International Journal of Pharmaceutics (v.522, #1-2)

Development of an inducible platform for intercellular protein delivery by Richard Siller; Eric Dufour; Max Lycke; Ian Wilmut; Yong-Wook Jung; In Hyun Park; Gareth J. Sullivan (1-10).
Display OmittedA challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.
Keywords: Poly-arginine; Cell penetrating peptides; Protein delivery; Cell therapy; Gene therapy; Stem cells; Cellular reprogramming;

Development of advanced biantibiotic loaded bone cement spacers for arthroplasty associated infections by F.J. Parra-Ruíz; A. González-Gómez; M. Fernández-Gutiérrez; J. Parra; J. García-García; G. Azuara; B. De la Torre; J. Buján; B. Ibarra; L. Duocastella-Codina; M. Molina-Crisol; B. Vázquez-Lasa; J. San Román (11-20).
Display OmittedThe incidence increase of infections in patients with hip or knee implants with resistant pathogens (mainly some S. coagulase-negative and gram positive bacteria) demands advanced antibiotic loaded formulations. In this paper, we report the design of new biantibiotic acrylic bone cements for in situ delivery. They include a last generation antibiotic (daptomycin or linezolid) in combination with vancomycin and are performed based on a novel modification of the Palacos R® acrylic bone cement, which is based on two components, a liquid (methyl methacrylate) and a solid (polymeric phase). Hence, the solid component of the experimental formulations include 45 wt% of microparticles of poly(D,L-lactic-co-glycolic) acid, 55 wt% of poly(methyl methacrylate) beads and supplements (10 wt-% each) of antibiotics. These formulations provide a selective and excellent control of the local release of antibiotics during a long time period (up to 2 months), avoiding systemic dissemination. The antimicrobial activity of the advanced spacers tested against S. aureus shows that single doses would be enough for the control of the infection. In vitro biocompatibility of cements on human osteoblasts is ensured. This paper is mainly focused on the preparation and characterization of cements and the studies of elution kinetics and bactericidal effects. Developed formulations are proposed as spacers for the treatment of infected arthroplasties, but also, they could be applied in other antibiotic devices to treat relevant bone-related infection diseases.
Keywords: Acrylic bone cement spacers; Daptomycin; Linezolid; Infection; S. aureus.;

Triphenylphosphonium-modified poly(ethylene glycol)-poly(ε-caprolactone) micelles for mitochondria- targeted gambogic acid delivery by Yingqi Xu; Shengpeng Wang; Hon Fai Chan; Yuling Liu; Hui Li; Chengwei He; Zeyong Li; Meiwan Chen (21-33).
Display OmittedMitochondria are important targets for the intracellular delivery of drugs and DNA. For mitochondria-targeted delivery, a mitochondriotropic molecule, triphenylphosphonium (TPP), was applied to the synthesis of amphiphilic TPP-poly(ethylene glycol)-poly(ε-caprolactone) (TPP-PEG-PCL) polymers. The TPP-PEG-PCL polymer was used to prepare micelles using a solvent evaporation method for the delivery of gambogic acid (GA) (GA-TPP). The micelles were obtained with a favorable particle size of 150.07 ± 11.71 nm and an encapsulation efficiency of 80.78 ± 1.36%, and they displayed homogeneous spherical shapes. The GA-TPP micelles exerted enhanced cytotoxic and pro-apoptotic effect against A549 cells compared to free GA and GA-loaded PEG-PCL (GA-PP) micelles, due to the inhibition of the expression of apoptosis-related proteins and promotion of caspase 3/7 and caspase 9 activity. Notably, the mitochondria-targeting GA-TPP micelles selectively accumulated in the mitochondria, inducing the loss of mitochondrial membrane potential and the release of cytochrome c, thereby achieving improved mitochondria-targeting effects. In conclusion, the GA-TPP micelle system shows great promise for lung cancer treatment by inducing an apoptotic effect via the mitochondrial signaling pathway.
Keywords: Gambogic acid; Polymeric micelles; Mitochondrial targeting; Lung cancer;

The application of novel nano-thermal and imaging techniques for monitoring drug microstructure and distribution within PLGA microspheres by Fan Yang; De Chen; Zhe-fei Guo; Yong-ming Zhang; Yi Liu; Sean Askin; Duncan Q.M. Craig; Min Zhao (34-49).
Display OmittedPoly (d,l-lactic-co-glycolic) acid (PLGA) based microspheres have been extensively used as controlled drug release systems. However, the burst effect has been a persistent issue associated with such systems, especially for those prepared by the double emulsion technique. An effective approach to preventing the burst effect and achieving a more ideal drug release profile is to improve the drug distribution within the polymeric matrix. Therefore, it is of great importance to establish a rapid and robust tool for screening and optimizing the drug distribution during pre-formulation. Transition Temperature Microscopy (TTM), a novel nano-thermal and imaging technique, is an extension of nano-thermal analysis (nano-TA) whereby a transition temperature is detected at a localized region of a sample and then designated a color based on a particular temperature/color palette, finally resulting in a coded map based on transition temperatures detected by carrying out a series of nanoTA measurements across the surface of the sample. In this study, we investigate the feasibility of applying the aforementioned technique combined with other thermal, imaging and structural techniques for monitoring the drug microstructure and spatial distribution within bovine serum albumin (BSA) loaded and nimodipine loaded PLGA microspheres, with a view to better predicting the in vitro drug release performance.
Keywords: Transition temperature microscopy; Microsphere; Bovine serum albumin; Nimodipine; Poly (d,l-lactic-co-glycolic) acid; Controlled drug release;

Evaluation of a dynamic dissolution/permeation model: Mutual influence of dissolution and barrier-flux under non-steady state conditions by Daniel Sironi; Mette Christensen; Jörg Rosenberg; Annette Bauer-Brandl; Martin Brandl (50-57).
Display OmittedCombined dissolution/permeation testing is gaining increasing attention as an in vitro tool for predictive performance ranking of enabling oral formulations. The current aim was to study how in vitro drug permeation evolves under conditions, where the donor concentration is changing (non-steady state). To this end, a model case was construed: compacts of pure crystalline hydrocortisone methanolate (HC·MeOH) of slow release rates were prepared, and their dissolution and permeation determined simultaneously in a side-by-side setup, separated by a biomimetic barrier (Permeapad®). This was compared to a corresponding setup for a suspension of micronized hydrocortisone (HC). The HC suspension showed constant dissolved HC concentration and constant flux across the barrier, representing the permeation-limited situation. For the HC·MeOH compacts, various dynamic scenarios were observed, where dissolution rate and flux influenced each other. Interestingly, for all the dynamic scenarios, the incremental flux values obtained correlated nicely with the corresponding actual donor concentrations. Furthermore, donor depletion was tested using a HC solution. The dynamic interplay between decrease in donor concentration (down to less than 10% of the initial concentration) and flux was studied. The experiences gained are discussed in terms of further developing combined dissolution/permeation setups.
Keywords: Hydrocortisone; Dissolution/permeation; Flux; Apparent permeability; Permeapad; Non-steady state;

Display OmittedMetastable crystals and the amorphous state of poorly water-soluble drugs in solid dispersions (SDs), are subject to a solid-liquid interface reaction upon exposure to a solvent. The dissolution behavior during the solid-liquid interface reaction often shows that the concentration of drugs is supersaturated, with a high initial drug concentration compared with the solubility of stable crystals but finally approaching the latter solubility with time. However, a method for measuring the precipitation rate of stable crystals and/or the potential solubility of metastable crystals or amorphous drugs has not been established. In this study, a novel mathematical model that can represent the dissolution behavior of the solid-liquid interface reaction for metastable crystals or amorphous drug was developed and its validity was evaluated. The theory for this model was based on the Noyes-Whitney equation and assumes that the precipitation of stable crystals at the solid-liquid interface occurs through a first-order reaction. Moreover, two models were developed, one assuming that the surface area of the drug remains constant because of the presence of excess drug in the bulk and the other that the surface area changes in time-dependency because of agglomeration of the drug. SDs of Ibuprofen (IB)/polyvinylpyrrolidone (PVP) were prepared and their dissolution behaviors under non-sink conditions were fitted by the models to evaluate improvements in solubility. The model assuming time-dependent surface area showed good agreement with experimental values. Furthermore, by applying the model to the dissolution profile, parameters such as the precipitation rate and the potential solubility of the amorphous drug were successfully calculated. In addition, it was shown that the improvement in solubility with supersaturation was able to be evaluated quantitatively using this model. Therefore, this mathematical model would be a useful tool to quantitatively determine the supersaturation concentration of a metastable drug from solid dispersions.
Keywords: Solid dispersions; Solid-liquid interfaces; Supersaturation; Mathematical model; Dissolution; Agglomeration; Ibuprofen; Polyvinylpyrrolidone;

Enhanced ocular efficacy of topically-delivered dorzolamide with nanostructured mucoadhesive microparticles by Chun Gwon Park; Young Kook Kim; Se-Na Kim; Seung Ho Lee; Beom Kang Huh; Min-A Park; Hyein Won; Ki Ho Park; Young Bin Choy (66-73).
Display OmittedDorzolamide eye drops are widely prescribed to reduce intraocular pressure (IOP) in the treatment of ocular hypertension and glaucoma. However, in an eye drop formulation, dorzolamide is rapidly cleared from the preocular space, hence requiring multiple daily administrations. Here, we sought to increase the preocular retention of dorzolamide using nanostructured, mucoadhesive microparticles (MUCO_NM) as carriers for topical delivery to the eye. MUCO_NM were prepared by freeze-milling dorzolamide-loaded, electrospun nanofibers composed of poly(lactic-co-glycolic acid) and polyethylene glycol. The microparticles were embedded in a rapidly-dissolving tablet of polyvinyl alcohol. To assess in vivo efficacy, the MUCO_NM were administered topically to the eyes of rabbits, and IOP was measured and compared to that in eyes treated with Trusopt®, a marketed eye drop of dorzolamide. The MUCO_NM showed a 35% greater maximum IOP decrease and a > 2-fold increase in the duration of the IOP decrease, compared to Trusopt®. This enhanced efficacy was comparable to that obtained with a single administration of 4 drops of Trusopt® or 2 administrations of Trusopt® at a 4-h interval. Our findings suggest that this MUCO_NM preparation is a promising carrier for topical delivery of dorzolamide to the eye, with enhanced drug efficacy and the potential to reduce administration frequency.
Keywords: Dorzolamide; Glaucoma; Intraocular pressure; Mucoadhesion; Microparticles; Nanostructures;

Amorphous is not always better—A dissolution study on solid state forms of carbamazepine by Linda G. Jensen; Frederik B. Skautrup; Anette Müllertz; Bertil Abrahamsson; Thomas Rades; Petra A. Priemel (74-79).
Display OmittedPoor aqueous solubility is a major concern for many new drugs. One possibility to overcome this issue is to formulate the drug as a high energy form, i.e. a metastable polymorph, an amorphous neat drug or a glass solution with polymers. In this study the dissolution properties of different solid state forms of carbamazepine, crystalline or amorphous drug, with or without either polyvinylpyrrolidone (PVP) or hydroxypropylmethylcellulose (HPMC) and glass solutions of the drug with both polymers (2:1, 4:1 and 10:1 (w/w) drug-to-polymer ratio) were tested with respect to their dissolution behaviour in a biorelevant gastric medium (for 30 min) and subsequently in intestinal conditions (for 2 h). Carbamazepine form III in the absence of polymer dissolved to a drug concentration of 540 μg/ml, but the concentration decreased after around 70 min due to precipitation of the dihydrate form, and reached 436 μg/ml after 2.5 h dissolution testing. The presence of PVP led to a similar dissolution profile with a slightly earlier onset of decrease in drug concentration, while in the presence of HPMC no decline in dissolved drug concentration was observed. Surprisingly, amorphous carbamazepine did not result in any supersaturation and the drug concentration was lower than that measured for crystalline carbamazepine. The addition of polymers further decreased the concentration of dissolved drug (290–310 μg/ml, depending on polymer type and concentration). Amorphous drug converted quickly into the dihydrate form and thus no supersaturation was achieved. Glass solutions of carbamazepine with PVP reached drug concentrations between 348 and 408 μg/ml after 2.5 h, i.e. lower than for the crystalline drug, whilst glass solutions with HPMC reached concentrations similar to the crystalline drug.
Keywords: Amorphous; Dihydrate; Dissolution; Supersaturation; Carbamazepine;

Display OmittedThe aim of this study was to evaluate the stability and solubility of the polymorphic forms of the ethenzamide (ET) – gentisic acid (GA) cocrystals during standard technological processes leading to tablet formation, such as compression and excipient addition. In this work two polymorphic forms of pharmaceutical cocrystals (ETGA) were characterized by 13C and 15N solid-state nuclear magnetic resonance and Fourier transformed infrared spectroscopy. Spectroscopic studies were supported by gauge including projector augmented wave (GIPAW) calculations of chemical shielding constants.Polymorphs of cocrystals were easily identified and characterized on the basis of solid-state spectroscopic studies. ETGA cocrystals behaviour during direct compressionand tabletting with excipient addition were tested. In order to choose the best tablet composition with suitable properties for the pharmaceutical industry dissolution profile studies of tablets containing polymorphic forms of cocrystals with selected excipients were carried out.
Keywords: Cocrystals; ssNMR; Polymorphs; Ethenzamide; Stability studies; Solubility;

Display OmittedThe diffusion dynamics of nanocarriers in the vitreous and the influence of nanocarrier physicochemical properties on these dynamics is an important aspect of the efficacy of intravitreal administered nanomedicines for the treatment of posterior segment eye diseases. Here we use fluorescence correlation spectroscopy (FCS) to determine liposome diffusion coefficients in the intact vitreous (DVit) of ex vivo porcine eyes using a modified Miyake-Apple technique to minimize the disruption of the vitreous fine structure. We chose to investigate whether the zeta potential of polyethylene glycol functionalized (i.e. PEGylated) liposomes altered liposome in situ diffusion dynamics in the vitreous. Non-PEGylated cationic nanocarriers have previously shown little to no diffusion in the vitreous, whilst neutral and anionic have shown diffusion. The liposomes investigated had diameters below 150 nm and zeta potentials ranging from −20 to +12 mV. We observed that PEGylated cationic liposomes had significantly lower DVit values (1.14 μm2s−1) than PEGylated neutral and anionic liposomes (2.78 and 2.87 μm2  s−1). However, PEGylated cationic liposomes had a similar biodistribution profile across the vitreous to the other systems. These results show that PEGylated cationic liposomes with limited cationic charge can diffuse across the vitreous and indicate that the vitreous as a barrier to nanocarriers (Ø < 500 nm) is more complicated than simply an electrostatic barrier as previously suggested.
Keywords: Ocular; Liposomes; Vitreous; Fluorescence; Diffusion;

Characterisation of aggregates of cyclodextrin-drug complexes using Taylor Dispersion Analysis by Hadar Zaman; Andrew G. Bright; Kevin Adams; David M. Goodall; Robert T. Forbes (98-109).
Display OmittedThere is a need to understand the nature of aggregation of cyclodextrins (CDs) with guest molecules in increasingly complex formulation systems. To this end an innovative application of Taylor dispersion analysis (TDA) and comparison with dynamic light scattering (DLS) have been carried out to probe the nature of ICT01-2588 (ICT-2588), a novel tumor-targeted vascular disrupting agent, in solvents including a potential buffered formulation containing 10% hydroxypropyl-β-cyclodextrin. The two hydrodynamic sizing techniques give measurement responses are that fundamentally different for aggregated solutions containing the target molecule, and the benefits of using TDA in conjunction with DLS are that systems are characterised through measurement of both mass- and z-average hydrodynamic radii. Whereas DLS measurements primarily resolve the large aggregates of ICT01-2588 in its formulation medium, methodology for TDA is described to determine the size and notably to quantify the proportion of monomers in the presence of large aggregates, and at the same time measure the formulation viscosity. Interestingly TDA and DLS have also distinguished between aggregate profiles formed using HP-β-CD samples from different suppliers. The approach is expected to be widely applicable to this important class of drug formulations where drug solubility is enhanced by cyclodextrin and other excipients.
Keywords: Taylor Dispersion Analysis; Peptide prodrug; Aggregation; Cyclodextrin; Solubility enhancement; Formulation;

Display OmittedGambogic acid (GA) is a potential anti-cancer agent with poor water-solubility, whereas heparin has anti-angiogenesis effects with good hydrophilicity. In this study, GA grafted low molecular weight heparin (GA-LMWH) was prepared and self-assembled into micelles in aqueous solution to improve the solubility and antitumor effects against hepatocellular carcinoma. The substitution of GA-LMWH is 27.5 ± 0.2%. The micelles had a mean size of 190.4 ± 10.8 nm, a low critical micelle concentration of 2.4 ± 0.2 μg mL−1, and the highest area under the concentration-time curve and mean retention time in the liver compared to the heart, spleen, lung and kidney (p <  0.05). The targeting efficiency of micelles to the liver is 2.1-times higher than that of the GA solution. GA-LMWH micelles were administered intravenously and significantly improved liver function, decreased cell lesions in hepatic tissue, inhibited the expression of CD105 and prolonged survival time of hepatocellular carcinoma model compared with groups treated with normal saline or GA solution. These results suggest that GA-LMWH micelles may improve anti-cancer effects by targeting the delivery of GA to the liver and enhancing the anti-angiogenesis effect.
Keywords: Low molecular weight heparin (PubChem CID: 772); Gambogic acid (PubChem CID: 9852185); Micelles; Ant-angiogenesis;

Sustained release ophthalmic dexamethasone: In vitro in vivo correlations derived from the PK-Eye by Sahar Awwad; Richard M. Day; Peng T. Khaw; Steve Brocchini; Hala M. Fadda (119-127).
Display OmittedCorticosteroids have long been used to treat intraocular inflammation by intravitreal injection. We describe dexamethasone loaded poly-DL-lactide-co-glycolide (PLGA) microparticles that were fabricated by thermally induced phase separation (TIPS). The dexamethasone loaded microparticles were evaluated using a two-compartment, in vitro aqueous outflow model of the eye (PK-Eye) that estimates drug clearance time from the back of the eye via aqueous outflow by the anterior route. A dexamethasone dose of 0.20 ± 0.02 mg in a 50 μL volume of TIPS microparticles resulted in a clearance t 1/2 of 9.6 ± 0.3 days using simulated vitreous in the PK-Eye. Since corticosteroids can also clear through the retina, it is necessary to account for clearance through the back of the eye. Retinal permeability data, published human ocular pharmacokinetics (PK) and the PK-Eye clearance times were then used to establish in vitro in vivo correlations (IVIVCs) for intraocular clearance times of corticosteroid formulations. A t 1/2 of 48 h was estimated for the dexamethasone-TIPS microparticles, which is almost 9 times longer than that reported for dexamethasone suspension in humans. The prediction of human clearance times of permeable molecules from the vitreous compartment can be determined by accounting for drug retinal permeation and determining the experimental clearance via the anterior aqueous outflow pathway using the PK-Eye.
Keywords: Ocular drug delivery; Pharmacokinetics; In vitro in vivo correlations; Sustained release; PLGA;

Display OmittedThe active tumor targeting ligands, hyaluronic acid (HA) and human serum albumin (HSA), are considered promising targeting moieties of drug carriers for cancer therapy. The chitosan nanoparticles loaded with methotrexate (MTX-CsNPs) were employed as the core for subsequent coating process. HA and HSA coating solutions were used at different concentrations. The effect of different HA Mw (1000, 360, 10 kDa) was also investigated. The coated MTX-CsNPs was characterized proving the success of surface functionalization. The antitumor activity of the prepared MTX-CsNPs was evaluated on MCF-7 breast cancer cell lines. Results showed that both 360 and 10 kDa HA allowed for successful HA adsorption, while its Mw and concentration determined negative charge density. HSA coating was accompanied by a slight increase in nanoparticles (NPs) size and a final positive surface charge. The in vitro cytotoxicity proved that HA and HSA coated MTX-CsNPs improved the antitumor activity compared to uncoated NPs and free drug.
Keywords: Chitosan; Nanoparticles; Hyaluronic acid; Human serum albumin; Methotrexate; In vitro cytotoxicity;

The aerodynamic dose emission characteristics of a fixed dose combination of a 200 μg budesonide and 6 μg formoterol Turbuhaler have been measured using methodology designed to use inhalation profiles of patients to identify the real life dose they would have inhaled. The results show that the dose emission characteristics were solely dependent on the peak inhalation flow (PIF) of each inhalation profile as shown in Figs. 1 and 2. The inhaled volume and acceleration of the inhalation flow had only a small, non-significant effect on dose emissions.The formoterol (Fig. 1) and budesonide (Fig. 2) fine particle dose (FPD) for the peak inhalation flow (PIF) of each inhalation profile.Display OmittedThe dose emitted from dry powder inhalers (DPI) is inhalation flow dependent and so varies with the peak inhalation flow (PIF) of a patient’s inhalation maneuver (IM). Dose emission could also be affected by other IM parameters-the inhaled volume (Vin) and the initial acceleration rate of the IM (ACIM). We have adapted the compendial method for in-vitro DPI determinations so that inhalation profiles replace the inhalation square profile generated by a vacuum pump. These real-life patient inhalation profiles were measured when 18 COPD patients inhaled through an empty placebo Symbicort® Turbuhaler®. They have been used to identify the dose emission characteristics from a fixed dosed combination of 200 μg budesonide plus 6 μg formoterol Turbuhaler®. To isolate each inhalation parameter some profiles were modified to provide a further 9 profiles to study the influence of Vin and 27 to identify the effect of ACIM. The fine particle dose, total emitted dose and mass median aerodynamic diameter were significantly (p < 0.05) influenced by PIF (p < 0.05) whereas ACIM and Vin had only a small effect. The results show the value of this ex-vivo methodology to provide an insight into the dose that each patient would have inhaled during real-life use.
Keywords: Turbuhaler®; Inhalation profiles; Peak inhalation flow; Fine particle dose; Total emitted dose; Mass median aerodynamic diameter;

Display OmittedVirus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34 nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.
Keywords: HBcAg; VLPs; Oral delivery; Proteins; Gastrointestinal fluids; Gastrointestinal stability;

Display OmittedThe aim of this research was to encapsulate ciprofloxacin, a broad spectrum fluoroquinolone antibiotic, into Span 60 based nano-elastic vesicles, nano-spanlastics, for accomplishing improved non invasive trans-tympanic delivery, providing means for ototopical treatment of acute otitis media (AOM). To achieve this purpose, ciprofloxacin-loaded nano-spanlastics were prepared by thin film hydration (TFH) technique, using several non-ionic edge activators (EAs) according to full factorial design (32). The investigation of the effect of formulation variables on nano-spanlastic characteristics and selection of the optimum formula were performed using Design-Expert® software. The selected formulation was also subjected to comparative ex-vivo permeation studies through tympanic membrane (TM) of rabbits. Results revealed that the optimal nano-spanlastic formulation (S-2; containing 20% Brij 35 as an EA) exhibited nano-sized spherical vesicles (287.55 ± 9.97 nm), relatively high entrapment efficiency percent (51.81 ± 1.57%), and good physical stability after six months of storage at 4–8 °C. Ex-vivo TM permeation studies demonstrated the superiority of the optimal nano-spanlastic formulation over the commercial Ciprocin® drops. However, when compared to lipid-based elastic vesicles, nano-transfersomes, nano-spanlastics exhibited lower drug permeation through the TM. Concisely, the obtained results suggested that nano-spanlastics can be promising for improving trans-tympanic delivery of ciprofloxacin.
Keywords: Span 60; Elastic vesicles; Thin film hydration; Tympanic membrane permeation; Hydrophilic edge activator;

Preparation and characterization of gastrointestinal wafer formulations by Kirsten Kirsch; Ulrike Hanke; Werner Weitschies (165-171).
Display OmittedMany active pharmaceutical ingredients (API) have a very poor or highly variable bioavailability after oral administration. One possibility to overcome this problem might be found in the application of mucoadhesive dosage forms like gastrointestinal wafers. However, a currently unsolved challenge is the control of the adhesion of the wafer to the intestinal mucus. One suggested solution might be the combination of gastrointestinal wafers and expanding systems. Such a combination requires thin and elastic wafers which are further characterized by an unidirectional drug release. In this study gastrointestinal, twolayered wafers containing a water-insoluble backing layer and a drug-loaded, mucoadhesive layer were fabricated by casting solvent technique. The backing layer consists of Ethocel™ Standard 10 Premium and the mucoadhesive layer was prepared using a mixture of Methocel™ E15 Premium LV, polyvinyl alcohol and Macrogol 400. The wafers were characterized regarding their appearance, mechanical properties and dissolution profiles as well as the influence of backing layer thickness on drug transfer and their ability of unidirectional drug release. The wafers with backing layer thickness of 500 μg Ethocel™/cm2 presented adequate mechanical properties, a drug transfer about 73% and unidirectional drug release.
Keywords: Gastrointestinal wafer; Mucoadhesion; Unidirectional drug release; Methocel™ E15LV;

Protein-based nanoparticles: From preparation to encapsulation of active molecules by Mohamad Tarhini; Hélène Greige-Gerges; Abdelhamid Elaissari (172-197).
Preparation methods of nanoparticles and proteins used with them. Red cases: animal protein, green cases: plant protein.▪Nowadays, nanotechnology has become very integrated in the domain of pharmaceutical sciences since nanoparticle dispersions show various advantages as drug carriers. Among nanoparticles, the protein-based ones are of paramount importance. In fact, protein nanoparticles show many advantages over other types of nanoparticles, they are often non-toxic and biodegradable. In this review, the most common preparation methods of protein nanoparticles were targeted. In addition, the factors affecting their dispersions and the concepts of drug loading and drug release are also highlighted. It was obvious that each method can be optimized for a given protein. This issue was discussed in depth in the light of the current state of art, and supported by evidences for each method from the literature. In addition, it was concluded that the processing parameters strongly affect the properties of nanoparticles dispersion.
Keywords: Nanoparticles; Protein; Encapsulation; Active molecules;

Display OmittedGene therapy bears great potential for the cure of a multitude of human diseases. Research efforts focussed on the use of viral delivery vectors in the past decades, neglecting non-viral gene therapies of physical or chemical origin due to low transfection efficiency. However, side effects such as activation of oncogenes and inflammatory reactions upon immune cell activation are major obstacles impeding the clinical applicability of viral gene therapy vectors. The aim of this study was the development of a non-viral gene delivery system based on plasmid-loaded human serum albumin nanoparticles, which are biocompatible, biodegradable, and non-toxic in relevant concentrations. The surface of said nanoparticles was modified with different cell penetrating peptides, namely Tat, nona-arginine R9, and the penetratin analogue EB1. We hypothesise that the surface modified nanoparticles can effectively enter HEK 293T cells based on the cell penetrating properties of the different peptides attached. A variety of inhibitors were used targeting distinct uptake pathways in an effort to understand the mechanisms utilized by the various cell penetrating peptides on the surface of the nanoparticles. A significant increase in transfection efficiency compared to free DNA or polyplexes was seen for these novel delivery vectors.
Keywords: Cell penetrating peptides; Human serum albumin (HSA); Plasmid DNA; Transfection; Cellular uptake; Cell transfection;

A pH-responsive glycolipid-like nanocarrier for optimising the time-dependent distribution of free chemical drugs in focal cells by Bolin Cheng; Binbin Lu; Xuan Liu; Tingting Meng; Yanan Tan; Yun Zhu; Na Liu; Hong Yuan; Xuan Huang; Fuqiang Hu (210-221).
Display OmittedThough Drug delivery systems have achieved accumulation at tumor sites via passive targeting and active targeting, the therapeutic effects are far from perfect. The unsatisfactory results are mainly due to limited drug release from the nanocarriers at tumor sites, while the pharmacological activities of the drug are attributed to the concentration of the free drug and the time maintained at the pharmacological targets. A pH-responsive chitosan based glycolipid-like nanocarrier (CSO-FBA-SA) was fabricated by conjugating stearyl alcohol (SA) to chitosan oligosaccharide (CSO) with the linkage of 4-formylbenzoic acid (FBA). FBA was a kind of aromatic aldehyde carbonyl compounds, which can form the benzoic-imine bond. In the presence of a Schiff’s base structure, the carrier showed improved properties and could be quickly degraded in an acidic environment. In order to explore the process and mechanism of the nanocarriers in focal cells, the method for determining the intracellular concentration of released free doxorubicin was established, and the time-dependent change of the DOX-loaded micelles was revealed. The sight of drug release was also obtained with CLSM. The cytotoxicity of the CSO-FBA-SA/DOX against human breast cancer MCF-7 cells increased by 2.75-fold and 3.77-fold in comparison with the CSO-SA/DOX and DOX, respectively. Furthermore, the CSO-FBA-SA/DOX showed a 2.12-fold higher cytotoxicity against the MCF-7 cells than that treated against human ovarian cancer SKOV-3 cells with lower intracellular pH value, which indicated that the cellular inhibition positively correlated with the intracellular pH value. High tumor accumulation and fast drug release of the CSO-FBA-SA/DOX in tumor was responsible for the remarkable tumor growth inhibitory effect. Moreover, the CSO-FBA-SA/DOX could selectively respond to the acidic environment and release DOX in tumor only, which had relatively minimal cytotoxicity towards normal tissues. The results showed that this newly developed glycolipid-like nanocarrier could act as a potential vector for delivering the drug effectively with a low systemic toxicity.
Keywords: PH-responsive; Glycolipid-like nanocarriers; Selectively drug release; Time-dependent distribution;

Quantitative prediction of ionization effect on human skin permeability by Hiromi Baba; Yusuke Ueno; Mitsuru Hashida; Fumiyoshi Yamashita (222-233).
Display OmittedAlthough skin permeability of an active ingredient can be severely affected by its ionization in a dose solution, most of the existing prediction models cannot predict such impacts. To provide reliable predictors, we curated a novel large dataset of in vitro human skin permeability coefficients for 322 entries comprising chemically diverse permeants whose ionization fractions can be calculated. Subsequently, we generated thousands of computational descriptors, including Log D (octanol–water distribution coefficient at a specific pH), and analyzed the dataset using nonlinear support vector regression (SVR) and Gaussian process regression (GPR) combined with greedy descriptor selection. The SVR model was slightly superior to the GPR model, with externally validated squared correlation coefficient, root mean square error, and mean absolute error values of 0.94, 0.29, and 0.21, respectively. These models indicate that Log D is effective for a comprehensive prediction of ionization effects on skin permeability. In addition, the proposed models satisfied the statistical criteria endorsed in recent model validation studies. These models can evaluate virtually generated compounds at any pH; therefore, they can be used for high-throughput evaluations of numerous active ingredients and optimization of their skin permeability with respect to permeant ionization.
Keywords: Skin permeability; Transdermal absorption; Ionization effect; Support vector regression; Gaussian process; QSPR;

Display OmittedThe overall objective of this work is to understand how excipient characteristics influence the process and product performance for a continuous twin-screw wet granulation process. The knowledge gained through this study is intended to be used for a Quality by Design (QbD)-based formulation design approach and formulation optimization. A total of 9 preferred fillers and 9 preferred binders were selected for this study. The selected fillers and binders were extensively characterized regarding their physico-chemical and solid state properties using 21 material characterization techniques. Subsequently, principal component analysis (PCA) was performed on the data sets of filler and binder characteristics in order to reduce the variety of single characteristics to a limited number of overarching properties. Four principal components (PC) explained 98.4% of the overall variability in the fillers data set, while three principal components explained 93.4% of the overall variability in the data set of binders. Both PCA models allowed in-depth evaluation of similarities and differences in the excipient properties.
Keywords: Excipient characterization; Principal component analysis (PCA); Multivariate data analysis; Quality by Design (QbD); Continuous twin-screw wet granulation;