International Journal of Pharmaceutics (v.455, #1-2)

The water-induced ionic charge transport in compacted microcrystalline cellulose (MCC) has been reported to be governed by the densification behaviour. Hence, mechanical properties were expected to correlate with conductivity behaviour of MCC compacts. Both in-die and out-of-die compaction behaviour of MCC powder was investigated using a fully instrumented rotary tablet press. The dielectric measurements were carried out using a Novocontrol Concept 40 broadband dielectric spectrometer and dc conductivity (σ dc) was extracted from the low frequency conductivity data at room temperature. As postulated, compaction pressure corresponding to maximum conductivity (σ dc max) was observed to correlate with yield strength of MCC, determined using in-die and out-of-die Heckel analysis. Although Heckel transformation is most commonly used in pharmaceutical technology, its general use to characterise the mechanical properties of organic pharmaceutical materials has been criticized. The present study has provided experimental evidence that Heckel equation is practically useful to describe plastic deformation of organic pharmaceutical powders.
Keywords: Yield strength; Heckel analysis; Conductivity; MCC; Compaction; Plastic deformation;

Synthesis and micellization of a pH-sensitive diblock copolymer for drug delivery by Konstantinos P. Koutroumanis; Richard G. Holdich; Stella Georgiadou (5-13).
A diblock copolymer constituting of a poly(ethylene glycol) (PEG) and a polycaprolactone (PCL) segment, linked with a pH-sensitive hydrazone bond (Hyd), was synthesized. Micelles formed from this copolymer, offer the advantage of encapsulating hydrophobic drugs without the need for conjugation sites. All synthesized polymers were characterized using gel permeation chromatography, infrared and proton nuclear spectroscopies. PEG-Hyd-PCL micelles were prepared using the solvent-displacement method and α-tocopherol was used as a model drug due to its high hydrophobicity. The micelle size and drug loading efficiency were studied with regards to the hydrophilic ratio, f, molecular weight, and the polymer/drug ratio. Dynamic light scattering and transmission electron microscopy showed that the PEG-Hyd-PCL micelles had sizes ranging from 50 to 200 nm. Aqueous micellar dispersions exhibited significantly higher values of turbidity in mildy acidic pH than in neutral, indicating pH-sensitivity for the PEG-Hyd-PCL micelles. The zeta potential of the micellar solutions decreased and the molecular weight distribution became bimodal at mildly acidic pH also supporting the pH sensitive properties of the copolymer. The critical micelle concentration was calculated using fluorescence spectroscopy.
Keywords: Block copolymers; Micelles; pH-sensitive drug delivery; Poly(ethylene glycol); Hydrazone; Polycaprolactone;

Overcoming the absorption barriers of skin is important to deliver medications either via diffusion based passive transport or with the help of active transdermal delivery approaches such as energy enhanced microporation technologies or with mechanically permeating the skin with microneedles. Alternatives to needle based injections will set new standards of care for pediatric use for vaccines as well as for the delivery of active therapeutic molecules. Novel patient centric drug delivery methods that appeal to children and caregivers in providing safety and efficacy will open up new therapies for pediatric use.Administration of medications via the parenteral route directly to the systemic circulation is an effective way of overcoming the first pass effect, obtaining quicker onset of action, and achieving higher bioavailability. However, needle phobia and the pain perceived during the injection process often make this a less preferred route than oral in terms of patient acceptance and compliance, particularly for pediatrics. Needleless injection technologies that deliver medications via the transdermal interface have been an active area of pharmaceutical research for many years. This review summarizes the various emerging technologies in the area of active transdermal delivery that can be potentially extended to pediatric applications.
Keywords: Microporation; Transdermal; Drug delivery; Pediatrics;

Biopharmaceutical profiling of a pyrido[4,3-d] pyrimidine compound library by Benjamin Wuyts; Janneke Keemink; Steven De Jonghe; Pieter Annaert; Patrick Augustijns (19-30).
The pyrido-pyrimidine structure is associated with different biological activities, including kinase inhibition and antibacterial activity. However, drug-like properties of this scaffold have not been explored thoroughly. Therefore, the biopharmaceutical profile of ten pyrido[4,3-d]pyrimidines with different substitution pattern was determined at the intestinal and hepatic level.The compounds’ biopharmaceutical properties were significantly affected by the substitution pattern, resulting in a broad range in fasted state simulated intestinal fluid solubility values (1.9 μM–4.2 mM) and Caco-2 permeability coefficients (0.17 × 10−6  cm/s to 52 × 10−6  cm/s). A phenylhydrazido group was responsible for the low FaSSIF solubility. Caco-2 permeability was impaired by a dimethoxyphenyl substituent. All analogs were metabolically stable in human intestinal microsomes. The hepatic metabolism, reflected by the extraction ratio, was intermediate to high (ER > 0.3). Aliphatic chains, methoxy groups on a phenyl substituent, ketone and amine substituents were predicted as most susceptible sites for hepatic metabolism. Correlations were found between polar surface area of the compound and Caco-2 permeability (R  = 0.86) and metabolic stability (R  = 0.76). No toxicity was seen for the pyrido[4,3-d]pyrimidines with Caco-2 cells and sandwich-cultured rat hepatocytes.In conclusion, the large diversity of substituents on the pyrido[4,3-d]pyrimidine core highly influenced the compounds’ drug-like properties.
Keywords: Pyrido[4,3-d]pyrimidines; Biopharmaceutical profiling; Caco-2; Suspended rat hepatocytes; Sandwich cultured rat hepatocytes; Clearance prediction;

In vitro and in vivo correlation of disintegration time and bitter taste masking effect of orally disintegrating tablet (ODT) formulations containing ion exchange resin-drug complex (IRDC) were established for bitter model drug, donepezil HCl.Although the taste-masking of bitter drug using ion exchange resin has been recognized, in vitro testing using an electronic tongue (e-Tongue) and in vivo bitterness test by human panel test was not fully understood. In case of orally disintegrating tablet (ODT) containing bitter medicine, in vitro and in vivo disintegration is also importance for dosage performance. Donepezil hydrochloride was chosen as a model drug due to its bitterness and requires rapid disintegration for the preparation of ODT. In this study, ion exchange resin drug complex (IRDC) at three different ratios (1:2, 1:1, 2:1) was prepared using a spray-drying method and then IRDC-loaded ODT containing superdisintegrants (crospovidone, croscarmellose sodium, and sodium starch glycolate) were prepared by the direct compression method. The physical properties and morphologies were then characterized by scanning electron microscopy (SEM), X-ray powder diffraction (PXRD) and electrophoretic laser scattering (ELS), respectively. The in vitro taste-masking efficiency was measured with an electronic tongue (e-Tongue). In vivo bitterness scale was also evaluated by human volunteers and then we defined new term, “bitterness index (BI)” to link in vitro e-Tongue. There was a good correlation of IRDC between in vitro e-Tongue values and in vivo BI. Furthermore, IRDC-loaded ODT showed good in vitro/in vivo correlation in the disintegration time. The optimal IRDC-loaded ODTs displayed similar drug release profiles to the reference tablet (Aricept® ODT) in release media of pH 1.2, pH 4.0, pH 6.8 and distilled water but had significantly better palatability in vivo taste-masking evaluation. The current IRDC-loaded ODT according to the in vitro and in vivo correlation of disintegration and bitter taste masking could provide platforms in ODT dosage formulations of donepezil hydrochloride for improved patient compliances.
Keywords: Donepezil hydrochloride; Ion exchange-resin drug complex; Orally disintegrating tablet; In vitro/in vivo correlation; Electronic tongue; Bitterness index;

Cytoplasm-responsive nanocarriers conjugated with a functional cell-penetrating peptide for systemic siRNA delivery by Ko Tanaka; Takanori Kanazawa; Shogo Horiuchi; Taichi Ando; Ken Sugawara; Yuuki Takashima; Yasuo Seta; Hiroaki Okada (40-47).
To develop a gene carrier for cancer therapy by systemic injection, we synthesized methoxypolyethylene glycol–polycaprolactone (MPEG–PCL) diblock copolymers conjugated with a cytoplasm-responsive cell-penetrating peptide (CPP), CH2R4H2C (C, Cys; H, His; R, Arg). The carrier/small interfering RNA (siRNA) complexes (N/P ratio of 20) had a particle size of approximately 50 nm and stabilized the siRNA against RNase. The cellular uptake ability of the carrier/FAM-siRNA complexes with fetal bovine serum was significantly higher than that of naked FAM-siRNA. In addition, the carrier/anti-vascular endothelial growth factor siRNA (siVEGF) complexes attained a significantly greater silencing effect than naked siVEGF with low cytotoxicity, resulting from higher uptake, early endosomal escape, and efficient release from the complexes in the cytoplasm. Furthermore, intravenous injection of MPEG–PCL–CH2R4H2C/siVEGF complexes had a significantly higher anti-tumor effect in S-180 tumor-bearing mice, which could be attributed to the rigid compaction of siRNA by ionic interactions and disulfide linkages in the CPP polymer micelles in the blood, as well as higher release following cleavage of the disulfide bonds in the reductive cytosol. Taken together, our data demonstrated that these cytoplasm-responsive polymer micelles conjugated with multi-functional CPP, could facilitate siVEGF delivery to tumor tissues after systemic injection and could exert an extremely strong anti-tumor effect.
Keywords: Anti-VEGF siRNA; Systemic delivery polymer micelles; Cell-penetrating peptide; Cytoplasm-responsive nanocarriers; Disulfide linkage; Anti-tumor effect;

Novel bioadhesive hyaluronan–itaconic acid crosslinked films for ocular therapy by J.A. Calles; L.I. Tártara; A. Lopez-García; Y. Diebold; S.D. Palma; E.M. Vallés (48-56).
New hyaluronic acid (HA)–itaconic acid (IT) films have been previously synthesized and used as potential topical drug delivery systems (DDS) for ocular administration. In this study we explored homogeneous and heterogeneous crosslinking reactions of HA using glutaraldehyde (GTA) and polyethylene glycol diglycidyl ether (PEGDE) in the presence of IT, a naturally occurring compound that is non-toxic and readily biodegradable.We have studied the morphology, mechanical properties and in vitro biocompatibility between these new materials and ocular surface cells (human corneal epithelial cell line) and evaluated the biopharmaceutical performance of the designed formulations. Although all the synthesized materials exhibited good mechanical properties, the PEGDE modified films exhibited the best biocompatibility, with in vivo assays showing good adhesive performance and minimal irritation. PEGDE films were also tested for their effects in the treatment of intraocular pressure (IOP) in rabbits using timolol maleate (TM) as the model drug.These results may be useful for further design of novel bioadhesive matrix containing drugs by topical application in ophthalmology.
Keywords: Hyaluronan; Glaucoma; Bioadhesive; Polymer; Drug delivery;

Pressurized metered dose inhalers (pMDIs) are widely used for the treatment of diseases of the lung, including asthma and chronic obstructive pulmonary disease. The mass median aerodynamic diameter of the residual particles (MMADR) delivered from a pMDI plays a key role in determining the amount and location of drug deposition in the lung and thereby the efficacy of the inhaler. The mass median diameter of the initial droplets (MMDI), upon atomization of a formulation, is a significant factor influencing the final particle size. The purpose of this study was to evaluate the extent that MMDI and initial droplet geometric standard deviation (GSD) influence the residual aerodynamic particle size distribution (APSDR) of solution and suspension formulations. From 48 solution pMDI configurations with varying ethanol concentrations, valve sizes and actuator orifice diameters, it was experimentally found that the effective MMDI ranged from 7.8 to 13.3 μm. Subsequently, computational methods were utilized to determine the influence of MMDI on MMADR, by modulating the MMDI for solution and suspension pMDIs. For solution HFA-134a formulations of 0.5% drug in 10% ethanol, varying the MMDI from 7.5 to 13.5 μm increased the MMADR from 1.4 to 2.5 μm. For a suspension formulation with a representative particle size distribution of micronized drug (MMAD = 2.5 μm, GSD = 1.8), the same increase in MMDI resulted in an increase in the MMADR from 2.7 to only 3.3 μm. Hence, the same increase in MMDI resulted in a 79% increase in MMADR for the solution formulation compared to only a 22% increase for the suspension formulation. Similar trends were obtained for a range of drug concentrations and input micronized drug sizes. Thus, APSDR is more sensitive to changes in MMDI for solution formulations than suspension formulations; however, there are situations in which hypothetically small micronized drug in suspension (e.g. 500 nm MMAD) could resemble trends observed for solution formulations. Furthermore, the relationship between APSDR and drug concentration and MMDI is predictable for solution pMDIs, but this is not as straightforward for suspension formulations. In addition, the MMADR was relatively insensitive to changes in initial droplet GSD (from 1.6 to 2.0) and the solution and suspension pMDI residual particle GSDs were essentially identical to the initial droplet GSDs.
Keywords: Pressurized metered dose inhaler (pMDI); Atomized droplets; Solution formulation; Suspension formulation; Initial droplet diameter; Aerodynamic particle size;

Glycerosomes: A new tool for effective dermal and transdermal drug delivery by Maria Letizia Manca; Marco Zaru; Maria Manconi; Francesco Lai; Donatella Valenti; Chiara Sinico; Anna Maria Fadda (66-74).
This work describes glycerosomes, vesicles composed of phospholipids, glycerol, and water, as novel vesicular carriers for (trans)dermal drug delivery. In this work, glycerosomes were prepared by hydrating dipalmitoylglycerophosphatidylcholine-cholesterol films with glycerol aqueous solutions (10–30%, v/v). The model drug was diclofenac sodium salt and conventional liposomes were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, and vesicle deformability. Glycerosomes and liposomes were oligo/multilamellar vesicles, spherical in shape with a mean diameter ranging between 81 and 97 nm and a fairly narrow distribution (P.I. = 0.14–0.19), negative zeta potential values (from −35 to −48) and drug loading capacity between 64 and 73%. Deformability index of both conventional liposomes and glycerosomes showed that glycerol is able to act as edge activator for dipalmitoylglycerophosphatidylcholine bilayers when used in concentration higher than 10%. DSC studies suggested that glycerosomes are in a more fluid state than conventional liposomes. In vitro transdermal delivery experiments showed an improved skin deposition and permeation of diclofenac when 20 and 30% glycerosomes were used. MTT test demonstrated that glycerosomes were able to reduce the in vitro drug toxicity versus keratinocytes.
Keywords: Phospholipids; Glycerol; Liposomes; DSC; Keratinocytes; Cell viability;

A potential carrier based on liquid crystal nanoparticles for ophthalmic delivery of pilocarpine nitrate by Jing Li; Lin Wu; Weijun Wu; Baoyan Wang; Zhongyuan Wang; Hongliang Xin; Qunwei Xu (75-84).
Poor corneal penetration and short preocular retention of a clinical hydrophilic drug, pilocarpine nitrate (PN), for the treatment of open-angle glaucoma and acute angle-closure glaucoma, limit its ocular application. The purpose of this study was to investigate the potential of liquid crystal nanoparticles (LCNPs) for ocular delivery of PN. LCNPs were developed by a top-down method using glyceryl monoolein (GMO) and water in the presence of stabilizer Poloxamer 407. They were characterized by transmission electron microscopy (TEM) and small angle X-ray diffraction (SAXS). The size of LCNP is 202.28 ± 19.32 nm and the encapsulation efficiency reached 61.03%. The in vitro release profiles indicated that PN could keep sustained release from PN-loaded LCNPs for 8 h. An ex vivo corneal permeation study revealed that the apparent permeability coefficient of PN-loaded LCNPs was 2.05-fold higher than that of commercial eye drops. In addition, the topical administration test showed that PN-loaded LCNPs had a prolonged effect on decreasing intraocular pressure (IOP) of rabbits compared with commercial drug and physiological saline. In conclusion, LCNPs had been demonstrated to be potential for controlled-release ocular drug delivery.
Keywords: Pilocarpine nitrate; Liquid crystal nanoparticle; Glycerol monooleate; Reverse hexagonal phase; Corneal permeability;

Preparation, characterization, biodistribution and antitumor efficacy of hydroxycamptothecin nanosuspensions by Meihua Han; Xiaoli Liu; Yifei Guo; Yanhong Wang; Xiangtao Wang (85-92).
HCPT nanosuspensions shown remarkable liver targetability, compared to commercial HCPT injection.Hydroxycamptothecin (HCPT) has shown activity against a broad spectrum of cancers, but its therapeutic efficacy is impaired by its poor solubility and delivery challenges. In this study, HCPT nanosuspensions were prepared with precipitation-combined ultrasonication and characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM) and X-ray powder diffraction (XRD). The HCPT nanosuspensions were spherical with a smooth surface and a small size of 150–200 nm. The lyophilized powders for the HCPT nanosuspensions were amorphous and displayed sustained release in vitro. Compared to commercial HCPT injection, in vivo experiments with HCPT nanosuspensions showed significantly increased HCPT concentrations in the blood and all tissues of the tested as well as improved tumor targetability and liver targetability. Meanwhile, nanosuspensions displayed better anticancer efficacy than injection on H22 bearing mice (81.20% vs. 56.39%, in tumor inhibition rate). Therefore, HCPT nanosuspensions seem very promising for the treatment of hepatic cancer.
Keywords: Hydroxycamptothecin (HCPT); Nanosuspensions; Antitumor efficacy; Liver targetability; Biodistribution;

Fast drying of biocompatible polymer films loaded with poorly water-soluble drug nano-particles via low temperature forced convection by Ramana Susarla; Lucas Sievens-Figueroa; Anagha Bhakay; Yueyang Shen; Jackeline I. Jerez-Rozo; William Engen; Boris Khusid; Ecevit Bilgili; Rodolfo J. Romañach; Kenneth R. Morris; Bozena Michniak-Kohn; Rajesh N. Davé (93-103).
Fast drying of nano-drug particle laden strip-films formed using water-soluble biocompatible polymers via forced convection is investigated in order to form films having uniform drug distribution and fast dissolution. Films were produced by casting and drying a mixture of poorly water soluble griseofulvin (GF) nanosuspensions produced via media milling with aqueous hydroxypropyl methylcellulose (HPMC E15LV) solutions containing glycerin as a plasticizer. The effects of convective drying parameters, temperature and air velocity, and film-precursor viscosity on film properties were investigated. Two major drying regimes, a constant rate period as a function of the drying conditions, followed by a single slower falling rate period, were observed. Films dried in an hour or less without any irreversible aggregation of GF nanoparticles with low residual water content. Near-infrared chemical imaging (NIR-CI) and the content uniformity analysis indicated a better drug particle distribution when higher viscosity film-precursors were used. Powder X-ray diffraction showed that the GF in the films retained crystallinity and the polymorphic form. USP IV dissolution tests showed immediate release (∼20 min) of GF. Overall, the films fabricated from polymer-based suspensions at higher viscosity dried at different conditions exhibited similar mechanical properties, improved drug content uniformity, and achieved fast drug dissolution.
Keywords: Nanosuspensions; BCS class II drugs; Pharmaceutical films; Convective drying; Fast dissolution;

Examination of the stability of clonazepam, diazepam, alprazolam, haloperidol, and doxepin in basic solutions was performed, together with an assessment of the kinetic (k, t 0.1 i t 0.5) and thermodynamic (E a, ΔH ++ i ΔS ++) stability-indicating parameters, which were compared with the lipophilicity (log  P) of the studied drugs. It was observed that the calculated values of E a, ΔH ++ and ΔS ++ for the studied drugs increased from 41.04 kJ/mol to 125.50 kJ/mol, from 37.82 kJ/mol to 122.24 kJ/mol and from −167.09 J/Kmol to 53.02 J/Kmol, respectively, along with an increase of lipophilicity (log  P) from 2.12 to 4.30 for the most hydrophilic alprazolam to the most lipophilic haloperidol. The degradation products were identified using UPLC/MS/MS method.
Keywords: Stability studies; Lipophilicity; TLC; LC/MS;

Nimodipine (NM) commercial formulation has been recalled due to drug crystallization in the product. Aim of present investigation was to systematically evaluate NM ternary cosolvents systems, characterize the crystallized drug and develop discriminating dissolution method that could detect the drug crystallization in the product. Mixture design was constructed using independent components namely water (X 1), glycerin (X 2) and polyethylene glycol 400 (X 3, PEG-400). Nineteen formulations were developed using various level of cosolvents mixture while drug concentration was kept constant. The response selected was the drug crystallized in the formulations kept at four storage conditions 5 °C, 15 °C, 25 °C and 25 °C/60% RH for four weeks. The crystallized drug was characterized by Fourier transformed infrared (FTIR), near infrared (NIR), NIR-chemical imaging and Raman spectroscopies, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and scanning electron microscopy. Dissolution of formulation and modification was tested by USP method 2 in 0.25 and 0.50% sodium lauryl sulphate (SLS) aqueous media and run at 50 and 75 rpm. X 1 promoted drug crystallization at all conditions of storage and reverse was true for X 3. Characterization data indicated that the crystallized drug in most of the formulations were modification II, but a few formulations contained significant proportion of the modification I. Dissolution in 0.25% (w/v) SLS at 75 rpm was more discriminating in detecting the crystallization in the product compared to dissolution in 0.5% (w/v) SLS media. In summary, cosolvents system of NM was prone to crystallization depending upon the cosolvents composition and storage conditions. A more rational approach to develop NM formulation would entail a then through understanding of the causes of crystallization and their characterization in a variety of storage conditions.
Keywords: Nimodipine; Polymorph; Crystallization; Cosolvents; Dissolution; NIR-chemical imaging;

Characterization of novel lactoferrin loaded capsules prepared with polyelectrolyte complexes by Qing-Xi Wu; Qi-Lei Zhang; Dong-Qiang Lin; Shan-Jing Yao (124-131).
Novel capsules loaded with lactoferrin (LF) were prepared using polyelectrolyte complexes that were formed by water soluble chitosan (WSC), sodium cellulose sulfate (NaCS) and sodium polyphosphate (PPS). Normal chitosan (soluble in acidic conditions) was chosen as a control to prepare similar capsules with NaCS and PPS. 1H NMR and FTIR spectra analysis showed that WSC was in a form of chitosan hydrochloride which can be directly dissolved and protonated in acid-free water. SEM results showed that the capsules had a typical wall-capsule structure with a regular spherical shape and an average diameter of 1.97 mm. TGA studies revealed that the thermal stability of the capsules were enhanced and the moisture content of the drug-free/loaded capsules were 6.3% and 3.2%. SDS-PAGE results showed that the primary structures of the processed LF in the capsules were unchanged. Drug loading (LE%) and encapsulation efficiency (EE%) analysis showed that the capsules had a higher LE% (45.6%) and EE% (70.7%) than that of the control. In vitro release studies showed that the capsules had a regular and sustainable release profiles in simulated colonic fluid. All of these results indicated that the capsules prepared could be used as a candidate protein drug carrier for colon.
Keywords: Capsules; Lactoferrin; Water soluble chitosan; Sodium cellulose sulfate; Polyelectrolyte complexes; Drug release;

A completely solvent-free process for the improvement of erythritol compactibility by Yohei Hoashi; Yuichi Tozuka; Hirofumi Takeuchi (132-137).
We obtained improvement of erythritol compactibility by formulating composite particles composed of erythritol and porous silica using a twin-screw kneader.Erythritol-based tablets formulated with composite particles were directly compacted, and we estimated their hardness and the friability. The compression properties of the erythritol powder bed including composite particles were estimated using a Heckel analysis and force-displacement profiles, and we investigated the physical states of the composite particles by powder X-ray diffractometry, a thermal analysis and a nitrogen gas adsorption study.A direct-compacted erythritol tablet formulated with composite particles, prepared at the melting temperature of erythritol (120 °C), exhibited high hardness and low friability. A pressure transmission study revealed the higher plasticity and lower elasticity of an erythritol powder bed formulated with composite particles prepared at 120 °C. Physical states of the composite particles indicated that erythritol in the composite particles was adsorbed onto porous silica with a subsequent decrease in the erythritol crystallinity as a result of high mechanical force at the melting temperature of erythritol.The improvement of the erythritol compactibility formulated with composite particles through processing with a twin-screw kneader at 120 °C. This was affected by the reduction of the erythritol crystallinity in the composite particles.
Keywords: Erythritol; Porous silica; Twin-screw kneader; Solvent-free process; Rapidly disintegrating tablet; Direct compression;

Effect of type and ratio of solubilising polymer on characteristics of hot-melt extruded orodispersible films by Ariana Qiao Jun Low; Johannes Parmentier; Yuet Mei Khong; Cherry Chang Er Chai; Thin Yu Tun; Jacqueline E. Berania; Xueming Liu; Rajeev Gokhale; Sui Yung Chan (138-147).
In formulating an orodispersible film (ODF), it is important for polymer choice to strike a balance between mechanical properties and release rates. Studies have been done to study polymer combinations. However, there is a lack of a systematic study to determine key factors affecting these properties. We studied the effect of varying the ratios of a solubilising polymer (Kollidon® VA 64 or Soluplus®) to a film forming polymer, hydroxypropyl cellulose (HPC), on mechanical properties and release rates of hot-melt extruded ODFs using a 23 factorial design. The two drugs evaluated were chlorpheniramine and indomethacin. The main effects impacting mechanical properties were the drug and two-way interaction between drug and solubilising polymer. For dissolution, the main effects were the solubilising polymer; the drug; and the two-way interaction between solubilising polymer and ratio of solubilising to film forming polymer. Both drugs exhibited plasticising effects on the polymer matrix and had higher film ductility and lower film stiffness. Kollidon® VA 64-containing films performed better in terms of drug release whereas Soluplus®-containing films had better mechanical properties. The dissolution rate can be improved by decreasing film thickness. The findings of our study will be crucial to forming a robust ODF formulation.
Keywords: Soluplus®; Copovidone; Mechanical properties; Hot-melt extrusion; Orodispersible film; Solubilising polymer ratio;

Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury by Eduardo Ansorena; Pauline De Berdt; Bernard Ucakar; Teresa Simón-Yarza; Damien Jacobs; Olivier Schakman; Aleksandar Jankovski; Ronald Deumens; Maria J. Blanco-Prieto; Véronique Préat; Anne des Rieux (148-158).
Injection of free GDNF-loaded hydrogel in the lesion improved rat functional outcome, starting 4 weeks post spinal cord hemisection.We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere-loaded hydrogels. Intense GFAP (astrocytes), low βIII tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals.
Keywords: GDNF; Microspheres; Spinal cord injury; Injectable hydrogel; Alginate;

Inline monitoring and a PAT strategy for pharmaceutical hot melt extrusion by Patrick R. Wahl; Daniel Treffer; Stefan Mohr; Eva Roblegg; Gerold Koscher; Johannes G. Khinast (159-168).
Monitoring of extrusion dynamics with in-line NIR spectroscopy.Implementation of continuous manufacturing in the pharmaceutical industry requires tight process control. This study focuses on a PAT strategy for hot melt extrusion of vegetable calcium stearate (CaSt) as matrix carrier and paracetamol as active pharmaceutical ingredient (API). The extrusion was monitored using in-line near-infrared (NIR) spectroscopy. A NIR probe was located in the section between the extrusion screws and the die, using a novel design of the die channel. A chemometric model was developed based on premixes at defined concentrations and was implemented in SIPAT for real time API concentration monitoring. Subsequently, step experiments were performed for different API concentrations, screw speeds and screw designs.The predicted API concentration was in good agreement with the pre-set concentrations. The transition from one API plateau to another was a smooth curve due to the mixing behaviour of the extruder. The accuracy of the model was confirmed via offline HPLC analysis. The screw design was determined as the main influential factor on content uniformity (CU). Additionally the influence of multiple feeders had a significant impact on CU.The results demonstrate that in-line NIR measurements is a powerful tool for process development (e.g., mixing characterization), monitoring and further control strategies.
Keywords: Hot melt extrusion; Process Analytical Technology (PAT); Near-infrared spectroscopy (NIRS); API content; Quality control;

Risk based approach for design and optimization of stomach specific delivery of rifampicin by Chintan Vora; Riddhish Patadia; Karan Mittal; Rajashree Mashru (169-181).
The research envisaged focuses on risk management approach for better recognizing the risks, ways to mitigate them and propose a control strategy for the development of rifampicin gastroretentive tablets. Risk assessment using failure mode and effects analysis (FMEA) was done to depict the effects of specific failure modes related to respective formulation/process variable. A Box–Behnken design was used to investigate the effect of amount of sodium bicarbonate (X1), pore former HPMC (X2) and glyceryl behenate (X3) on percent drug release at 1st hour (Q1), 4th hour (Q4), 8th hour (Q8) and floating lag time (min). Main effects and interaction plots were generated to study effects of variables. Selection of the optimized formulation was done using desirability function and overlay contour plots. The optimized formulation exhibited Q1 of 20.9%, Q4 of 59.1%, Q8 of 94.8% and floating lag time of 4.0 min. Akaike information criteria and Model selection criteria revealed that the model was best described by Korsmeyer–Peppas power law. The residual plots demonstrated no existence of non-normality, skewness or outliers. The composite desirability for optimized formulation computed using equations and software were 0.84 and 0.86 respectively. FTIR, DSC and PXRD studies ruled out drug polymer interaction due to thermal treatment.
Keywords: Risk management; Gastroretention; Failure mode and effects analysis; Box–Behnken design; Desirability function; Overlay contour plots;

New insights in the production of aerosol antibiotics. Evaluation of the optimal aerosol production system for ampicillin-sulbactam, meropenem, ceftazidime, cefepime and piperacillin-tazobactam by Paul Zarogoulidis; Ioannis Kioumis; Christos Ritzoulis; Dimitris Petridis; Kaid Darwiche; Konstantinos Porpodis; Dionysis Spyratos; Scott Parrish; Robert Browning; Qiang Li; J. Francis Turner; Lutz Freitag; Konstantinos Zarogoulidis (182-188).
Several aerosol antibiotics are on the market and several others are currently being evaluated. Aim of the study was to evaluate the aerosol droplet size of five different antibiotics for future evaluation as an aerosol administration.The nebulizers Sunmist®, Maxineb® and Invacare® were used in combination with four different “small <6 ml” residual cups and two “large <10 ml” with different loadings 2–4–6–8 ml (8 ml only for large residual cups) with five different antibiotic drugs (ampicilln-sulbactam, meropenem, ceftazidime, cefepime and piperacillin-tazobactam). The Mastersizer 2000 (Malvern) was used to evaluate the produced droplet size from each combinationSignificant effect on the droplet size produced the different antibiotic (F  = 96.657, p  < 0.001) and the residual cup design (F  = 68.535, p  < 0.001) but not the different loading amount (p  = 0.127) and the nebulizer (p  = 0.715). Interactions effects were found significant only between antibiotic and residual cup (F  = 16.736, p  < 0.001). No second order interactions were found statistically significant.Our results firstly indicate us indirectly that the chemical formulation of the drug is the main factor affecting the produced droplet size and secondly but closely the residual cup design.
Keywords: Aerosol; Antibiotics; Infection;

Dry foam technology was developed to overcome insufficient oral bioavailability of poorly soluble and wettable active pharmaceutical ingredients (APIs). It is intended to enable a faster and more efficient dissolution by avoiding API agglomeration and floating of non-wetted API particles. The aim of this study was to investigate the influence of process parameters, such as paste water content and type of equipment used on dry foam morphology, granule characteristics and dissolution behavior of the corresponding tablets using indomethacin as model compound. Multiple analytical methods, namely scanning electron microscopy, X-ray micro-computed tomography and mercury porosimetry, specific surface area analysis and sieve analysis were employed. Dissolution of dry foam formulation tablets was compared to a reference formulation in biorelevant media. Process parameters proved to have a distinct influence on dry foam morphology and granule characteristics, correlations between paste viscosity and pore size distribution could be observed. Dissolution behavior of indomethacin was improved by dry foam technology compared to the reference formulation. Variation of process parameters within the studied ranges did not alter the characteristics of the dry foam formulation dissolution behavior. Therefore, dry foam technology seems a promising future technology with the option of continuous manufacturing.
Keywords: Dry foam; Vacuum drying; Process parameters; Indomethacin; Dissolution rate; Maltodextrin;

Monolayered multipolymeric buccal films with drug and polymers of opposing solubilities for ARV therapy: Physico-mechanical evaluation and molecular mechanics modelling by Elsabé Jones; Elizabeth Ojewole; Viness Pillay; Pradeep Kumar; Sanjeev Rambharose; Thirumala Govender (197-212).
Although buccal permeation investigations with antiretroviral drug solutions have confirmed their transbuccal delivery potential, studies on their formulation into delivery systems are lacking. Multipolymeric monolayered films (MMFs) with drugs and polymers of opposing solubilities will offer several advantages for the controlled release delivery of didanosine (DDI) via the buccal route. The aim of this study was to employ a co-blending-co-plasticization technique for preparation of MMFs containing Eudragit® RS 100 (EUD) and Hydroxypropyl methylcellulose (HPMC) and to undertake molecular modelling and in vitro characterizations. Uniform drug content (91–105%) with low variability was obtained for all films. Co-blending of DDI:HPMC:EUD (1:1:10) was required to achieve controlled drug release. The buccal permeability potential of DDI from the MMFs was successfully demonstrated with a permeability coefficient of 0.72 ± 0.14 × 10−2  cm/h and a steady state flux of 71.63 ± 13.54 μg/cm2  h. Films had acceptable mucoadhesivity (2184 mN), mechanical strength (0.698 N/mm2) and surface pH (6.63). The mechanism inherent to the mucoadhesive and drug release profile performance of the MMFs was elucidated via static lattice molecular mechanics simulations wherein a close corroboration among the in vitroin silico (IVIS) data was observed. These extensive physico-mechanical and molecular atomistic studies have confirmed the use of MMFs containing DDI, HPMC and EUD as a buccal delivery system.
Keywords: Didanosine; Buccal; Films; Co-blended polymers; Physico-mechanical properties; Static lattice atomistic simulations;

The dose emitted from a dry powder inhaler is dependent on the pressure change generated by the interaction between inhalation flow and the device's resistance (a). Aerodynamic dose characteristics can be measured at low flows using the above in vitro methodology (b). Consideration should be given to the aerodynamic dose characteristics of the emitted dose from a DPI using a 2 L inhalation volume and a lower pressure change than the recommended 4 kPa (c and d).An interaction between device resistance and inhalation flow provides the ‘energy’ to de-aggregate the metered dose of dry powder inhalers (DPIs). Hence all dry powder inhalers demonstrate flow dependent dose emission but information on this at low flows is not available. We have adapted the compendial method for the Andersen Cascade Impactor (ACI) to include a mixing inlet to determine the aerodynamic dose emission characteristics of a salbutamol Diskus® [DSK], Easyhaler® [EASY] and Clickhaler® [CLICK] and the terbutaline Turbuhaler® [TBH] using flows of 10–60 L/min and inhalation volumes of 2 and 4 L. All DPIs demonstrated flow dependent dose emission (p  < 0.001) but there was no difference in the measurements between 2 and 4 L. The flow dependent dose emission properties of each DPI started to plateau when the pressure change inside each device, during an inhalation, was between 1 and 1.5 kPa. This corresponds to inhalation flows of 40.1–49.1, 25.4–28.9, 23.6–28.9 and 29.7–36.3 L/min through DSK, CLICK, EASY and TBH. The adapted methodology allows measurements at low flows. The results highlight that the compendial methodology to use an inhaled volume of 4 L with the ACI could be replaced by 2 L and that the recommendation to make measurements using a pressure drop of 4 kPa should be revised.
Keywords: Dry powder inhaler; Dose emission; Low flows; Different volumes;

Chitosan nanoparticles: Preparation, size evolution and stability by Antonio Rampino; Massimiliano Borgogna; Paolo Blasi; Barbara Bellich; Attilio Cesàro (219-228).
Characterisation of chitosan-tripolyphosphate nanoparticles is presented with the aim of correlating particle shape and morphology, size distribution, surface chemistry, and production automatisation with preparation procedure, chitosan molecular weight and loaded protein.Nanoparticles were prepared by adding drop wise a tripolyphosphate-pentasodium solution to chitosan solutions under stirring. Trehalose, mannitol and polyethylene-glycol as bioprotectants were used to prevent particle aggregation and to reduce mechanical stress during freezing and drying processes.As a novel result, time evolution of the particle size distribution curve showed the presence of a bimodal population composed of a fraction of small particles and of a second fraction of larger particles attributed to the rearrangement of particles after the addition of tripolyphosphate. Storage for 4 weeks resulted in a slight increase in average size, due to the continuous rearrangement of small particles. Improvement of nanoparticle stability after lyophilisation and spray-drying was observed in the presence of all bioprotectants. Trehalose was the best protectant for both methods. Finally, in vivo tests using chick embryos assessed the biocompatibility of chitosan, tripolyphosphate and the nanoparticles.The simple ionotropic gelation method with low-MW chitosan was effective in achieving reproducible nanoparticles with the desired physico-chemical and safety characteristics.
Keywords: Chitosan nanoparticles; Ionotropic gelation; Nanoparticle ageing; Freeze-drying; Spray drying; Protein carriers;

Evaluation of novel lipid based formulation of β-Artemether and Lumefantrine in murine malaria model by Sushant Patil; Shital Suryavanshi; Sulabha Pathak; Shobhona Sharma; Vandana Patravale (229-234).
The present investigation aims at formulating lipid based drug delivery system of β-Artemether and Lumefantrine and comparative pharmacological evaluation with innovator formulation. Commercial modified oil and indigenous natural fatty acids comprised the oily phase in developing lipidic formulation of β-Artemether and Lumefantrine. The developed system was characterized for mean globule size, stability by freeze thaw cycles, and birefringence. Developed formulation and innovator formulation were compared for their in vivo anti-malarial activity at different dose levels in male Swiss mice, infected with lethal ANKA strain of Plasmodium berghei. The percent parasitemia, activity against time and animal survival period were examined.On fourth day of antimalarial studies, at normal and ½ dose levels, formulations revealed zero percent parasitemia while control showed 33.92 ± 6.00% parasitemia. At 1/10 dose level, developed and innovator formulations revealed zero percent parasitemia upto 11th day, however, three mice from innovator formulation demonstrated recrudescence after 12th day. Both the formulations at normal dose and ½ dose levels showed 100% activity and survival whereas at 1/10 dose level, innovator formulation showed, 62.5% survival. The developed lipidic system of β-Artemether and Lumefantrine exhibited excellent antimalarial activity with 100% survival.
Keywords: Lipid; Artemether; Lumefantrine; Parasitemia; Survival; Recrudescence;

Gelatin–oleic acid nanoparticles distributed with poorly water-soluble drug molecules in hydrophobic inner cores.Recently, we synthesized novel amphiphilic gelatin–oleic acid (GO) conjugate to prepare self-assembled nanoparticles for drug delivery. The aim of this study was to investigate pharmaceutical potentialities of self-assembled GO nanoparticles for solubility enhancement and modified release of poorly water-soluble drugs. Three poorly water-soluble model drugs with different pH-dependent solubility (valsartan and aceclofenac, insoluble at pH 1.2; telmisartan, insoluble at pH 6.8) were chosen to investigate the potential contributions of self-assembled GO nanoparticles to solubility enhancement and controlled release. The particle size of the drug-loaded nanoparticles was 200–250 nm. Zeta potential was calculated, and instrumental analysis such as powder X-ray diffraction (PXRD) and Fourier transform infrared (FT-IR) spectroscopy were used to investigate the physicochemical properties of the drug-loaded nanoparticles. Compared to the drug alone, the drug-loaded nanoparticles showed enhanced solubility. Furthermore, the release profiles of the model drugs were modified in a controlled manner. The current self-assembled GO nanoparticles can provide a versatile potential in drug delivery and tumor targeting.
Keywords: Gelatin–oleic acid conjugate; Self-assembled nanoparticles; pH-dependent solubility; Poorly water-soluble drugs; Enhanced solubility; Modified release;

Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery by Ketan Sharma; Satyanarayana Somavarapu; Agnes Colombani; Nayna Govind; Kevin M.G. Taylor (241-247).
To explore the potential of crosslinked chitosan nanoparticles as carriers for delivery of siRNA using a jet nebuliser.Nanoparticles encapsulating siRNA were prepared using an ionic crosslinking technique at chitosan to siRNA weight/weight ratios of 10:1, 30:1 and 50:1. Particles were characterised for their size, charge, morphology, pH stability and siRNA encapsulation efficiency. Gel electrophoresis was used to assess the association and stability of siRNA with nanoparticles, including after aerosolisation using a Pari LC Sprint jet nebuliser. The aerosolisation properties of FITC labelled chitosan nanoparticles were investigated using a two-stage impinger. Cell viability was performed with H-292 cells using a WST-1 assay.Positively charged spherical nanoparticles were produced with mean diameters less than 150 nm, at all chitosan to siRNA ratios. Nanoparticles were non-aggregated at the pH of the airways and showed high siRNA encapsulation efficiency (>96%). Complete binding of siRNA to chitosan nanoparticles was observed when the w/w ratio was 50:1. Nebulisation produced fine particle fractions of 54 ± 11% and 57.3 ± 1.9% for chitosan and chitosan:siRNA (10:1 w/w) nanoparticles respectively. The stability of chitosan-encapsulated siRNA was maintained after nebulisation. Cell viability was high (>85%) at the highest chitosan concentration (83 μg/ml).The results suggest that crosslinked chitosan nanoparticles have potential for siRNA delivery to the lungs using a jet nebuliser.
Keywords: Chitosan; Inhalation; Nanocarrier; Nanoparticle; Nebuliser; siRNA;

Sildenafil citrate monohydrate–cyclodextrin nanosuspension complexes for use in metered-dose inhalers by Somchai Sawatdee; Hirihattaya Phetmung; Teerapol Srichana (248-258).
Sildenafil is a selective phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction and pulmonary hypertension. Sildenafil citrate monohydrate was complexed with α-, hydroxypropyl-β- and γ-cyclodextrin (α-CD, HP-β-CD and γ-CD, respectively) to enhance its water solubility. The complexes of sildenafil citrate monohydrate with all types of CDs were characterized by phase solubility diagrams, 1H and 13C NMR, and dielectric constants. Sildenafil citrate monohydrate complexed with CDs was developed as nanosuspensions for use in a pressurized metered-dose inhaler (pMDI). Sildenafil citrate monohydrate pMDI formulations were prepared by a bottom-up process using dried ethanol as a solvent and HFA-134a as an antisolvent and propellant in order to form nanosuspensions. A 3 × 3 factorial design was applied for the contents of the dried ethanol and HFA-134a propellant. The phase solubility profiles of the sildenafil and cyclodextrins were described as AL type with a mole ratio 1:1. The piperazine moiety of sildenafil formed an inclusion in the cavity of the CDs. The particle diameters of the sildenafil citrate monohydrate suspensions in pMDIs were all within a nanosuspension size range. An assay of the sildenafil content showed that the formation of complexes with CDs was close to 100%. In the case of the formulations with CDs, the emitted doses varied within 97.4 ± 10.8%, the fine particle fractions (FPFs) were in a range of 45–81%, the fine particle dose (FPD) was 12.6 ± 2.0 μg and the mass median aerodynamic diameters (MMADs) were 1.86 ± 0.41 μm. In contrast, the formulations without CDs produced a low emitted dose of sildenafil (<60%). Therefore, only sildenafil citrate monohydrate pMDI formulations containing CDs were suitable for use as aerosols.
Keywords: Sildenafil citrate; Cyclodextrin; Sildenafil–cyclodextrin complex; Metered-dose inhaler; Nanosuspension; Aerosols;

Poly(ester amide) blend microspheres for oral insulin delivery by Pan He; Huaiyu Liu; Zhaohui Tang; Mingxiao Deng; Yan Yang; Xuan Pang; Xuesi Chen (259-266).
This study developed a novel oral insulin formulation centered on microspheres consisting of a blend of biodegradable poly(ester amide) (PEA). In the formulation, l-lysine-/l-leucine-based PEA with pendant COOH groups (PEA-COOH) was used as a pH-responsive material for the protection of insulin from the harsh environmental conditions of the stomach. Arginine-based PEA (Arg-PEA) was introduced to improve the intestinal absorption of the drug. The influence of both the hydrophobicity of PEA-COOH and the content of Arg-PEA was investigated in detail on microsphere surface morphology, drug loading, and the in vitro release profile of insulin. The PEA-COOH/Arg-PEA blend microspheres protected the loaded insulin in simulated gastric fluid and released insulin in a fast and sustained manner in simulated intestinal fluid. The in vivo test demonstrated that the oral administration of insulin-loaded PEA blend microspheres could effectively suppress the blood glucose level in diabetic rats for 10 h, and the oral bioavailability was improved to 5.89 + 1.84% in healthy rats. These results indicate that the PEA blend microspheres are promising vehicles for the oral delivery of insulin.
Keywords: Poly(ester amide); Blend microspheres; Oral insulin delivery; pH sensitivity;

Preparation and in vitro/in vivo evaluation of fenofibrate nanocrystals by Baoyan Zuo; Yinghua Sun; Hui Li; Xiaohong Liu; Yinglei Zhai; Jin Sun; Zhonggui He (267-275).
The majority of the candidate drug entities exhibit solubility-limiting absorption. Nanocrystal suspensions with particle size in the nanometer scale (nanonization) can increase aqueous solubility and improve oral bioavailability. Regarding the importance of nanosuspension solidification, this study intended to study the critical parameters on redispersed particle size of dried nanocrystals as pretabletting material during spray drying process, such as supporting agents, inlet temperature and feed rate. Fenofibrate with poor water solubility and low melting point was used as a model drug. Nanocrystals of fenofibrate were prepared by a bead-milling method. Five types of hydrophilic excipients in combination with sodium dodecyl sulfate (SDS) were studied as supporting agents during spray drying. The resultant products were characterized by particle size analysis, scanning electron microscopy imaging, differential scanning calorimetry, X-ray powder diffraction and dissolution testing. Spray dried powder with a mean redispersed particle size of 699 nm was produced by using mannitol and SDS as supporting agent. Weight ratio (R F/m) of fenofibrate:mannitol and inlet temperature strongly influenced the particle size of the nanocrystals. The optimal inlet temperature and feed rate was optimized as 75 °C and 4 mL min−1, respectively. Partially transformation of fenofibrate crystalline to the amorphous form was observed. The dissolution profiles of tablets prepared with the spray dried powder were similar to the commercial nanocrystal formulation Lipidil™ ez, and faster than that of the micronized formulation. The relative bioavailability of the spray-dried formulation was determined to be 89.6% taking Lipidil™ ez as the reference. There were no significant statistic differences of AUC0–72 and C max between the two formulations.
Keywords: Fenofibrate; Spray drying; Particle size; Dissolution; Pharmacokinetic;

Liposomes interiorly thickened with thermosensitive nanogels as novel drug delivery systems by Bei Zhang; Jian Chen; Yi Lu; Jianping Qi; Wei Wu (276-284).
The fundamental structure of liposomes suffers from drawbacks of physical instability. To overcome this problem, the hypothesis of this study was to thicken the liposomal interior by incorporating thermosensitive in situ gel. The so called gelliposomes (GLs) were prepared by a thin-film method using poloxamer solutions as interior aqueous phase. Interior thermosensitive gelation was proved by observation of sustained dissolving of the poloxamer gel after destroying the lipid bilayers with Triton X-100; structural transformation as observed under optical microscopy in a heating–cooling circle also proved the fact of interior gelling. The sol–gel transition temperatures of GLs were in good correlation with those of the bulk poloxamer solution counterparts, which could be easily tailored by adjusting the concentration and ratio of poloxamer 407 (P407) to poloxamer 188 (P188). Membrane anisotropy measurement indicated increased membrane rigidity. In vitro release of the model drug cytosine arabinoside from GLs showed sustained release characteristics for at least one week with typical biphasic kinetics. Study on storage stability and protection against the destroying effect by membrane destroyers indicated improved physical stability in comparison with conventional liposomes. In situ evading of phagocytic uptake by macrophages was observed for GLs, which however should be attributed to the effect of exteriorly adsorbed poloxamers. In conclusion, GLs present distinct characteristics to be used as potential drug delivery systems.
Keywords: Liposomes; In situ gel; Gelliposomes; Drug delivery; Poloxamer; Cytosine arabinoside;

A novel lab-scale screen extruder for studying extrusion-spheronisation by M. Zhang; S. Mascia; S.L. Rough; R. Ward; C. Seiler; D.I. Wilson (285-297).
A novel apparatus, the laboratory roller screen extruder (termed the LRS), was developed to replicate key aspects of the geometry and shear strain rates generated near the screen of industrial screen extruders. The configuration of the LRS is reported alongside a commissioning study employing a cohesive 45 wt% water/microcrystalline cellulose paste. The key operating parameters which controlled the extrudate mass flowrate, force on the screen and roller torque were (i) the size of the gap between the top of the roller blade and the screen, and (ii) the roller rotational speed. The data suggest that the apparent shear rate, based on the blade-screen clearance, provides a quantitative criterion for scale-up. The amount of screen flex showed good agreement with a simple bending deformation model. Spheronisation of the extrudates gave pellets with a narrow size distribution and acceptable sphericity which would be acceptable for capsule filling. Optimisation of the pellet shape was not performed. The results indicate that the LRS can be used to assess formulations for industrial screen extrusion-spheronisation.
Keywords: Extrusion; Granulation; Paste; Screen; Spheronisation;

In vitro evaluation of TiO2 nanotubes as cefuroxime carriers on orthopaedic implants for the prevention of periprosthetic joint infections by P. Chennell; E. Feschet-Chassot; T. Devers; K.O. Awitor; S. Descamps; V. Sautou (298-305).
The prevention of periprosthetic joint infections requires an antibiotic prophylactic therapy, which could be delivered locally using titanium dioxide nanotubes as novel reservoirs created directly on the orthopaedic implant titanium surface.In this study, the influence of several parameters that could impact the use of titanium dioxide nanotubes as cefuroxime carriers was investigated.Cefuroxime loading and release was studied for 90 min with three nano-topography conditions (nano-smooth, nano-rugged and nano-tubular), two cefuroxime loading solution concentrations (150 mg/mL and 25 mg/mL) and two nano-tubular crystalline structures.In all tested conditions, maximum amount of cefuroxime was obtained within 2 min. For both cefuroxime loading solution concentrations, nano-smooth samples released the least cefuroxime, and the nano-tubular samples released the most, and a six-fold increase in the concentration of the cefuroxime loading increased the amount of cefuroxime quantified by more than seven times, for all tested nano-topographies. However, the nano-tubes’ crystalline structure did not have any influence on the amount of cefuroxime quantified.The results demonstrated that the surface nano-topography and loading solution concentration influence the efficiency of titanium dioxide nanotubes as cefuroxime carriers and need to be optimized for use as novel reservoirs for local delivery of cefuroxime to prevent periprosthetic infections.
Keywords: Periprosthetic joint infections; Drug delivery systems; Titanium dioxide; Nanotubes; Cefuroxime;

A physical and chemical comparison of material from a conventional spray-dried system and a single particle spray-dried system by Paul T. Whiteside; Jianxin Zhang; Andrew P. Parker; Claire E. Madden-Smith; Nikin Patel; Jesper Jensen; Jakob Sloth; Clive J. Roberts (306-311).
When assessing the suitability of potential drug/polymer systems for improving drug bioavailability, substantial efficiency gains can be achieved through the development and application of rapid miniaturised screening methods. For this to be possible new methods of small-scale formulation manufacture that produce materials equivalent to full-scale manufacture are urgently required. In this work, we use Atomic Force Microscopy (AFM) and Confocal Raman Microscopy (CRM) to investigate the potential physical and chemical equivalence of individually dried particles generated using a DRYING KINETICS ANALYZER™ (DKA) with material from a conventional spray-drier. For our model system of griseofulvin (at loadings of 2.5%, w/w and 20%, w/w) and PEG 6000, the results demonstrate physicochemical equivalence between the two spray-drying methods for the same drug loading. Thus we suggest that single particle spray drying offers a viable and novel route to the production of materials for miniaturised methods of screening candidate drug/polymer formulations.
Keywords: Spray-drying; Bioavailability; Griseofulvin; Screening; Solid-dispersion;

An ovarian cell microcapsule system simulating follicle structure for providing endogenous female hormones by Cong Liu; Xiaoping Xia; Wang Miao; Xiaolu Luan; Liangliang Sun; Yi Jin; Longxiao Liu (312-319).
The aim of this study was to create a microcapsule system simulating native follicle structure by introducing microcarrier culture to microencapsulation for providing endogenous female hormones. Granulosa and theca cells of rat follicles were isolated. Granulosa cells were grown on microcarriers and enclosed together with theca cells in alginate–chitosan–alginate microcapsules. The cell viability and female hormone secretion were investigated in vitro. The microcapsules were transplanted to ovariectomized rats and the serum levels of estradiol and progesterone were measured for 60 days. The microencapsulated granulosa cells growing on microcarriers exhibited enhanced viability and promoted secreting ability of estradiol and progesterone compared with those without the microcarriers. Co-microencapsulation of granulosa cells and theca cells markedly elevated estradiol secretion in vitro. Transplantation of co-microencapsulated granulosa cells on microcarriers and theca cells maintained serum estradiol and progesterone at normal levels for 60 days. Microcarrier cell culture has been proved to be an effective method to enhance the viability of granulosa cells in microcapsules. Moreover, the transplantation of microcapsules enclosing granulosa cells on microcarriers and theca cells may be promising to provide endogenous female hormones for menopausal syndrome treatment.
Keywords: Granulosa cell; Theca cell; Microcarrier culture; Microencapsulation; Estradiol; Progesterone;

Sirolimus-loaded stealth colloidal systems attenuate neointimal hyperplasia after balloon injury: A comparison of phospholipid micelles and liposomes by Azadeh Haeri; Saeed Sadeghian; Shahram Rabbani; Maryam Sotoudeh Anvari; Afsaneh Lavasanifar; Mohsen Amini; Simin Dadashzadeh (320-330).
%Stenosis and number of Ki67 positive cells (indicative of proliferation) were significantly reduced by local therapy with sirolimus-loaded micelles and liposomes. Small phospholipid micelles provided better antirestenotic effects than larger stealth liposomes.Restenosis after angioplasty remains a serious complication in clinical cardiology. This study aims to investigate the stealth colloidal systems for local intra-arterial drug delivery. Micelles from polyethylene glycol conjugated with phosphatidylethanolamine and PEGylated liposomes loaded with sirolimus were prepared and characterized with regard to their loading efficiency, particle size distribution, zeta potential, morphology, nuclear magnetic resonance spectroscopy, drug release profile and stability. The antirestenotic effects of the sirolimus-loaded micelles (14 nm) and liposomes (90 nm) were evaluated and compared in the rat carotid injury model following local intravascular delivery. In comparison to control groups, treatment of balloon injured rats with drug loaded micelles and nanoliposomes significantly reduced vascular stenosis by 42% and 19%, respectively (P  < 0.05). In addition, the luminal area was significantly enlarged by 39% and 60% following treatment with sirolimus-loaded liposomes and micelles, respectively (P  < 0.05). Immunohistochemistry revealed that sirolimus-loaded nanocarriers suppressed cell proliferation (Ki67-positive cells) as compared to control groups without affecting the density of smooth muscle actin staining. These results suggest that both colloidal nanocarriers could serve as effective intramural drug delivery systems for the treatment of restenosis; however, phospholipid based micelles provided better antirestenotic effects than PEGylated liposomes.
Keywords: DSPE–PEG micelles; PEGylated liposomes; Sirolimus; Restenosis; Local delivery;

Ocular delivery systems for poorly soluble drugs: An in-vivo evaluation by C. Luschmann; W. Herrmann; O. Strauß; K. Luschmann; A. Goepferich (331-337).
For highly potent but poorly water-soluble drugs like cyclosporine A, the development of aqueous formulations providing an increase of corneal drug tissue levels, and thus of bioavailability, to increase patient compliance is still a challenge. Therefore, we designed two water-based liquid application systems, an in-situ nanosuspension (INS) and a micellar solution (MS), and tested both formulations in vivo at the rabbit cornea for tolerability and the tissue uptake of CsA. The evaluation of the biological tolerability by periodical eye examination during 180 min and quantification in a defined grading system revealed that the INS evoked minimal to no irritations whereas the MS was perfectly tolerated. After the observation period, the rabbits were sacrificed and the corneal tissue levels of CsA were analyzed. The INS and the MS both showed high levels of 1683 ± 430 ngCsA/gcornea and 826 ± 163 ngCsA/gcornea, respectively, and exceeded drug tissue levels reported for Restasis® (350 ngCsA/gcornea) and cationic emulsions (750 ngCsA/gcornea). These results marked our INS and MS as outstanding novel approaches for the treatment of inflammatory corneal diseases.
Keywords: Surfactant micelles; In-situ nanosuspension; Cyclosporine A; Dry eye disease; Biocompatibility; Bioavailability;

The design trend in tissue-engineering scaffolds based on nanomechanical properties of individual electrospun nanofibers by Biljana Janković; Jan Pelipenko; Miha Škarabot; Igor Muševič; Julijana Kristl (338-347).
This paper especially highlights the finding that the mechanical properties of polymeric nanofibers can be tuned by changing the fiber size as well as the composition. For this purpose, the bending Young's modulus was determined using atomic force microscope by involving single-material (polyvinyl alcohol (PVA), polyethylene oxide (PEO 400K)) and composite nanofibers (polyvinyl alcohol/hyaluronic acid (PVA/HA), polyethylene oxide/chitosan (PEO 400K/CS)). The mechanical property, namely the bending Young's modulus, increases as the diameter of the fibers decreases from the bulk down to the nanometer regime (less than 200 nm). The ranking of increasing stiffness according to the AFM measurements of the three-point beam bending test are in agreement, and can be ranked: PEO 400K < PVA/HA ≈ PVA < PEO < 400K/CS. According to our results, CS-based nanofibers are the stiffest (15 GPa) and the most resilient to erosion in an aqueous medium. Consequently, they possess the most appropriate attributes for bone, tendon, and cartilage tissue scaffold engineering. Nanofibers based on PVA (6 GPa) and PEO (3 GPa) are more elastic (a smaller bending Young's modulus) and therefore are the most suitable for skin and wound tissue scaffolds.
Keywords: Electrospinning; Nanofiber; Atomic force microscopy; Tissue engineering; Elasticity;

Nanostructured lipid carriers loaded with CoQ10: Effect on human dermal fibroblasts under normal and UVA-mediated oxidative conditions by Francesca Brugè; Elisabetta Damiani; Carmelo Puglia; Alessia Offerta; Tatiana Armeni; Gian Paolo Littarru; Luca Tiano (348-356).
Nanostructured lipid carriers (NLC) represent an emerging tool for drug delivery and are characterized by important features which promote increased bioavailability and epithelial penetration of lipophilic compounds. However, despite these advantages, their potential cytotoxicity should not be underestimated, especially under in vivo usage conditions. Here we analyzed the viability, intracellular reactive oxygen species (ROS), oxidative DNA damage and mitochondrial functionality in human dermal fibroblasts (HDF) in the presence of NLC either empty or loaded with the reduced or oxidized form of Coenzyme Q10. Experiments were carried out under standard culture conditions and under oxidative stress induced by UVA irradiation, where the latter treatment significantly affected all the endpoints tested above compared to the non-UVA condition. The data show that NLC alone, whether exposed or not exposed to UVA, produce a slight, though significant decrease in cell viability associated with enhanced oxidative stress, which did not however lead to oxidative DNA damage nor mitochondrial impairment. Reduced CoQ10-NLC, differently from oxidized CoQ10-NLC, were able to efficiently counteract UVA-associated mitochondrial depolarization suggesting a potential role of this molecule in antiageing cosmetological formulations. In conclusion, our results suggest that interactions of NLC with cells and biomolecules should be routinely assessed for understanding their compatibility and toxicity, not only under normal conditions, but also under any chemical or physical stress which these delivery systems might be subjected to during their employment.
Keywords: Nanostructured lipid carriers (NLC); Coenzyme Q10; Oxidative stress; Cytotoxicity; Mitochondrial functionality; UVA;

In rapidly disintegrating tablets (RDTs) manufactured by freeze-drying processes, the % crystallinity of the freeze-dried matrix underpins the physical properties such as mechanical strength and dissolution profile. This study examines the feasibility of using terahertz pulsed spectroscopy (TPS) as an off-line tool (in the first instance) for assessing the degree of crystallinity in co-freeze dried amino-acid/gelatine mixtures.Three amino acids (l-alanine, serine and proline) were studied by TPS (in the wave number range of 3–100 cm−1) both in the pure crystalline form and in the form of a co-freeze-dried matrix with gelatin (in weight fractions of 10:90, 30:70, 50:50, and 70:30).In co-freeze dried proline/gelatin matrix no crystallinity was observed using TPS, whereas ∼62 ± 1% (n  = 3) and 72 ± 0.5% (n  = 3) crystallinity was observed in the co-freeze dried alanine/gelatin matrix (50% w/w and 70% w/w, respectively). Similarly, ∼41 ± 0.5% (n  = 3) and ∼80 ± 0.5% (n  = 3) crystallinity was observed in the serine/gelatin matrix (30% w/w and 50% w/w respectively).The potential for using TPS as a quantitative in-line tool for determining the degree of crystallinity in a range of complex systems such as RDTs, conventional tablets, sprayed dried micro particles, is suggested from these results.
Keywords: Terahertz pulsed spectroscopy; Rapidly disintegrating tablets; Crystallinity; Amino acid; Gelatin; Partial least squares;

Bioavailability enhancement of glucosamine hydrochloride by chitosan by Shuai Qian; Qizhi Zhang; Yanfeng Wang; Benjamin Lee; Guru V. Betageri; Moses S.S. Chow; Min Huang; Zhong Zuo (365-373).
Glucosamine, as a dietary supplement for management of osteoarthritis, has a low and erratic oral bioavailability due to its transport-mediated absorption and presystemic loss in liver and GI tract. The present study described an effective approach to improve glucosamine intestinal absorption and hence its bioavailability using chitosan. Effects of chitosan on intestinal permeability and pharmacokinetics of glucosamine were evaluated in Caco-2 cell monolayer and rats, respectively. In addition, randomized crossover pharmacokinetic studies in beagle dogs were performed to evaluate the oral bioavailabilities of the developed glucosamine oral formulations containing chitosan (QD-Glu solution and QD-Glu tablet) in comparison to its commercial products. Caco-2 permeability studies demonstrated that chitosan could enhance the absorptive transport of glucosamine by 1.9–4.0-fold via the reversible opening of the cell tight junction. After oral administration of glucosamine solutions containing chitosan in rats, it was found that 0.5% (w/v) chitosan exhibited the highest enhancement in C max (2.8-fold) and AUC0−∞ (2.5-fold) of glucosamine. Further pharmacokinetic studies in beagle dogs demonstrated that QD-Glu solution and QD-Glu tablet showed much higher relative bioavailabilities of 313% and 186%, when comparing with Wellesse™ solution and Voltaflex™ tablet, respectively. In conclusion, chitosan could serve as a promising oral absorption enhancer for glucosamine.
Keywords: Glucosamine; Chitosan; Caco-2 cell model; Permeability; Bioavailability;

Respirable microparticles/nanoparticles of the antibiotics vancomycin (VCM) and clarithromycin (CLM) were successfully designed and developed by novel organic solution advanced spray drying from methanol solution. Formulation optimization was achieved through statistical experimental design of pump feeding rates of 25% (Low P), 50% (Medium P) and 75% (High P). Systematic and comprehensive physicochemical characterization and imaging were carried out using scanning electron microscopy (SEM), hot-stage microscopy (HSM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), Karl Fischer titration (KFT), laser size diffraction (LSD), gravimetric vapor sorption (GVS), confocal Raman microscopy (CRM) and spectroscopy for chemical imaging mapping. These novel spray-dried (SD) microparticulate/nanoparticulate dry powders displayed excellent aerosol dispersion performance as dry powder inhalers (DPIs) with high values in emitted dose (ED), respirable fraction (RF), and fine particle fraction (FPF). VCM DPIs displayed better aerosol dispersion performance compared to CLM DPIs which was related to differences in the physicochemical and particle properties of VCM and CLM.In addition, organic solution advanced co-spray drying particle engineering design was employed to successfully produce co-spray-dried (co-SD) multifunctional microparticulate/nanoparticulate aerosol powder formulations of VCM and CLM with the essential lung surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPC), for controlled release pulmonary nanomedicine delivery as inhalable dry powder aerosols. Formulation optimization was achieved through statistical experimental design of molar ratios of co-SD VCM:DPPC and co-SD CLM:DPPC. XRPD and DSC confirmed that the phospholipid bilayer structure in the solid-state was preserved following spray drying. Co-SD VCM:DPPC and co-SD CLM:DPPC dry powder aerosols demonstrated controlled release of antibiotic drug that was fitted to various controlled release mathematical fitting models. The Korsmeyer–Peppas model described the best data fit for all powders suggesting super case-II transport mechanism of controlled release. Excellent aerosol dispersion performance for all co-SD microparticulate/nanoparticulate DPIs was higher than the SD antibiotic drugs suggesting that DPPC acts as an aerosol performance enhancer for these antibiotic aerosol dry powders. Co-SD VCM:DPPC DPIs had higher aerosol dispersion parameters compared to co-SD CLM:DPPC which was related to differences in the physicochemical properties of VCM and CLM.
Keywords: Antibiotic; Solid-state; Particle engineering design; Controlled release lung delivery; Aerosol performance enhancer; Dipalmitoylphosphatidylcholine (DPPC);

Letter to the Editor by Jeff Holst (393).