target receptors on the cancer cell surface, after which it is internalized via receptor-mediated endocytosis. Intracellular cleavage of cyclic disulfide bonds allows separation of the photosensitizers from the tryptophan residue, after which they emit near-infrared (NIR) fluorescence and produce a phototoxic effect in the target cells. This strategy enabled us to accomplish simultaneous real-time NIR fluorescence imaging of EGFR-overexpressing cancer cells with high contrast and selective photodynamic therapy
Keywords: Activatable; Theranostics; EGFR target; Fluorescence imaging; Photodynamic therapy;

Aerosol delivery of folate-decorated hyperbranched polyspermine complexes to suppress lung tumorigenesis via Akt signaling pathway by Cheng-Qiong Luo; Yoonjeong Jang; Lei Xing; Peng-Fei Cui; Jian-Bin Qiao; Ah Young Lee; Hyeon-Jeong Kim; Myung-Haing Cho; Hu-Lin Jiang (591-601).
Schemetic presentation of shAkt1 deivery using biocompatible catonic polymer FA-HPSPE via aerosol administration and proposed mechanish for Akt1 protein knock-down in lung cancer cells.Display OmittedLung cancer has been a leading cause of cancer mortality worldwide and aerosol-mediated gene therapy endows numerous advantages compared to other traditional modalities. Here, we reported a folic acid (FA)-modified hyperbranched polyspermine (HPSPE) with prominent biocompatibility for lung cancer cell targeted gene therapy. FA was decorated to the HPSPE via an amidation reaction and the physicochemical properties of nanoplexes formed with DNA were characterized. Gel electrophoresis study elucidated that the designed polymer was capable to condense DNA and protect it from degradation by DNase I. Cell viability and transfection efficiency assay in vitro and in vivo indicated its increased transfection performance with lower toxicity. Furthermore, reduced tumor numbers and down-regulation of Akt1 protein after aerosol treatment containing FA-HPSPE/shAkt1 complexes proved its therapeutic potential for lung cancer suppression. Results obtained in this study suggested that FA-HPSPE with highly biocompatibility and targeting capability while forming complexes with shAkt1 and administrated through noninvasive aerosol could be prospective for inhibiting lung tumorigenesis.
Keywords: shAkt1; Aerosol delivery; Lung cancer; Biocompatibility; Cancer cell targeting; Gene therapy;

A comparative study between melt granulation/compression and hot melt extrusion/injection molding for the manufacturing of oral sustained release thermoplastic polyurethane matrices by G. Verstraete; P. Mertens; W. Grymonpré; P.J. Van Bockstal; T. De Beer; M.N. Boone; L. Van Hoorebeke; J.P. Remon; C. Vervaet (602-611).
Display OmittedDuring this project 3 techniques (twin screw melt granulation/compression (TSMG), hot melt extrusion (HME) and injection molding (IM)) were evaluated for the manufacturing of thermoplastic polyurethane (TPU)-based oral sustained release matrices, containing a high dose of the highly soluble metformin hydrochloride.Whereas formulations with a drug load between 0 and 70% (w/w) could be processed via HME/(IM), the drug content of granules prepared via melt granulation could only be varied between 85 and 90% (w/w) as these formulations contained the proper concentration of binder (i.e. TPU) to obtain a good size distribution of the granules. While release from HME matrices and IM tablets could be sustained over 24 h, release from the TPU-based TSMG tablets was too fast (complete release within about 6 h) linked to their higher drug load and porosity. By mixing hydrophilic and hydrophobic TPUs the in vitro release kinetics of both formulations could be adjusted: a higher content of hydrophobic TPU was correlated with a slower release rate. Although mini-matrices showed faster release kinetics than IM tablets, this observation was successfully countered by changing the hydrophobic/hydrophilic TPU ratio. In vivo experiments via oral administration to dogs confirmed the versatile potential of the TPU platform as intermediate-strong and low-intermediate sustained characteristics were obtained for the IM tablets and HME mini-matrices, respectively.
Keywords: Hot melt extrusion; Twin screw melt granulation; Matrices; High drug load; Sustained release; Thermoplastic polyurethanes; Metformin hydrochloride;

One-step assembly of polymeric demethylcantharate prodrug/Akt1 shRNA complexes for enhanced cancer therapy by Jia-Liang Zhang; Jia-Hui Gong; Lei Xing; Peng-Fei Cui; Jian-Bin Qiao; Yu-Jing He; Mei Zhang; Jin-Yuan Lyu; Cheng-Qiong Luo; Shun-Ai Che; Tuo Jin; Hu-Lin Jiang (612-627).
Co-delivery of Akt1 shRNA and demethylcantharate prodrug using polymeric demethylcantharate prodrug/Akt1 shRNA complexes for enhanced cancer therapy.Display OmittedThis report demonstrated a one-step assembly for co-delivering chemotherapeutics and therapeutic nucleic acids, constructed by integrating drug molecules into a nucleic acid condensing polymeric prodrug through degradable linkages. Demethylcantharate was selected as the model drug and pre-modified by esterifying its two carboxylic groups with 2-hydroxyethyl acrylate. The synthesized demethylcantharate diacrylate was then used to polymerize with linear polyethyleneimine (PEI 423) through a one-step Michael-addition reaction. The obtained cationic polymeric demethylcantharate prodrug was used to pack Akt1 shRNA into complexes through a one-step assembly. The formed complexes could release the parent drug demethylcantharate and Akt1 shRNA through the hydrolysis of ester bonds. Cellular assays involving cell uptake, cytotoxicity, and cell migration indicated that demethylcantharate and Akt1 shRNA co-delivered in the present form significantly and synergistically suppress the growth and metastasis of three human cancer cells. This work suggests that incorporating drug molecules into a nucleic acid-packing cationic polymer as a polymeric prodrug in a degradable form is a highly convenient and efficient way to co-deliver drugs and nucleic acids for cancer therapy.
Keywords: Cancer therapy; Polymeric prodrug; Co-delivery; Akt1 shRNA; Demethylcantharate;

Display OmittedWe have developed a nano drug delivery system for the treatment of tuberculosis (TB) using rifampicin (RF) encapsulated in poly(ester amide)s nanoparticles (PEA-RF-NPs), which are biocompatible polymers. In this study, biodegradable amino acid based poly(ester amide)s (PEAs) were synthesized by the poly condensation reaction and RF-loaded NPs were fabricated by the dialysis method. The surface morphology and in-vitro drug release efficiency were examined. The effect of time and temperature on the cellular uptake of PEA-RF-NPs in NR8383 cells was evaluated. Fluorescence microscopic results of PEA-RF-NPs from NR8383 cell lines suggest its potential application in treating TB. The antibacterial activity of RF against Mycobacterium smegmatis was also evaluated. Based on these results, this approach provides a new means for controlled and efficient release of RF using the PEA-NPs delivery system and is promising for the treatment of TB.
Keywords: Alveolar macrophages; Mycobacterium smegmatis; Nanoparticles; Polyesteramides; Rifampicin; Tuberculosis;

A novel chitosan-polyethylene oxide nanofibrous mat designed for controlled co-release of hydrocortisone and imipenem/cilastatin drugs by Yousef Fazli; Zahra Shariatinia; Iraj Kohsari; Amirreza Azadmehr; Seied Mahdi Pourmortazavi (636-647).
Display OmittedAntimicrobial chitosan–polyethylene oxide (CS-PEO) nanofibrous mats containing ZnO nanoparticles (NPs) and hydrocortisone-imipenem/cilastatin-loaded ZnO NPs were produced by electrospinning technique. The FE-SEM images displayed that the spherical ZnO NPs were ∼70–200 nm in size and the CS-PEO nanofibers were very uniform and free of any beads which had average diameters within the range of ∼20–130 nm. For all of the nanofibrous mats, the water uptakes were the highest in acidic medium but they were decreased in the buffer and the least swellings were obtained in the alkaline environment. The drug incorporated mat preserved its bactericidal activity even after it was utilized in the release experiment for 8 days in the PBS buffer. The hydrocortisone release was increased to 82% within first 12 h while the release rate of imipenem/cilastatin was very much slower so that 20% of the drug was released during this period of time suggesting this nanofibrous mat is very suitable to inhibit inflammation (by hydrocortisone) and infection (using imipenem/cilastatin antibiotic and ZnO NPs) principally for the wound dressing purposes.
Keywords: Electrospinning; Chitosan–polyethylene oxide nanofibrous mat; Antimicrobial activity; Hydrocortisone; Imipenem/cilastatin; Zinc oxide nanoparticles;

Gold nanoparticles enhance 5-fluorouracil anticancer efficacy against colorectal cancer cells by Mohamed A. Safwat; Ghareb M. Soliman; Douaa Sayed; Mohamed A. Attia (648-658).
Gold nanoparticles (GNPs) functionalized with two bifunctional ligands, thioglycolic acid (TGA) and glutathione (GSH) were successfully used as delivery system for 5-FU. 5-FU/GSH-GNPs achieved about 1.5-2-fold enhancement in 5-FU cytotoxic effect against colorectal cancer cells compared with free 5-FU.Display Omitted5-Fluorouracil (5-FU), an antimetabolite drug, is extensively used in the treatment solid tumors. However, its severe side effects limit its clinical benefits. To enhance 5-FU anticancer efficacy and reduce its side effects it was loaded onto gold nanoparticles (GNPs) using two thiol containing ligands, thioglycolic acid (TGA) and glutathione (GSH). The GNPs were prepared at different 5-FU/ligand molar ratios and evaluated using different techniques. Anticancer efficacy of 5-FU/GSH-GNPs was studied using flow cytometry in cancerous tissue obtained from patients having colorectal cancer. The GNPs were spherical in shape and had a size of ∼9–17 nm. Stability of the GNPs and drug release were studied as a function of salt concentration and solution pH. Maximum 5-FU loading was achieved at 5-FU/ligand molar ratio of 1:1 and 2:1 for TGA-GNPs and GSH-GNPs, respectively. GNPs coating with pluronic F127 improved their stability against salinity. 5-FU release from GNPs was slow and pH-dependent. 5-FU/GSH-GNPs induced apoptosis and stopped the cell cycle progression in colorectal cancer cells. They also had a 2-fold higher anticancer effect compared with free 5-FU. These results confirm the potential of GNPs to enhance 5-FU anticancer efficacy.
Keywords: Gold nanoparticles; Colorectal cancer; 5-Fluorouracil; Thioglycolic acid; Glutathione;

Adaptation of pharmaceutical excipients to FDM 3D printing for the fabrication of patient-tailored immediate release tablets by Muzna Sadia; Agata Sośnicka; Basel Arafat; Abdullah Isreb; Waqar Ahmed; Antonios Kelarakis; Mohamed A Alhnan (659-668).
Display OmittedThis work aims to employ fused deposition modelling 3D printing to fabricate immediate release pharmaceutical tablets with several model drugs. It investigates the addition of non-melting filler to methacrylic matrix to facilitate FDM 3D printing and explore the impact of (i) the nature of filler, (ii) compatibility with the gears of the 3D printer and iii) polymer: filler ratio on the 3D printing process. Amongst the investigated fillers in this work, directly compressible lactose, spray-dried lactose and microcrystalline cellulose showed a level of degradation at 135 °C whilst talc and TCP allowed consistent flow of the filament and a successful 3D printing of the tablet. A specially developed universal filament based on pharmaceutically approved methacrylic polymer (Eudragit EPO) and thermally stable filler, TCP (tribasic calcium phosphate) was optimised. Four model drugs with different physicochemical properties were included into ready-to-use mechanically stable tablets with immediate release properties. Following the two thermal processes (hot melt extrusion (HME) and fused deposition modelling (FDM) 3D printing), drug contents were 94.22%, 88.53%, 96.51% and 93.04% for 5-ASA, captopril, theophylline and prednisolone respectively. XRPD indicated that a fraction of 5-ASA, theophylline and prednisolone remained crystalline whilst captopril was in amorphous form. By combining the advantages of thermally stable pharmaceutically approved polymers and fillers, this unique approach provides a low cost production method for on demand manufacturing of individualised dosage forms.
Keywords: Fused filament fabrication; Personalized; Patient-specific; Three dimensional printing;

Reevaluation of the diametral compression test for tablets using the flattened disc geometry by V. Mazel; S. Guerard; B. Croquelois; J.B. Kopp; J. Girardot; H. Diarra; V. Busignies; P. Tchoreloff (669-677).
Display OmittedMechanical strength is an important critical quality attribute for tablets. It is classically measured, in the pharmaceutical field, using the diametral compression test. Nevertheless, due to small contact area between the tablet and the platens, some authors suggested that during the test, the failure could occur in tension away from the center which would invalidate the test and the calculation of the tensile strength. In this study, the flattened disc geometry was used as an alternative to avoid contact problems. The diametral compression on both flattened and standard geometries was first studied using finite element method (FEM) simulation. It was found that, for the flattened geometry, both maximum tensile strain and stress were located at the center of the tablet, which was not the case for the standard geometry. Experimental observations using digital image correlation (DIC) confirmed the numerical results. The experimental tensile strength obtained using both geometries were compared and it was found that the standard geometry always gave lower tensile strength than the flattened geometry. Finally, high-speed video capture of the test made it possible to detect that for the standard geometry the crack initiation was always away from the center of the tablet.
Keywords: Tablet; Diametral compression; Tensile strength; Compression; Numerical simulation;

Targeted release of transcription factors for cell reprogramming by a natural micro-syringe by Lionel Berthoin; Bertrand Toussaint; Frédéric Garban; Audrey Le Gouellec; Benjamin Caulier; Benoît Polack; David Laurin (678-687).
Display OmittedEctopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34+ hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34+ hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming.
Keywords: Type 3 secretion system; Pseudomonas aeruginosa; Embryonic transcription factors; Reprogramming cell fate; Induced pluripotent stem cells; Protein delivery systems;

Display OmittedThe objective of this work was to encapsulate terconazole (TCZ), a water insoluble antifungal drug, into novel ultradeformable bilosomes (UBs) for achieving enhanced ocular delivery. In addition to the constituents of the conventional bilosomes; namely, Span 60, cholesterol, and the bile salts, UBs contain an edge activator which imparts extra elasticity to the vesicles and consequently hypothesized to result in improved corneal permeation. In this study, TCZ loaded UBs were prepared utilizing ethanol injection method according to 23 full factorial design. The investigation of the influence of different formulation variables on UBs properties and selection of the optimum formulation was done using Design-Expert® software. The selected UBs formulation (UB1; containing 10 mg bile salt and 5 mg Cremophor EL as an edge activator) showed nanosized spherical vesicles (273.15 ± 2.90 nm) and high entrapment efficiency percent (95.47 ± 2.57%). Results also revealed that the optimum UBs formulation exhibited superior ex vivo drug flux through rabbit cornea when compared with conventional bilosomes, niosomes, and drug suspension. Furthermore, in vivo ocular tolerance and histopathological studies conducted using male albino rabbits proved the safety of the fabricated UBs after topical ocular application. Overall, the obtained results confirmed that UBs could be promising for ocular drug delivery.

Can Sophorolipids prevent biofilm formation on silicone catheter tubes? by Cristiana Pontes; Marta Alves; Catarina Santos; Maria H. Ribeiro; Lídia Gonçalves; Ana F. Bettencourt; Isabel A.C. Ribeiro (697-708).
Display OmittedGiven the impact of biofilms in health care environment and the increasing antibiotic resistance and/or tolerance, new strategies for preventing that occurrence in medical devices are obligatory. Thus, biomaterials surface functionalization with active compounds can be a valuable approach.In the present study the ability of the biosurfactants sophorolipids to prevent biofilms formation on silicone rubber aimed for medical catheters was investigated.Sophorolipids produced by Starmerella bombicola, identified by HPLC–MS/MS were used to cover silicone and surface characterization was evaluated through contact angle measurements and FTIR-ATR.Results revealed that sophorolipids presence on silicone surface decreased the hydrophobicity of the material and biofilm formation of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Antibiofilm activity was evaluated through different methods and was more pronounced against S. aureus. Furthermore, biocompatibility of silicone specimens with HaCaT cells was also obtained.From this study it was possible to conclude that sophorolipids seem to be a favourable approach for coating silicone catheters. Such compounds may represent a novel source of antibiofilm agents for technological development passing through strategies of permanent functionalization of surfaces.
Keywords: Biosurfactants; Sophorolipids; Antimicrobial; S. aureus; E. coli; Medical devices;

Comparison of breaking tests for the characterization of the interfacial strength of bilayer tablets by Luca Castrati; Vincent Mazel; Virginie Busignies; Harona Diarra; Alessandra Rossi; Paolo Colombo; Pierre Tchoreloff (709-716).
Display OmittedThe bilayer tableting technology is gaining more acceptance in the drug industry, due to its ability to improve the drug delivery strategies. It is currently assessed by the European Pharmacopoeia, that the mechanical strength of tablets can be evaluated using a diametral breaking tester. This device applies a force diametrically, and records the tablet breaking point. This approach has been used to measure the structural integrity of single layer tablets as well as bilayer (and multi-layer) tablets. The latter ones, however, have a much complex structure. Therefore, testing a bilayer tablet with the currently used breaking test methodology might not be appropriate.The aim of this work was to compare results from several tests that have been proposed to quantify the interfacial strength of bilayer tablets. The obtained results would provide an indication on which tests are appropriate to evaluate the robustness of a bilayer tablet.Bilayer tablets were fabricated using a model formulation: Microcrystalline Cellulose (MCC) for the first layer, and spray dried lactose (SDLac) as second layer.Each set of tablets were tested using the following tests: Diametral Test, Shear Test and Indentation Test.The tablets were examined before and after the breaking test using Scanning Electron Microscopy (SEM). When a bilayer tablet was subjected to shearing or indentation, it showed signs of clear delamination. Differently, using the diametral test system, the tablets showed no clear difference, before and after the testing. However, when examining each layer via SEM, it was clear that a fracture occurred in the layer made of SDLac. Thus, the diametral test is a measure of the strength of one of the two layers and therefore it is not suited to test the mechanical strength of bilayer tablets.
Keywords: Bilayer; Tablet; Diametral compression; Indentation; Shear; FEM; Breaking test;

Fabrication of non-dissolving analgesic suppositories using 3D printed moulds by Yuanyuan Sun; Xucong Ruan; Hairui Li; Himanshu Kathuria; Guang Du; Lifeng Kang (717-724).
Display OmittedConventional suppositories sometimes fail in exerting their therapeutic activity as the base materials melt inside body cavities. Also they are not suitable to provide long term treatment. Biomedical grade silicone elastomers may be used to fabricate non-dissolvable suppositories to overcome these disadvantages. We kneaded 4 analgesics into the 2 kinds of silicone polymers at 1%, 5% and 10% drug loading, respectively, to test their mechanical properties and drug release profiles. The optimized drug-polymer combinations were used to fabricate suppositories, and three dimensional printing (3DP) was used to create the suppository moulds. Subsequently, the drug release profiles and biocompatibility of the suppositories were studied. It was found that, the mechanical properties of the drug laden silicone elastomers and the rate of drug release from the elastomers can be tuned by varying drug-polymer combinations. The silicone elastomers containing 1% (w/w) and 5% (w/w) diclofenac sodium were the optimal formulations with prolonged drug release and biocompatibility at cellular level. These properties, together with complex geometries offered by 3DP technique, potentially made the non-dissolving suppositories promising therapeutic agents for personalized medicine.
Keywords: Non-dissolving suppository; Silicone elastomer; Sustained release; Three dimensional printing; Moulding; Analgesic;