International Journal of Pharmaceutics (v.519, #1-2)

Distinct biodistribution of doxorubicin and the altered dispositions mediated by different liposomal formulations by Ruijuan Luo; Yan Li; Miao He; Huixia Zhang; Hebao Yuan; Mark Johnson; Maria Palmisano; Simon Zhou; Duxin Sun (1-10).
Driving force for tissue distribution of doxorubicin solution (blue), Doxil (red) and Myocet (green) was distinct among three formulations. The difference in doxorubicin tissue distribution may suggest different toxicity risk vs. benefit of different liposomal doxorubicin formulations.Display OmittedThe liposomal formulations of doxorubicin produced distinct efficacy and toxicity profiles compared to doxorubicin solution in cancer patients. This study aims to investigate the drug tissue distribution and the driving force for tissue distribution from doxorubicin solution and two liposomal delivery systems, Doxil and Myocet. These three formulations were intravenously administered to mice at a single dose of 5 mg/kg. Eleven organs, plasma and blood were collected at different time points. Total doxorubicin concentrations in each specimen were measured with LC–MS/MS. Compared to doxorubicin solution, both Doxil and Myocet produced distinct doxorubicin tissue exposure in all 11 tissues. Interestingly, the tissue exposure by Myocet was drastically different from that of Doxil and showed a formulation-dependent pattern. Cmax of doxorubicin in heart tissue by Doxil and Myocet was approximately 60% and 50% respectively of that by doxorubicin solution. The predominant driving force for doxorubicin tissue distribution is liposomal-doxorubicin deposition for Doxil and free drug concentration for doxorubicin solution. For Myocet, the driving force for tissue distribution is predominately liposomal-doxorubicin deposition into tissues within the first 4 h; as the non-PEGylated doxorubicin liposomal decomposes, the driving force for tissue distribution is gradually switched to the released free doxorubicin. Unique tissue distributions are correlated with their toxicity profiles.
Keywords: Liposome; Doxorubicin; Tissue distribution; Drug delivery;

Layer-by-layer assembly of hierarchical nanoarchitectures to enhance the systemic performance of nanoparticle albumin-bound paclitaxel by Hima Bindu Ruttala; Thiruganesh Ramasamy; Beom Soo Shin; Han-Gon Choi; Chul Soon Yong; Jong Oh Kim (11-21).
Display OmittedAlthough protein-bound paclitaxel (PTX, Abraxane®) has been established as a standard PTX-based therapy against multiple cancers, its clinical success is limited by unfavorable pharmacokinetics, suboptimal biodistribution, and acute toxicities. In the present study, we aimed to apply the principles of a layer-by-layer (LbL) technique to improve the poor colloidal stability and pharmacokinetic pattern of nanoparticle albumin-bound paclitaxel (nab-PTX). LbL-based nab-PTX was successfully fabricated by the alternate deposition of polyarginine (pARG) and poly(ethylene glycol)-block-poly (L-aspartic acid) (PEG-b-PLD) onto an albumin conjugate. The presence of protective entanglement by polyamino acids prevented the dissociation of nab-PTX and improved its colloidal stability even at a 100-fold dilution. The combined effect of high nanoparticle internalization and controlled release of PTX from LbL-nab-PTX increased its cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cells. LbL-nab-PTX consistently induced apoptosis in approximately 52% and 22% of MCF-7 and MDA-MB-231 cancer cells, respectively. LbL assembly of polypeptides effectively prevented exposure of PTX to the systemic environment and thereby inhibited drug-induced hemolysis. Most importantly, LbL assembly of polypeptides to nab-PTX effectively increased the blood circulation potential of PTX and improved therapeutic efficacy via a significantly higher area under the curve (AUC)0–∞. We report for the first time the application of LbL functional architectures for improving the systemic performance of nab-PTX with a view toward its clinical translation for cancer therapy.
Keywords: Albumin; Nanoparticle; Colloidal stability; Systemic performance; Breast cancer;

Display OmittedRubbing a topical formulation on skin is generally assumed to enhance drug penetration. The aim of this study was to demonstrate different techniques such as using glass rod, rheometer, and gloved finger for rubbing a 2% salicylic acid gel on skin and investigate their effect on in vitro permeation of salicylic acid through dermatomed porcine ear skin. The studies included evaluation of the gel’s rheological properties, gel distribution on skin surface, in vitro permeability, drug distribution in skin, skin extraction recovery, and mass balance. Rubbing with a gloved finger resulted in a uniform gel layer with a thickness of 49.61 ± 15.33 μm on the skin surface. No significant difference between the different test groups was observed in terms of the cumulative amount of drug that permeated in 24 h (p >  0.05). Drug levels in stratum corneum, epidermis, and dermis were also analyzed. Rubbing with gloved finger delivered significantly higher amount of drug into the skin layers as compared to other test groups (p <  0.05). Amount of drug extracted from skin was reliably correlated to the actual drug levels in skin (R2  = 0.99). Considering drug amounts in different compartments, mass balance ranged from 75.86 ± 2.90% to 80.44 ± 2.99%.
Keywords: Salicylic acid; Rubbing; Permeation; Delivery; Skin extraction; Topical formulation;

Display OmittedLipid nanoparticles based on ionizable lipids have been clinically validated as a means of delivery for RNA interference (RNAi) therapeutics. The ideal properties of RNAi carriers are efficient delivery of oligonucleotides into target cells and rapid elimination after the function is performed. Here, we report that degradable lipid nanoparticles are effective carriers of small interfering RNA (siRNA) and have a high therapeutic index. The newly developed degradable lipid nanoparticles carrying siRNA showed potent gene-silencing activity in mouse hepatocytes (ED50  ≈ 0.02 mg/kg siRNA). The ester bond in the lipid tail was hydrolyzed in the liver, resulting in rapid metabolism of the lipid. Toxicity assays showed that the degradable lipid was well-tolerated at siRNA doses of up to 16 mg/kg in rats (over 800-fold higher than ED50). A single intravenous injection of siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) in cynomolgus monkeys resulted in more than 90% protein silencing, and a 50% decrease in plasma low-density lipoprotein (LDL) cholesterol, with a measurable reduction for 2 months. Moreover, quantification of lipids in liver biopsies revealed rapid hepatic clearance of the degradable lipid in nonhuman primates. These degradable lipid nanoparticles with a high therapeutic index hold promise for RNA-based treatments.
Keywords: Biodegradable; Drug delivery; Gene silencing; siRNA; Lipid nanoparticles;

A rational approach towards development of amorphous solid dispersions: Experimental and computational techniques by Paroma Chakravarty; Joseph W. Lubach; Jonathan Hau; Karthik Nagapudi (44-57).
Display OmittedThe purpose of this study was to determine the drug-polymer miscibility of GENE-A, a Genentech molecule, and hydroxypropyl methylcellulose-acetate succinate (HPMC-AS), a polymer, using computational and experimental approaches. The Flory-Huggins interaction parameter,χ, was obtained by calculating the solubility parameters for GENE-A and HPMC-AS over the temperature range of 25–100 °C to obtain the free energy of mixing at different drug loadings (0–100%) using the Materials Studio modeling and simulation platform (thermodynamic approach). Solid-state nuclear magnetic spectroscopy (ssNMR) was used to measure the proton relaxation times for both drug and polymer at different drug loadings (up to 60%) at RT (kinetic approach). Thermodynamically, the drug and polymer were predicted to show favorable mixing as indicated by a negative Gibbs free energy of mixing from 25 to 100 °C. ssNMR showed near identical relaxation times for both drug and polymer in the solid dispersion at RT and 40 °C for a period up to 6 months showing phase mixing between the API and polymer on <10 nm scale. Orthogonal computational and experimental approaches indicate phase mixing of the system components.
Keywords: Miscibility; Solid-state NMR; Molecular modeling; Solubility parameter; Amorphous solid dispersion; Polymer;

Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range by Maryam Shah; Zahra Rattray; Katie Day; Shahid Uddin; Robin Curtis; Christopher F. van der Walle; Alain Pluen (58-66).
Display OmittedCharacterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1–10 μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates’ generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development.
Keywords: Particle; Monoclonal antibody; Protein aggregation; Silicone-oil; Primary packaging; Raster image correlation spectroscopy;

Synergistic antimicrobial potential of essential oils in combination with nanoparticles: Emerging trends and future perspectives by Mahendra Rai; Priti Paralikar; Priti Jogee; Gauravi Agarkar; Avinash P. Ingle; Marcos Derita; Susana Zacchino (67-78).
Display OmittedThe development of resistance to different antimicrobial agents by bacteria, fungi, viruses, parasites, etc. is a great challenge to the medical field for the treatment of infections caused by them, and therefore, there is a pressing need to search for new and novel antimicrobials. The antimicrobial activity of essential oils and biogenic nanoparticles is well known. Recent studies have demonstrated that nanoparticles functionalized with essential oils have significant antimicrobial potential against multidrug- resistant pathogens. The aim of the present review is to discuss various studies on the broad-spectrum antimicrobial activity of essential oils used singly and in combination with nanoparticles. The brief explanation of their mechanism has also been discussed.
Keywords: Essential oils; Nanoparticles; Nanotechnology; Synergistic effect; Antimicrobial potential;

Exploring gastrointestinal variables affecting drug and formulation behavior: Methodologies, challenges and opportunities by Bart Hens; Maura Corsetti; Robin Spiller; Luca Marciani; Tim Vanuytsel; Jan Tack; Arjang Talattof; Gordon L. Amidon; Mirko Koziolek; Werner Weitschies; Clive G. Wilson; Roelof J. Bennink; Joachim Brouwers; Patrick Augustijns (79-97).
Display OmittedVarious gastrointestinal (GI) factors affect drug and formulation behavior after oral administration, including GI transfer, motility, pH and GI fluid volume and composition. An in-depth understanding of these physiological and anatomical variables is critical for a continued progress in oral drug development. In this review, different methodologies (invasive versus non-invasive) to explore the impact of physiological variables on formulation behavior in the human GI tract are presented, revealing their strengths and limitations. The techniques mentioned allow for an improved understanding of the role of following GI variables: gastric emptying (magnetic resonance imaging (MRI), scintigraphy, acetaminophen absorption technique, ultrasonography, breath test, intraluminal sampling and telemetry), motility (MRI, small intestinal/colonic manometry and telemetry), GI volume changes (MRI and ultrasonography), temperature (telemetry) and intraluminal pH (intraluminal sampling and telemetry).
Keywords: Intestinal absorption; MRI; Scintigraphy; Manometry; Telemetry; Intraluminal profiling;

Display OmittedPharmaceutical powders are mainly organic materials and are likely to be charged due to repeated inter-particle and particle-wall contacts during industrial processes. This study experimentally investigated the effect of moisture content (ranging from approximately 1.8 to 30 wt.%) on tribocharging behaviour of pharmaceutical granules, as well as their apparent volume resistivity. The tribocharging behaviour of pharmaceutical granules was investigated using a rotating device and apparent volume resistivity was measured in a conventional volume resistivity test cell. Additional measurements were performed on individual ingredients, each having the same moisture content as that of the granules, in order to investigate the effect of each single ingredient on the apparent volume resistivity of granules. In this work, the individual ingredients used for granules were: α-Lactose Monohydrate (α-LMH), Microcrystalline Cellulose (MCC), Hydroxypropyl Methylcellulose (HPMC), and Croscarmellose Sodium (CCS). The results showed that the specific charge of granules began to increase at the moisture contents below 5 wt.%, which can be referred as critical moisture content of granules. The apparent volume resistivity showed the same behaviour, indicating that the specific charge could be due to an increase in apparent volume resistivity of granules at reduced moisture content. Finally, it was shown that the apparent volume resistivity measured for granules was mainly affected by that of the α-LMH, the major component of granules accounting for 40 wt.%.
Keywords: Tribocharging; Volume resistivity; Moisture content; Pharmaceutical granules;

Display OmittedEGF fragment (EGFfr) and doxorubicin were chemically co-decorated on single magnetic nanoparticles (MNPs) for concomitant cancer targeting and treatment. Magnetic nanoparticles were prepared by the precipitation of ferric chloride hydrates in an ammonia solution and subsequent surface-functionalization with amines. The terminal thiol group of the EGF fragment was first conjugated to surface amines of the MNPs using a heterofunctional crosslinker, and doxorubicin was sequentially conjugated to the MNPs via a hydrazone linker, where the degree of subsitution of the surface amines to EGFfr was varied from 1% to 40%. The decorated doxorubicin showed clear pH-dependency in the release profile, and doxorubicin showed fast release at pH 5.0 in comparison to pH 7.4. The EGF-decorated MNPs were tested for differential cellular uptakes against EGF overexpressing cells (A549), and the uptake levels gradually increased to 10% and saturated, which was quantified by ICP-OES. Internalized doxorubicin was also visualized by confocal microscopy, and A549 cells with EGF-decorated MNPs with EGF decoration showed higher fluorescence intensity of doxorubicin than those with non-decorated MNPs. Anti-cancer activity of the MNPs was compared at various concentrations of doxorubicin and EGFfr. Decoration of EGFfr significantly increased the anti-cancer activity of doxorubicin-incorporated MNPs in A549 cells; however, EGFfr alone did not affect the viability of the cells. Thus, we concluded that MNPs with optimized EGFfr and doxorubicin ratios showed higher targeting and drug payload against EGF receptor overexpressing cancer cells.
Keywords: Epidermal growth factor; Doxoruicin; Anticancer; pH-responsive; Nanoparticle;

Gold nanostar–polymer hybrids for siRNA delivery: Polymer design towards colloidal stability and in vitro studies on breast cancer cells by Carla Sardo; Barbara Bassi; Emanuela F. Craparo; Cinzia Scialabba; Elisa Cabrini; Giacomo Dacarro; Agnese D’Agostino; Angelo Taglietti; Gaetano Giammona; Piersandro Pallavicini; Gennara Cavallaro (113-124).
Display OmittedTo overcome the low bioavailability of siRNA (small interfering RNA) and to improve their transfection efficiency, the use of non-viral delivery carriers is today a feasible approach to transform the discovery of these incredibly potent and versatile drugs into clinical practice. Polymer-modified gold nanoconstructs (AuNCs) are currently viewed as efficient and safe intracellular delivery carriers for siRNA, as they have the possibility to conjugate the ability to stably entrap and deliver siRNAs inside cells with the advantages of gold nanoparticles, which can act as theranostic agents and radiotherapy enhancers through laser-induced hyperthermia.In this study, AuNCs were prepared by coating Gold Nano Stars (GNS) with suitable functionalised polymers, to give new insight on the choice of the coating in order to obtain colloidal stability, satisfying in vitro transfection behaviour and reliability in terms of homogeneous results upon GNS type changing. For this goal, GNS synthesized with three different sizes and shapes were coated with two different polymers: i) α-mercapto-ω-amino polyethylene glycol 3000 Da (SH-PEG3000-NH2), a hydrophilic linear polymer; ii) PHEA-PEG2000-EDA-LA (PPE-LA), an amphiphilic hydroxyethylaspartamide copolymer containing a PEG moiety. Both polymers contain ―SH or ―SS― groups for anchoring on gold surface and NH2 groups, which can be protonated in order to obtain a positive surface for successive siRNA layering. The effect of the features of the coating polymers on siRNA layering, and the extent of intracellular uptake and luciferase gene silencing effect were evaluated for each of the obtained coated GNS. The results highlight that amphiphilic biocompatible polymers with multi-grafting function are more suitable for ensuring the colloidal stability and the effectiveness of these colloidal systems, compared to the coating with linear PEG.
Keywords: PHEA; PEG; Lipoic acid; Gold nanostars; siRNA delivery; MCF-7;

Display OmittedAlthough the intestinal microbial community is still incompletely understood, there is strong evidence of the benefits of using probiotics to address some medical states or conditions. As a result, the probiotics oral supplements market has exploded during the last few years. However, while their sensitivity to gastric juices, acidic pH and bile is well known, most of these oral forms would not guarantee any survival of the strains in such conditions. In this work, we have studied the resistance to simulated gastric juices of several commercially available probiotics products. These included sixteen strains and ten oral forms such as enteric/non-enteric capsules/tablets and microencapsulated strains.Results demonstrated that all tested strains showed high sensitivity to acidic conditions and suggested that most of these microorganisms would not show any viability when immersed in the stomach at fasting. Most probiotics oral forms did not provide any protection to strains, unless these forms presented strong enteric protection. Consequently, the efficacy of non-enteric products to fully provide to the patient the benefits related to the consumption of probiotics supplement would be strongly questionable. This study underlines the chasm between the current opinion about probiotics protection needs and the products proposed by many companies in the dietary supplements area.
Keywords: Probiotics; Oral forms; Enteric; Gastric conditions; Supplements;

Docosahexaenoic acid loaded lipid nanoparticles with bactericidal activity against Helicobacter pylori by Catarina Leal Seabra; Cláudia Nunes; Maria Gomez-Lazaro; Marta Correia; José Carlos Machado; Inês C. Gonçalves; Celso A. Reis; Salette Reis; M. Cristina L. Martins (128-137).
Display OmittedDocosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid present in fish oil, has been described as a promising molecule to the treatment of Helicobacter pylori gastric infection. However, due to its highly unsaturated structure, DHA can be easily oxidized loosing part of its bioactivity. This work aims the nanoencapsulation of DHA to improve its bactericidal efficacy against H. pylori. DHA was loaded into nanostructured lipid carriers (NLC) produced by hot homogenization and ultrasonication using a blend of lipids (Precirol ATO5®, Miglyol-812®) and a surfactant (Tween 60®). Homogeneous NLC with 302 ± 14 nm diameter, −28 ± 3 mV surface charge (dynamic and electrophoretic light scattering) and containing 66 ± 7% DHA (UV/VIS spectroscopy) were successfully produced.Bacterial growth curves, performed over 24 h in the presence of different DHA concentrations (free or loaded into NLC), demonstrated that nanoencapsulation enhanced DHA bactericidal effect, since DHA-loaded NLC were able to inhibit H. pylori growth in a much lower concentrations (25 μM) than free DHA (>100 μM).Bioimaging studies, using scanning and transmission electron microscopy and also imaging flow cytometry, demonstrated that DHA-loaded NLC interact with H. pylori membrane, increasing their periplasmic space and disrupting membrane and allowing the leakage of cytoplasmic content. Furthermore, the developed nanoparticles are not cytotoxic to human gastric adenocarcinoma cells at bactericidal concentrations. DHA-loaded NLC should, therefore, be envisaged as an alternative to the current treatments for H. pylori infection.
Keywords: Docosahexaenoic acid (DHA); Helicobacter pylori; Nanostructured lipid carriers (NLC); Polyunsaturated fatty acid (PUFA);

In vivo pharmacokinetic studies and intracellular delivery of methotrexate by means of glycine-tethered PLGA-based polymeric micelles by Madhwi; Rajendra Kumar; Pramod Kumar; Bhupinder Singh; Gajanand Sharma; Om Prakash Katare; Kaisar Raza (138-144).
Display OmittedMethotrexate (MTX) is a widely used drug for the management of various kinds of cancers. However, numerous challenges are associated with MTX like poor aqueous solubility, dose-dependent side effects and poor-bioavailability. With an aim to explore the potential benefits in drug delivery of MTX, it was intended to fabricate glycine-PLGA-based polymeric micelles. Glycine was chemically linked to PLGA and the linkage was confirmed by FT-IR, and NMR-Spectroscopy. The developed polymeric micelles offered substantial loading to MTX with a pH-dependent drug release profile. The drug was released maximally at the cancer cell pH vis-à-vis blood plasma pH. The cytotoxicity of drug against MDA-MB-231 cell lines was enhanced by approx. 100% and the confocal laser scanning microscopy confirmed the localization of dye-tagged nanocarriers in the interiors of cancer cells. The bioavailable fraction of the drug was increased by approx. 4-folds, whereas elimination half-life was enhanced by around two-folds in Wistar rats. The novel approach offers a biodegradable and promising carrier for the better delivery of anticancer agents with immense promises of efficacy enhancement, improved delivery and better pharmacokinetic profile.
Keywords: Cytotoxicity; Bioavailability; MDA-MB-231 cells; Protein binding; Glycine; Nanoparticles; Drug delivery;

Display OmittedVitamin E refers to a group of saturated tocopherol (T) isomers and the biologically more active unsaturated tocotrienol (T3) isomers. PEGylated α-tocopherol, commercially known as Vitamin E TPGS, has been used as an emulsifier and therapeutic agent for children with vitamin E deficiency. Limited information, however, is available about the PEG conjugates of the tocotrienol isomers of vitamin E. The current work was therefore undertaken to synthesize and characterize the water soluble polyethylene glycol (PEG 350 and 1000) derivatives of T and T3. Yield and the identity of the synthesized products were confirmed by 1H NMR, mass spectroscopy, HPLC, and thermal analysis. The self-assembly of the PEGylated vitamin E isomers in water at critical micelle concentrations (CMC) was further confirmed by size, zeta, and Cryo-TEM image analysis. While stable at pH 7.4, PEG conjugates were found to rapidly hydrolyze at pH 1.2. Our data showed that PEGylated T3 isomers were significantly more active as inhibitors for P-glycoprotein than PEGylated T. The in vitro cytotoxicity of the conjugates was also tested against a large panel of normal and tumorigenic cells. Of the conjugates, γ-T3PGS 1000 and δ-T3PGS 1000 were found to have the least toxicity against non-tumorigenic breast and pancreatic cell lines, which may be advantageous for its use as functional excipients in drug delivery. The results from the current work have demonstrated the feasibility of synthesizing PEGylated conjugates of vitamin E isomers and highlighted the potential use of these conjugates in drug delivery as functional and safer excipients especially for γ-T3PGS 1000 and δ-T3PGS 1000 conjugate.
Keywords: Vitamin E; Vitamin E TPGS; Tocopherol; Tocotrienols; PEGylation; Characterization; Functional excipient; Anti-tumor activity;

The importance of the molecular weight of ethyl cellulose on the properties of aqueous-based controlled release coatings by Jurgita Kazlauske; Sofie Gårdebjer; Sara Almer; Anette Larsson (157-164).
Display OmittedPrevious investigations of aqueous based ethyl cellulose (EC) latex dispersions have mainly focused on the commercially available viscosity grade 20 cps. In this study, dispersions of EC with varying viscosity grades (which correspond to molecular weights), ranging from 4 to 100 cps, were produced and characterised. The dispersions showed particle sizes around 200 nm and highly negative ζ-potentials (approx. −100 mV), which indicated stable dispersions as confirmed by sedimentation studies. The different latexes were used to produce free-standing film coatings. We hypothesised that the different viscosity grades of EC would result in different properties of the films. We found that an increase in viscosity grade (and higher molecular weight) resulted in lower coalescence between the particles during film formation and thus to higher water permeability than in film coatings of lower molecular weight. After exposure to water the EC 4 cps and 20 cps film coatings had a more porous structure in the side facing the air during production and drying after immersion in water. Molecular weight is therefore a factor that should be considered when producing pharmaceutical coatings for controlled release.
Keywords: Ethylcellulose; Film coatings; Latex; Coalescence; Water permeability;

Use of the channel fill level in defining a design space for twin screw wet granulation by L.J. Gorringe; G.S. Kee; M.F. Saleh; N.H. Fa; R.G. Elkes (165-177).
Display OmittedTwin screw wet granulation is a key process in the continuous manufacture of oral solid dosage forms. Previous research has qualitatively suggested that the channel fill level influences the granules produced. In this paper a quantitative measure of the total volumetric fraction of the conveying element channels of the screw filled with powder (φ) was used. Experimental results are shown which demonstrate that very similar particle size distributions can be obtained at the same φ with the same material and screw configuration but radically different solids feed rates and screw speeds. Morphology of the granules also correlates with φ. This is consistent with previous observations in the literature correlating granule attributes with powder feed rate and screw speed but also considers the two parameters in combination. A process design space approach based on φ is proposed. This can be determined empirically, and potentially has value in setting process control strategies, assuring process robustness and allowing process flexibility during the product lifecycle.
Keywords: Continuous manufacturing; Channel fill; Twin screw granulation; Design space; Residence time;

Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients by Andreia Almeida; Bruno Sarmento; Francisca Rodrigues (178-185).
Display OmittedAccording to the current European legislation, the safety assessment of each individual cosmetic ingredient of any formulation is the basis for the safety evaluation of a cosmetic product. Also, animal testing in the European Union is prohibited for cosmetic ingredients and products since 2004 and 2009, respectively. Additionally, the commercialization of any cosmetic products containing ingredients tested on animal models was forbidden in 2009. In consequence of these boundaries, the European Centre for the Validation of Alternative Methods (ECVAM) proposes a list of validated cell-based in vitro models for predicting the safety and toxicity of cosmetic ingredients. These models have been demonstrated as valuable and effective tools to overcome the limitations of animal in vivo studies. Although the use of in vitro cell-based models for the evaluation of absorption and permeability of cosmetic ingredients is widespread, a detailed study on the properties of these platforms and the in vitro-in vivo correlation compared with human data are required. Moreover, additional efforts must be taken to develop in vitro models to predict carcinogenicity, repeat dose toxicity and reproductive toxicity, for which no alternative in vitro methods are currently available. This review paper summarizes and characterizes the most relevant in vitro models validated by ECVAM employed to predict the safety and toxicology of cosmetic ingredients.
Keywords: Cosmetics ingredients; Animal alternatives; In vitro models; Safety evaluation; Toxicity;

Coupling 3D printing with hot-melt extrusion to produce controlled-release tablets by Jiaxiang Zhang; Xin Feng; Hemlata Patil; Roshan V. Tiwari; Michael A. Repka (186-197).
Display OmittedThe main objective of this work was to explore the potential of coupling fused deposition modeling in three-dimensional (3D) printing with hot-melt extrusion (HME) technology to facilitate additive manufacturing, in order to fabricate tablets with enhanced extended release properties. Acetaminophen was used as the model drug and different grades and ratios of polymers were used to formulate tablets. Three-point bending and hardness tests were performed to determine the mechanical properties of the filaments and tablets. 3D-printed tablets, directly compressed mill-extruded tablets, and tablets prepared from a physical mixture were evaluated for drug release rates using a USP-II dissolution apparatus. The surface and cross-sectional morphology of the 3D-printed tablets were assessed by scanning electron microscopy. Differential scanning calorimetry and thermogravimetric analysis were used to characterize the crystal states and thermal properties of materials, respectively. The 3D-printed tablets had smooth surfaces and tight structures; therefore, they showed better extended drug release rates than the directly compressed tablets did. Further, this study clearly demonstrated the feasibility of coupling HME with 3D printing technology, which allows for the formulation of drug delivery systems using different grades and ratios of pharmaceutical polymers. In addition, formulations can be made based on the personal needs of patients.
Keywords: Hot-melt extrusion; 3D printing; Solid dispersion; Extended release; Acetaminophen; 3-Point bend test;

Distribution of phospholipid based formulations in the skin investigated by combined ATR-FTIR and tape stripping experiments by Martin Wolf; Maria Halper; Raffaela Pribyl; Dieter Baurecht; Claudia Valenta (198-205).
Display OmittedThe spatial distribution of exogenous substances in the stratum corneum (SC) could have an influence on their skin irritation potential. In this study it was possible to monitor the distribution of phospholipids with their phosphatidylcholine scaffold on porcine ear skin by combining tape stripping and in vitro ATR-FTIR spectroscopy. Significant vibrational modes in the spectra could be successfully assigned to the functional groups of the molecules. Thus it was possible to track the phospholipids without the need of their deuterated form by calculating difference spectra from the treated – untreated skin samples. The correlation between four characteristic bands (R2  ≥ 0.9909) revealed the excellent suitability of this semi-quantitative method for deep profiling analysis. The penetration capabilities of aqueous suspensions of the different phospholipid compositions as well as two monoacyl-phosphatidylcholine based liposome formulations were investigated using this method. Nevertheless, differences in the distribution of the investigated phospholipid species, having different amounts of monoacyl-phosphatidylcholine, could not be found. It could be clearly shown that the deepest skin penetration was seen in the irritating anionic SDS (sodium dodecyl sulfate) out of the aqueous solution. The aqueous suspensions based on different phospholipid surfactants showed the same range of penetration depth (10–15% of SC), whereas the smallest skin penetration depth was observed after the application of liposomal formulations.
Keywords: ATR-FTIR; Phospholipids; Tape-stripping; Undeuterated; Irritancy; Skin;

Improved mucoadhesive properties of self-nanoemulsifying drug delivery systems (SNEDDS) by introducing acyl chitosan by Nuri Ari Efiana; Arshad Mahmood; Hung Thanh Lam; Ožbej Zupančič; Gintare Leonaviciute; Andreas Bernkop-Schnürch (206-212).
Display OmittedThis study was aimed to improve the mucoadhesive properties of SNEDDS by the incorporation of acyl chitosan including octanoyl chitosan (OC), lauroyl chitosan (LC) and palmitoyl chitosan (PC). SNEDDS and acyl chitosan SNEDDS were characterized regarding droplet size and zeta potential. Their mucoadhesivity on porcine intestinal mucosa was evaluated by falling liquid film technique using Sudan Red G as marker. Degree of substitution of chitosan was determined to be 52.8%, 64.8 and 48.5% for OC, LC and PC, respectively. SNEDDS and acyl chitosan SNEDDS displayed a droplet size less than 50 nm and 80–300 nm as well as a zeta potential of −0.2 to −1.6 and 0.05 to 0.99 mV, respectively. Introducing 2% acyl chitosan into SNEDDS increased the residence time of SNEDDS on intestinal mucosa 2-fold. It is concluded that due to the incorporation of acyl chitosan into SNEDDS, their mucoadhesive properties can be increased.
Keywords: Acyl chitosan; Fatty acid chloride; Mucoadhesion; SNEDDS;

Amorphous solid dispersion of cyclosporine A prepared with fine droplet drying process: Physicochemical and pharmacokinetic characterization by Hiroki Suzuki; Tatsuru Moritani; Tadahiko Morinaga; Yoshiki Seto; Hideyuki Sato; Satomi Onoue (213-219).
Display OmittedThe present study aimed to develop an amorphous solid dispersion (ASD) of cyclosporine A (CsA) by a fine droplet drying (FDD) process for improvement in oral absorption of CsA. CsA and hydroxypropyl cellulose-SSL were dissolved in 1,4-dioxane, and the solution was powdered by the FDD process to obtain the ASD formulation of CsA (ASD/CsA). The ASD/CsA was characterized in terms of morphology, particle size distribution, crystallinity, dissolution behavior, physicochemical stability, and pharmacokinetic behavior in rats. The ASD/CsA was obtained in the form of uniform spherical particles, and the span factor was calculated to be ca. 0.4. CsA in the formulation existed in an amorphous state. The ASD/CsA exhibited a higher dissolution behavior of CsA than amorphous CsA, whereas storage of the ASD/CsA under accelerated conditions led to impairment in the dissolution behavior. The constant release of CsA from non-aged ASD/CsA was observed during dissolution testing. After oral administration of CsA samples (10 mg-CsA/kg) in rats, the ASD/CsA showed a high and sustained plasma concentration of CsA as evidenced by a 18-fold increase in the oral bioavailability of CsA compared with amorphous CsA. From these findings, the FDD process might be an efficacious option for the ASD formulation of CsA with enhanced biopharmaceutics properties.
Keywords: Amorphous solid dispersion; Cyclosporine A; Dissolution; Fine droplet drying process; Oral absorption;

Simulation and evaluation of rupturable coated capsules by finite element method by Yan Yang; Jie Fang; Lian Shen; Weiguang Shan (220-229).
Display OmittedThe objective of this study was to simulate and evaluate the burst behavior of rupturable coated capsules by finite element method (FEM). Film and coated capsules were prepared by dip-coating method and their dimensions were determined by stereomicroscope. Mechanical properties of the film were measured by tensile test and used as material properties of FEM models. Swelling pressure was determined by restrained expansion method and applied to the internal surface of FEM models. Water uptake of coated capsules was determined to study the formation of internal pressure. Burst test and in vitro dissolution was used to verify the FEM models, which were used to study and predict the coating burst behavior. Simulated results of coating burst behavior were well agreed with the experiment results. Swelling pressure, material properties and dimensions of coating had influence on the maximum stress. Burst pressure and critical L-HPC content were calculated for burst prediction and formulation optimization. FEM simulation was a feasible way to simulate and evaluate the burst behavior of coated capsules.
Keywords: Coated capsules; Finite element method; Mechanical properties; Swelling pressure; Burst behavior; Pulsatile drug release;

A novel methodology to study polymodal particle size distributions produced during continuous wet granulation by Carlota Mendez Torrecillas; Gavin W. Halbert; Dimitrios A. Lamprou (230-239).
Display OmittedIt is important during powder granulation to obtain particles of a homogeneous size especially in critical situations such as pharmaceutical manufacture. To date, homogeneity of particle size distribution has been defined by the use of the d50 combined with the span of the particle size distribution, which has been found ineffective for polymodal particle size distributions. This work focuses on demonstrating the limitations of the span parameter to quantify homogeneity and proposes a novel improved metric based on the transformation of a typical particle size distribution curve into a homogeneity factor which can vary from 0 to 100%. The potential of this method as a characterisation tool has been demonstrated through its application to the production of granules using two different materials. The workspace of an 11 mm twin screw granulator was defined for two common excipients (α-lactose monohydrate and microcrystalline cellulose). Homogeneity of the obtained granules varied dramatically from 0 to 95% in the same workspace, allowing identification of critical process parameters (e.g. feed rate, liquid/solid ratio, torque velocities). In addition it defined the operational conditions required to produce the most homogeneous product within the range 5 μm–2.2 mm from both materials.
Keywords: Twin screw granulation; Particle size distribution; Homogeneity factor; Quality by design; Granules;

Aerosolizable gold nano-in-micro dry powder formulations for theragnosis and lung delivery by A. Sofia Silva; Ana M. Sousa; Renato P. Cabral; Marta C. Silva; Clarinda Costa; Sónia P. Miguel; Vasco D.B. Bonifácio; Teresa Casimiro; Ilídio J. Correia; Ana Aguiar-Ricardo (240-249).
Display Omitted Nano-in-micro dry powder formulations were designed and prepared for delivery and release of theragnostic gold nanoprobes (AuNPs) to lung cancer cells. Respirable POxylated luminescent nanoparticles were micronized using chitosan through a lab scale supercritical CO2-assisted spray drying facility. Adequate aerodynamic performance, effective transport and adhesion to deep lung were also demonstrated.Functionalized gold nanoparticles (AuNPs) have been widely investigated as promising multifunctional nanosystems for the theragnosis of lung cancer, the most common and prominent cause of cancer death worldwide. Nevertheless, nanoparticles are not in appropriate sizes for an accurate deep lung delivery and the lack of locally and effective delivery of therapeutic biomolecules to the deep lungs is, in fact, the major cause of low therapeutic outcome. Herein we incorporate, for the first time, AuNPs into respirable microparticles. AuNPs were functionalized with biocompatible oligo(2-oxazoline)-based optically stable fluorescent coatings, and conjugated with a laminin peptide (YIGSR) for targeted lung cancer delivery. These POxylated AuNPs were then incorporated into a chitosan matrix by a clean process, supercritical CO2-assisted spray drying (SASD), yielding nano-in-micro clean ultrafine dry powder formulations. The engineered formulations present the adequate morphology and flowability to reach the deep lung, with aerodynamic sizes ranging 3.2–3.8 μm, and excellent fine particle fraction (FPF) (FPF of 47% for CHT-bearing targeted AuNPs). The optimal biodegradation and release profiles enabled a sustained and controlled release of the embedded nanoparticles, with enhanced cellular uptake, opening new prospects for future lung theragnosis.
Keywords: Pulmonary delivery; Gold nanoparticles; Nano-in-micro; Supercritical CO2; Spray drying; Dry powder formulation; Lung cancer;

RGD conjugated liposome-hollow silica hybrid nanovehicles for targeted and controlled delivery of arsenic trioxide against hepatic carcinoma by Weidong Fei; Yan Zhang; Shunping Han; Jiaoyang Tao; Hongyue Zheng; Yinghui Wei; Jiazhen Zhu; Fanzhu Li; Xuanshen Wang (250-262).
To construct a targeted and controlled arsenic trioxide (ATO, a poor pharmacokinetic drug with dose-limited toxicity) delivery core-shell nanovehicle which combined the features of hollow mesoporous silica nanoparticles (HMSN) and functional liposome (named as RGD-LP-CHMSN-ATO). The chlorodimethyloctadecylsilane was modified on the surface of HMSN for supporting phospholipid bilayer which enhanced the stability of nanoparticles and increased the affinity between nanoparticles and cell membrane. Improved PK features and significant hepatic tumor-specific distribution of ATO were achieved by RGD-LP-CHMSN-ATO. The antitumor efficacy in vitro (HepG2 cell line) and in vivo (H22 xenografts) was remarkably improved by RGD-LP-CHMSN-ATO. All the findings proved this favorable drug delivery system may significantly expand the potential use of arsenic compounds in treating solid tumor.Display OmittedThe aim of our study was to construct an Arg-Gly-Asp (RGD)-conjugated liposome-hollow silica hybrid nanovehicle for targeted delivery and controlled release of arsenic trioxide (ATO), whose anti-solid tumor effect was hampered by poor pharmacokinetics and dose-limited toxicity. Hydrophobic interactions were used to attach intact lipid membrane to the surface of chlorodimethyloctadecylsilane-modified hollow mesoporous silica nanoparticles. The prepared nanovehicles (RGD-LP-CHMSN) were characterized for uniform structure (silica core of ∼140 nm in diameter and liposomal shell of ∼6 nm), comparable drug loading efficiency (6.76%), desirable stability and strengthened controlled release. In vitro, RGD-LP-CHMSN showed good biocompatibility and low toxicity on HepG2, MCF-7 and LO2 cells. The targeted delivery of ATO by nanocarriers (RGD-LP-CHMSN-ATO) was demonstrated by an enhanced cellular uptake and a reduced half maximal inhibitory concentration (IC50) value. In pharmacokinetic studies, the RGD-LP-CHMSN-ATO group, compared to the free ATO group, prolonged the half time (t1/2β) by 1.7 times and increased the area under curve (AUC) by 2.4 times. In addition, in a H22 tumor-xenograft mouse model, nanovehicles improved the targeting efficiency and anticancer potential of ATO. In conclusion, the strategy of constructing a nanocarrier with targeted delivery and controlled release characteristics is prospective to enhance the antitumor effect of ATO.
Keywords: Arsenic trioxide; Hollow mesoporous silica nanoparticles; Liposomal shell; Arg-Gly-Asp; Controlled release; Hepatic carcinoma;

Nanoaggregation of inclusion complexes of glibenclamide with cyclodextrins by David Lucio; Juan Manuel Irache; María Font; María Cristina Martínez-Ohárriz (263-271).
Display OmittedGlibenclamide is a sulfonylurea used for the oral treatment of type II diabetes mellitus. This drug shows low bioavailability as consequence of its low solubility. In order to solve this problem, the interaction with cyclodextrin has been proposed. This study tries to provide an explanation about the processes involved in the formation of GB-βCDs complexes, which have been interpreted in different ways by several authors. Among native cyclodextrins, βCD presents the most appropriate cavity to host glibenclamide molecules showing AL solubility diagrams (K1:1  ≈ 1700 M−1).However, A L − solubility profiles were found for βCD derivatives, highlighting the coexistence of several phenomena involved in the drug solubility enhancement. At low CD concentration, the formation of inclusion complexes can be studied and the stability constants can be calculated (K1:1  ≈ 1400 M−1). Whereas at high CD concentration, the enhancement of GB solubility would be mainly attributed to the formation of nanoaggregates of CD and GB-CD complexes (sizes between 100 and 300 nm). The inclusion mode into βCD occurs through the cyclohexyl ring of GB, adopting a semi-folded conformation which maximizes the hydrogen bond network.As consequence of all these phenomena, a 150-fold enhancement of drug solubility has been achieved using β-cyclodextrin derivatives. Thus, its use has proven to be an interesting tool to improve the oral administration of glibenclamide in accordance with dosage bulk and dose/solubility ratio requirements.
Keywords: Glibenclamide; Cyclodextrins; Inclusion complexes; Non-inclusion phenomena; Self-association; Nanoaggregates;

Composite particles based on particle engineering for direct compaction by Zhe Li; Xiao Lin; Lan Shen; YanLong Hong; Yi Feng (272-286).
Display OmittedDirect compaction (DC) is the preferred method for tablet production. However, only a minority of the active pharmaceutical ingredients (APIs) can be truly manufactured into tablets by DC so far due to that most of APIs lack sufficient functional properties required for DC. Particle engineering with co-processing provides a promising way to obtain various composite API and/or excipient particles with markedly improved functional properties, which makes successful tableting of them by DC possible. This review, as an informative update and supplement, covers the improvement of functional properties of composite API and/or excipient particles via co-processing based on recent developments and researches in the area of particle engineering for DC. The improved functionality of co-processed particles and corresponding mechanisms were summarized and discussed from the perspective of structure characteristics (Crystal level and Particle level) as the properties of particles are markedly affected by their structure.
Keywords: Particle engineering; Direct compaction; Composite particles; Co-processing; Functional properties; Tablet;

Biocompatible polymersomes-based cancer theranostics: Towards multifunctional nanomedicine by Marzieh Mohammadi; Mohammad Ramezani; Khalil Abnous; Mona Alibolandi (287-303).
Display OmittedPolymersomes are polymeric vesicles that have numerous advantages for theranostics, the integrated approach of therapeutics and diagnostics. Polymersomes possess core- shell structures which encapsulate hydrophilic molecules in the aqueous compartment and hydrophobic molecules in the bilayer of the vesicles. Polymersomes are made of different amphiphilic block copolymers. Thus, in the process of designing polymersomes, a variety of amphiphilic block copolymers with different molecular weights are used to develop intelligent or sustained released formulations and to modify the stability of the system and bilayer thickness or to functionalize the particle with targeting moieties to improve the delivery efficiency.In addition, biocompatible and/or biodegradable polymersomes are diverse in size and charge which show low toxicity in vivo. Polymersomes are increasingly being used as platforms for simultaneous drug delivery and imaging and are therefore becoming popular theranostic nanoparticles. This review focuses on the methods of nanoparticle formation when polymersomes for theranostic nanomedicine are engineered. We highlight recent examples of polymersome theranostic systems from literature and their potential for use in the clinic, particularly biodegradable or biocompatible-based NPs.
Keywords: Polymersome; Theranostics; Cancer; Diagnostics; Copolymer;

A novel injectable formulation of 6-fluoro-l-DOPA imaging agent for diagnosis of neuroendocrine tumors and Parkinson’s disease by Adriana Trapani; Domenico Tricarico; Antonietta Mele; Fatima Maqoud; Delia Mandracchia; Paola Vitale; Vito Capriati; Giuseppe Trapani; Vincenzo Dimiccoli; Anna Tolomeo; Antonio Scilimati (304-313).
Display OmittedTwo [19F]F-l-DOPA (F-DOPA) new β-cyclodextrin (CD)-based dosage forms (FA and FB, respectively) have been studied and their physico-chemical and pharmacological features determined to overcome the administration site reactions showed by the currently used [18F]F-l-DOPA formulation (IASOdopa®) to perform PET-CT diagnosis in oncology (neuroendocrine tumors) and neurological (Parkinson’s disease) field. Chemical stability of FA and FB was found to be longer than IASOdopa® by adding the thiol-antioxidant agent, L-Cysteine. 1H and 19F NMR investigations suggest the formation of an inclusion complex of F-DOPA with β-CD. In vitro experiments on the effects of FA and FB on mouse skeletal muscle fibers and on the human neuroblastoma SH-SY5Y and embryonal kidney tsA201 cell lines viability showed that FA was the most performant formulation compared to F-DOPA solutions. In vivo tolerability tests of FA on adult male rat showed no significant effects on body weight and no change in their dried organs weight. In addition, their metabolic and physiological parameters were not affected. In conclusion, [18F]F-l-DOPA, formulated as FA, constitutes a promising dosage form for PET-CT diagnosis of both neuroendocrine tumors and Parkinson’s disease.
Keywords: F-DOPA; Cyclodextrins; NMR; Antioxidant agents; Pharmacological assays;

Micro-feeding and dosing of powders via a small-scale powder pump by M.O. Besenhard; S. Fathollahi; E. Siegmann; E. Slama; E. Faulhammer; J.G. Khinast (314-322).
Display OmittedRobust and accurate powder micro-feeding ( < 100 m g / s ) and micro-dosing ( < 5  mg ) are major challenges, especially with regard to regulatory limitations applicable to pharmaceutical development and production. Since known micro-feeders that yield feed rates below 5 m g / s use gravimetric feeding principles, feed rates depend primarily on powder properties. In contrast, volumetric powder feeders do not require regular calibration because their feed rates are primarily determined by the feeder’s characteristic volume replacement.In this paper, we present a volumetric micro-feeder based on a cylinder piston system (i.e., a powder pump), which allows accurate micro-feeding and feed rates of a few grams per hours even for very fine powders. Our experimental studies addressed the influence of cylinder geometries, the initial conditions of bulk powder, and the piston speeds. Additional computational studies via Discrete Element Method simulations offered a better understanding of the feeding process, its possible limitations and ways to overcome them.The powder pump is a simple yet valuable tool for accurate powder feeding at feed rates of several orders of magnitude.
Keywords: Powder feeding; Low dose; Micro-feeding; Micro-dosing; Cylinder piston; Feed rate robustness;

Injection of SDF-1 loaded nanoparticles following traumatic brain injury stimulates neural stem cell recruitment by Laura N. Zamproni; Mayara V. Mundim; Marimelia A. Porcionatto; Anne des Rieux (323-331).
Display OmittedRecruiting neural stem cell (NSC) at the lesion site is essential for central nervous system repair. This process could be triggered by the local delivery of the chemokine SDF-1. We compared two PLGA formulations for local brain SDF-1 delivery: SDF-1 loaded microspheres (MS) and SDF-1 loaded nanoparticles (NP). Both formulations were able to encapsulate more than 80% of SDF-1 but presented different release profiles, with 100% of SDF-1 released after 6 days for the MS and with 25% of SDF-1 released after 2 weeks for NP. SDF-1 bioactivity was demonstrated by a chemotactic assay. When injected in mouse brain after traumatic brain injury, only SDF-1 nanoparticles induced NSC migration to the damage area. More neuroblasts (DCX+ cells) could be visualized around the lesions treated with NP SDF-1 compared to the other conditions. Rostral migratory stream destabilization with massive migration of DCX+ cell toward the perilesional area was observed 2 weeks after NP SDF-1 injection. Local injection of SDF-1-loaded nanoparticles induces recruitment of NSC and could be promising for brain injury lesion.
Keywords: Neural stem cells; SDF-1; Nanoparticles; Brain injury;

Spray dried hyaluronic acid microparticles for adhesion controlled aggregation and potential stimulation of stem cells by Fabio S. Palumbo; Stefano Agnello; Calogero Fiorica; Giovanna Pitarresi; Roberto Puleio; Guido R. Loria; Gaetano Giammona (332-342).
Display OmittedSpray-dried microparticles of a derivative of hyaluronic acid (HA) have been engineered to obtain a controlled aggregation with Human Mesenchymal Stem Cells (hMSCs) into 3D constructs. We demonstrated the utility of chemical functionalization of a native constituent of the extracellular matrix to improve processing performances and to control on stem cell adhesion and differentiation. Native hyaluronic acid (HA), cell adhesive peptides (RGD), transforming growth factor β3, dexamethasone are biological agents potentially suitable for chondrogenic stimulation of hMSCS. However unmodified HA suffers of drawbacks in terms of stability and versatility of processing. Functionalization strategies are needed to overcome these drawbacks. In this paper microparticles were produced by spray-drying of an aliphatic and amino functionalized HA derivative. Hydrophobic derivatization of HA allowed the production of microparticles stabilized by physical crosslinking and to load and to control dexamethasone release. The presence of pendant amino groups was exploited to tether cyclic RGD and transforming growth factor β3 via maleimide chemistry; cyRGDC functionalization controlled hMSCs/microparticles aggregation. Chondrogenic potential was preliminary assayed by qualitative immunohistological detection.
Keywords: Bottom-up approach; Hyaluronic acid; Microparticles; Stem cells;

Amorphization within the tablet: Using microwave irradiation to form a glass solution in situ by Maria Doreth; Murtadha Abdul Hussein; Petra A. Priemel; Holger Grohganz; René Holm; Heidi Lopez de Diego; Thomas Rades; Korbinian Löbmann (343-351).
Display Omitted In situ amorphization is a concept that allows to amorphize a given drug in its final dosage form right before administration. Hence, this approach can potentially be used to circumvent recrystallization issues that other amorphous formulation approaches are facing during storage. In this study, the feasibility of microwave irradiation to prepare amorphous solid dispersions (glass solutions) in situ was investigated. Indomethacin (IND) and polyvinylpyrrolidone K12 (PVP) were tableted at a 1:2 (w/w) ratio. In order to study the influence of moisture content and energy input on the degree of amorphization, tablet formulations were stored at different relative humidity (32, 43 and 54% RH) and subsequently microwaved using nine different power-time combinations up to a maximum energy input of 90 kJ. XRPD results showed that up to 80% (w/w) of IND could be amorphized within the tablet. mDSC measurements revealed that with increasing microwaving power and time, the fractions of crystalline IND and amorphous PVP reduced, whereas the amount of in situ formed IND-PVP glass solution increased. Intrinsic dissolution showed that the dissolution rate of the microwaved solid dispersion was similar to that of a quench cooled, fully amorphous glass solution even though the microwaved samples contained residual crystalline IND.
Keywords: Microwave radiation; Amorphization; In situ; Glass solution; Recrystallization; Tablet;

Smart AS1411-aptamer conjugated pegylated PAMAM dendrimer for the superior delivery of camptothecin to colon adenocarcinoma in vitro and in vivo by Mona Alibolandi; Seyed Mohammad Taghdisi; Pouria Ramezani; Fazileh Hosseini Shamili; Sara Amel Farzad; Khalil Abnous; Mohammad Ramezani (352-364).
Display OmittedIn the current study camptothecin-loaded pegylated PAMAM dendrimer were synthesized and were functionalized with AS1411 anti-nucleolin aptamers for site-specific targeting against colorectal cancer cells which over expresses nucleolin receptors.The morphological properties and size dispersity of the prepared nanoparticles were evaluated using transmission electron microscope (TEM) and DLS. The drug-loading content and encapsulation efficiency were obtained 8.1% and 93.67% respectively. The in vitro release of camptothecin from the formulation was provided the sustained release of encapsulated camptothecin during 4 days. Comparative in vitro cytotoxicity experiments demonstrated that the targeted camptothecin loaded-pegylated dendrimers had higher antiproliferation activity, towards nucleolin-positive HT29 and C26 colorectal cancer cells than nucleolin-negative CHO cell line.Fluorscence microscopy and flow cytometry also confirmed the enhanced cellular uptake of AS1411 targeted pegylated-dendrimer. In vivo study in C26 tumor-bearing BALB/C mice revealed that the AS1411-functionalized camptothecin loaded pegylated dendrimers improved antitumor activity and survival rate of the encapsulated camptothecin. Conjugation of AS1411 aptamer to the camptothecin loaded-pegylated dendrimer surface provides site-specific delivery of camptothecin, inhibit C26 tumor growth in vivo and significantly decrease systemic toxicity. These results suggested that the new nucleolin-targeted pegylated PAMAM dendrimer as a delivery system for camptothecin have the potential for the treatment of nucleolin-overexpressed colorectal cancer.
Keywords: Dendrimer; PAMAM; AS1411; Camptothecin; Targeted drug delivery;

Design of spray dried ternary solid dispersions comprising itraconazole, soluplus and HPMCP: Effect of constituent compositions by Mark T. Davis; Catherine B. Potter; Maryam Mohammadpour; Ahmad B. Albadarin; Gavin M. Walker (365-372).
Display OmittedA range of 17 ternary formulations of itraconazole (ITZ), HPMCP and Soluplus have been manufactured using spray drying. These amorphous solid dispersions (ASDs) were very stable against crystallisation and ITZ was found to be amorphous in all formulations after one year at 40 °C/75% RH. A number of solid state analytical techniques including PXRD, DSC, small angle X-ray scattering, FTIR and solid state NMR were used to characterise the physicochemical properties of the ASDs following processing and storage and to assess any interactions between components. Microtrac laser scattering analysis revealed a relationship between polymer levels and particle size distribution (PSD). Dissolution studies indicated that higher Soluplus content in the formulation resulted in higher concentrations of ITZ in acidic media.
Keywords: Itraconazole; Soluplus; HPMCP; Ternary; Amorphous; Spray drying;

Characterization and biocompatibility evaluation of cutaneous formulations containing lipid nanoparticles by F. Eiras; M.H. Amaral; R. Silva; E. Martins; J.M. Sousa Lobo; A.C. Silva (373-380).
Display OmittedNanostructured lipid carriers (NLC) are well-known systems that show effectiveness to improve skin hydration, being suggested for cosmetic and dermatological use. Nonetheless, NLC dispersions present low viscosity, which is non-attractive for cutaneous application. To circumvent this drawback, the dispersions can be gelled or incorporated in semisolid systems, increasing the final formulation consistency.In this study, we prepared a hydrogel based on NLC containing vitamin E (HG-NLCVE) and evaluated its suitability for cutaneous application. The experiments started with the HG-NLCVE characterization (organoleptic analysis, accelerated stability, particle size, morphology, pH, texture and rheology). Afterwards, in vitro experiments were carried out, evaluating the formulation biocompatibility (MTT and Neutral Red) and irritant potential (Hen’s egg test on the chorioallantoic membrane, HET-CAM) for cutaneous application. The results showed that the HG-NLCVE has adequate features for skin application, is biocompatible and non-irritant. From this study, it was predicted the in vivo irritant potential of the developed formulation, avoiding the need to perform a high number of tests on human volunteers. Regarding vitamin E and NLC potential to improve skin hydration, we suggest that the HG-NLCVE could be used in cosmetic (e.g. moisturizers and anti-aging) or dermatologic (e.g. xerosis and other skin disorders) products.
Keywords: Nanostructured lipid carriers; Vitamin E; Cutaneous application; Hydrogel; Biocompatibility; Irritant potential;

Display OmittedThis study reports design and evaluation of chitosan-based microparticle activity against Candida glabrata in vitro and in vivo in immunocompetent mice model artificially maintained in oestrus state. Because their flattened shape, chitosan microparticles are called here micro-platelets. They were obtained by self-association of oleoyl chitosan and α-cyclodextrin in water. A mixture of amphotericin B-deoxycholate (Fungizone®, AmB-DOC) and chitosan micro-platelets gelified with pluronic® F127 (20 wt%) completely cured C. glabrata vaginal infection. Colony factor unit counting and mycological analysis of mice vaginal mucosa after Grocott-Gomori methenamine-silver staining confirmed the absence of C. glabrata. Furthermore, in vitro evaluations revealed that IC50 and MIC90 of AmB-DOC were decreased 1.8 and 1.4-times respectively when associated with chitosan micro-platelets. Neither native chitosan nor oleoyl chitosan allowed improvement in AmB-DOC anti-C. glabrata activity. This work demonstrates for the first time that a simple mixing of chitosan micro-platelets with AmB-DOC enhanced its anti-C. glabrata activity.
Keywords: Candida glabrata; Chitosan; Amphotericin B; Micro-platelets; Self-assembly; Cyclodextrin;

Continuous direct compression as manufacturing platform for sustained release tablets by B. Van Snick; J. Holman; C. Cunningham; A. Kumar; J. Vercruysse; T. De Beer; J.P. Remon; C. Vervaet (390-407).
Display OmittedThis study presents a framework for process and product development on a continuous direct compression manufacturing platform. A challenging sustained release formulation with high content of a poorly flowing low density drug was selected. Two HPMC grades were evaluated as matrix former: standard Methocel CR and directly compressible Methocel DC2. The feeding behavior of each formulation component was investigated by deriving feed factor profiles. The maximum feed factor was used to estimate the drive command and depended strongly upon the density of the material. Furthermore, the shape of the feed factor profile allowed definition of a customized refill regime for each material. Inline NIRs was used to estimate the residence time distribution (RTD) in the mixer and monitor blend uniformity. Tablet content and weight variability were determined as additional measures of mixing performance. For Methocel CR, the best axial mixing (i.e. feeder fluctuation dampening) was achieved when an impeller with high number of radial mixing blades operated at low speed. However, the variability in tablet weight and content uniformity deteriorated under this condition. One can therefore conclude that balancing axial mixing with tablet quality is critical for Methocel CR. However, reformulating with the direct compressible Methocel DC2 as matrix former improved tablet quality vastly. Furthermore, both process and product were significantly more robust to changes in process and design variables. This observation underpins the importance of flowability during continuous blending and die-filling. At the compaction stage, blends with Methocel CR showed better tabletability driven by a higher compressibility as the smaller CR particles have a higher bonding area. However, tablets of similar strength were achieved using Methocel DC2 by targeting equal porosity. Compaction pressure impacted tablet properties and dissolution. Hence controlling thickness during continuous manufacturing of sustained release tablets was crucial to ensure reproducible dissolution.
Keywords: Continuous manufacturing; Raw material properties; Twin screw feeding; Continuous mixing; Continuous direct compression; Process analytical technology; Sustained release;

Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme by Josefine Morgenstern; Pascal Baumann; Carina Brunner; Jürgen Hubbuch (408-417).
Display OmittedDuring production, purification, formulation, and storage proteins for pharmaceutical or biotechnological applications face solution conditions that are unfavorable for their stability. Such harmful conditions include extreme pH changes, high ionic strengths or elevated temperatures. The characterization of the main influencing factors promoting undesired changes of protein conformation and aggregation, as well as the manipulation and selective control of protein stabilities are crucially important to biopharmaceutical research and process development. In this context PEGylation, i.e. the covalent attachment of polyethylene glycol (PEG) to proteins, represents a valuable strategy to improve the physico-chemical properties of proteins.In this work, the influence of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme is investigated. Specifically, conformational and colloidal properties were studied by means of high-throughput melting point determination and automated generation of protein phase diagrams, respectively. Lysozyme from chicken egg-white as a model protein was randomly conjugated to 2 kDa, 5 kDa and 10 kDa mPEG-aldehyde and resulting PEGamer species were purified by chromatographic separation. Besides protein stability assessment, residual enzyme activities were evaluated employing a Micrococcus lysodeikticus based activity assay. PEG molecules with lower molecular weights and lower PEGylation degrees resulted in higher residual activities. Changes in enzyme activities upon PEGylation have shown to result from a combination of steric hindrance and molecular flexibility. In contrast, higher PEG molecular weights and PEGylation degrees enhanced conformational and colloidal stability. By PEGylating lysozyme an increase of the protein solubility by more than 11-fold was achieved.
Keywords: PEGylation; Protein phase diagram; Melting point determination; Molecular flexibility; Enzyme activity;