International Journal of Pharmaceutics (v.434, #1-2)

Polysorbate 80 coated poly (ɛ-caprolactone)–poly (ethylene glycol)–poly (ɛ-caprolactone) micelles for paclitaxel delivery by YuJun Wang; Cheng Wang; ChangYang Gong; YingJing Wang; Gang Guo; Feng Luo; ZhiYong Qian (1-8).
PTX concentrations in different organs after intravenous injection of PTX formulations: (A) brain, (B) heart, (C) liver, (D) spleen, (E) lung, (F) kidney.In this article, polysorbate 80 coated poly (ɛ-caprolactone)–poly (ethylene glycol)–poly (ɛ-caprolactone) (PCEC) micelles were successfully prepared for paclitaxel (PTX) delivery. The particle size distribution, morphology, drug loading, encapsulation efficiency and sustained release profile of the micelles were studied in detail. The safety of the micelle formulation was evaluated by MTT assay on HEK293 cells. And the encapsulated PTX in the micelles remained potent antitumor effect on C6 glioma cells. The pharmacokinetic study showed that the PCEC micelles coated with polysorbate 80 altered the biodistribution pattern and increased PTX concentration in the brain significantly compared to the uncoated micelles and the free drug after intravenous injection. The results indicated that polysorbate 80 coated PCEC micelles might be a candidate for PTX delivery for brain tumor chemotherapy.
Keywords: Poly (ɛ-caprolactone)–poly (ethylene glycol)–poly (ɛ-caprolactone); Paclitaxel; Micelles; Polysorbate 80; Blood–brain barrier;

Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment by Lígia C. Gomes-da-Silva; Adriana O. Santos; Luís M. Bimbo; Vera Moura; José S. Ramalho; Maria C. Pedroso de Lima; Sérgio Simões; João N. Moreira (9-19).
The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety.The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes.Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level.
Keywords: Dual-targeted delivery; Ligand-mediated targeting; Stable nucleic acid lipid particles (SNALP); siRNA; Breast cancer;

Toxicity assessment of extracts from infusion sets in cEND brain endothelial cells by George R. Dakwar; Veronika Kaplun; Lena Kojukarov; Pavel Gorenbein; Ilana Schumacher; Diana Kontorovich; Carola Förster; Elie Beit-Yannai; David Stepensky (20-27).
In vitro safety assessment of disposable medical devices, including infusion sets, is usually performed using L-929 mouse keratinocytes. However, cells of different origin (endothelial, lymphoid and myeloid cells) are also exposed to infusion sets’ extractables during their clinical use. We studied whether the cEND mouse brain endothelial cells can be suitable for in vitro safety assessment of infusion sets. We analyzed infusion sets from different manufacturers that varied in design and storage time. cEND cells were incubated with extracts of individual parts of the infusion sets (tube, cup, latex), and relative toxicities were analyzed using MTT test, DCFH-DA-based analysis of reactive oxygen species formation, apoptosis and cell cycle analyses. We identified a pattern of yellowing of the infusion sets upon storage and revealed that it originated from the latex part. Extracts of the individual parts of the infusion sets, primarily of the latex, were toxic to the cEND cells leading to induction of apoptosis and cell death. We conclude that infusion sets release extractables that can be toxic to the endothelial cells of the patients that receive infusion. We suggest to use cEND cells for in vitro safety assessment of infusion sets and other medical devices that release extractables to the bloodstream.
Keywords: Infusion sets; Disposable medical equipment; Leachables and extractables; Safety; Shelf life;

The kinetics of in vitro drug release from nanoparticulate systems is extensive, though uncritically, being studied by dialysis. Evaluating the actual relevance of dialysis data to drug release was the purpose of this study. Diclofenac- or ofloxacin-loaded chitosan nanoparticles crosslinked with tripolyphosphate were prepared and characterized. With each drug, dynamic dialysis was applied to nanoparticle dispersion, solution containing dissolved chitosan·HCl, and solution of plain drug. Drug kinetics in receiving phase (KRP), nanoparticle matrix (KNM) and nanoparticle dispersion medium (KDM) were determined. Release of each drug from nanoparticles was also assessed by ultracentrifugation. Although KRP data may be interpreted in terms of sustained release from nanoparticles, KNM and KDM data show that, with both drugs, the process was in fact controlled by permeation across dialysis membrane. Analysis of KRP data reveals a reversible interaction of diclofenac with dispersed nanoparticle surface, similar to the interaction of this drug with dissolved chitosan·HCl. No such interactions are noticed with ofloxacin. The results from the ultracentrifugation method agree with the above interpretation of dialysis data. This case study shows that dialysis data from a nanoparticle dispersion is not necessarily descriptive of sustained-release from nanoparticles, hence, if interpreted uncritically, it may be misleading.
Keywords: Chitosan nanoparticles; Sustained release; Dynamic dialysis; Drug binding; Kinetic model;

In this paper we have evaluated the gene expression programming (GEP) methodology for modeling the effect of different variables (continuous and nominal) and their interactions on the properties of direct compression formulations.The effect of four variables was studied; variety of diluents, type and percentage of drug and maximum compression force, on the mechanical and drug release properties of direct compression tablets. The generated database (36 formulations) was used for mathematical and GEP modeling.GEP has been shown to have a high accuracy in prediction for four out five outputs studied including friability which had no replicate measurements. Compared to the traditional statistical treatment GEP is less time consuming and gives equations which are extremely helpful in understanding the interactions of the different variables and for establishing the design space in the development of direct compression formulations.GEP allows similar conclusions than traditional statistical treatment. The helpfulness of this methodology in establishing the design space has been demonstrated. The knowledge derived from GEP can easily be increased by including additional information or new inputs, such as additional drugs or combinations of excipients in the data set.
Keywords: Gene expression programming; Quality by design; Design space; Direct compression tablets; Response Surface Methodology;

Radio-decontamination efficacy and safety studies on optimized decontamination lotion formulation by S. Rana; S. Bhatt; M. Dutta; A.W. Khan; J. Ali; S. Sultana; S. Kotta; S.H. Ansari; R.K. Sharma (43-48).
Objective of the present study was to optimize decontamination lotion and to evaluate its relative decontamination efficacy using three radio-isotopes (Technetium-99m, Iodine-131 and Thallium-201) as contaminants with varying length of contaminant exposure (0–1 h). Experiments were performed on Sprague Dawley rat's intact skin and human tissue equivalent models. Rat's hair was removed by using depilator after trimming with scissors. Relative decontamination efficacy of the optimized lotion was investigated and compared with water as control. Static counts were recorded before and after decontamination using single photon emission computed tomography (SPECT). Measured decontamination efficacy (DE) values were analyzed using one way ANOVA and Student's t-test (p value < 0.05) and were found statistically significant. Decontamination efficacy of the lotion was observed to be 90 ± 5%, 80 ± 2% and 85 ± 2%, for the 131I, 201Tl and 99mTc radio-contaminants respectively on skin. Reduced contaminant removal was recorded for the skin which was cleaned by depilator (50–60%). Skin decontamination was found more efficacious for rat skin decontamination than the human tissue equivalent model. Decontamination efficacy of the lotion against 99mTc was recorded 70 ± 15% at 0–1 h on the tissue equivalent model. In vitro chelation efficacy of the lotion was also established by using the instant thin layer chromatography-slica gel (ITLC-SG) and >95% of 99mTc was recorded. Neither erythema nor edema was scored in the primary skin irritancy test visually observed for two weeks.
Keywords: Decontamination; Technetium-99m; Iodine-131; Thallium-201; Sprague Dawley; Human tissue equivalent;

Electrostatically-driven assembly of hybrid nanoparticles on micro-scale polysaccharide carrier particles and its application in inhaled drug delivery.Lipid–polymer hybrid nanoparticles have emerged as promising nanoscale carriers of therapeutics as they combine the attractive characteristics of liposomes and polymers. Herein we develop dry powder inhaler (DPI) formulation of hybrid nanoparticles composed of poly(lactic-co-glycolic acid) and soybean lecithin as the polymer and lipid constituents, respectively. The hybrid nanoparticles are transformed into inhalable microscale nanocomposite structures by a novel technique based on electrostatically-driven adsorption of nanoparticles onto polysaccharide carrier particles, which eliminates the drawbacks of conventional techniques based on controlled drying (e.g. nanoparticle-specific formulation, low yield). First, we engineer polysaccharide carrier particles made up of chitosan cross-linked with tripolyphosphate and dextran sulphate to exhibit the desired aerosolization characteristics and physical robustness. Second, we investigate the effects of nanoparticle to carrier mass ratio and salt inclusion on the adsorption efficiency, in terms of the nanoparticle loading and yield, from which the optimal formulation is determined. Desorption of the nanoparticles from the carrier particles in phosphate buffer saline is also examined. Lastly, we characterize aerosolization efficiency of the nanocomposite product in vitro, where the emitted dose and respirable fraction are found to be comparable to the values of conventional DPI formulations.
Keywords: Chitosan; Dextran sulphate; Hybrid nanoparticles; Pulmonary delivery; Lipids; Adsorption;

The airway deposition, aerosol behavior and pulmonary fate of inhaled terbutaline submicron particles were studied in experimental rodents by UHPLC/MS technique and these effects were compared with micron sized TBS.Terbutaline submicron particles (SμTBS) were prepared by nanoprecipitation technique followed by spray drying for deep lungs deposition. Inhalable SμTBS particles were 645.16 nm of diameter with 0.11 μm of MMAD, suggested for better aerosol effects. Both submicron and micron-sized TBS particles were administered in rodents administered via major delivery routes, and their biological effects were compared by using UHPLC/ESI-q-TOF-MS method. TBS was found stable in all exposed conditions with 96.28–99.0% of recovery and <4.34% of accuracy (CV). An inhalation device was designed and validated to deliver medicines to lungs, which was found best at dose level of 25 mg for 30 min of fluidization. Both submicron and micron particles were compared for in vivo lung deposition and a 1.67 fold increase in concentration was observed for SμTBS exposed by inhalation. Optimized DPI formulation contained lesser fraction of ultrafine particle (<500 nm) with the major fraction of submicron particles (>500 nm), advocated for better targeting to lungs. UHPLC/ESI-q-TOF-MS confirmed that designed submicron particles has been successfully delivered to the lungs. From tongue to lungs, the landing of pulmonary medicines can be improved by submicronization technology.
Keywords: Submicron terbutaline particle; UHPLC/ESI-q-TOF-MS; Stability effects; Inhalation device; Pulmonokinetics; Biodistribution;

The objective of present investigation was to study in vivo behavior of tacrolimus-loaded lipid-nanoparticles (T-LN) to understand its targeting potential for treatment of atopic-dermatitis-(AD). T-LN have shown significantly improved drug penetration to deeper epidermal and dermal skin-layers than commercial ointment-Protopic® and effectively reached target dendritic-immune-cells, responsible for immunopathogenesis of AD. Due to enhanced penetrability of T-LN, it became necessary to evaluate the toxicity of the nanocarrier and the drug at non-target tissues. This paper evaluates dermatopharmacokinetics (DPK), biodistribution, efficacy and safety of T-LN in comparison to Protopic® as reference. In vivo DPK in guinea pigs showed 3.02-fold higher bioavailability while γ-scintigraphy in albino-rats demonstrated 1.5-fold rapid penetration of radioactivity in skin for T-LN. Biodistribution in albino-rats revealed restricted localization at the target-skin-area with no general spreading to other body organs suggesting targeting potential of T-LN. In vivo efficacy studies in BALB/c mice showed highly efficient suppression of inflammatory AD-like skin-lesions with T-LN than reference and placebo. Dermal toxicity-studies revealed keratosis and collagenous mass-infiltration with repeated application of reference however interestingly, T-LN treated group showed no evident toxicity demonstrating significantly improved safety. Thus T-LN offered improved penetration to the target site without any toxic-effects and would represent an efficient and commercially viable alternative for AD treatment.
Keywords: Drug targeting; Dermatopharmacokinetics; Dermal penetration; Biodistribution; Safety;

Influence of skin model on in vitro performance of drug-loaded soluble microneedle arrays by Martin J. Garland; Katarzyna Migalska; Tuan-Mazlelaa Tuan-Mahmood; Thakur Raghu Raj Singh; Rita Majithija; Ester Caffarel-Salvador; Cian M. McCrudden; Helen O. McCarthy; A. David Woolfson; Ryan F. Donnelly (80-89).
Representative optical coherence tomography images of methylene blue loaded MN dissolving following application to rat skin in vivo. (A) Time = 0 min, (B) time = 3 min, (C) time = 5 min, (D) time = 7 min, (E) time = 10 min, (F) time = 15 min. Scale bar represents a length of 300 μm.A plethora of studies have described the in vitro assessment of dissolving microneedle (MN) arrays for enhanced transdermal drug delivery, utilising a wide variety of model membranes as a representation of the skin barrier. However, to date, no discussion has taken place with regard to the choice of model skin membrane and the impact this may have on the evaluation of MN performance. In this study, we have, for the first time, critically assessed the most common types of in vitro skin permeation models – a synthetic hydrophobic membrane (Silescol® of 75 μm) and neonatal porcine skin of definable thickness (300–350 μm and 700–750 μm) – for evaluating the performance of drug loaded dissolving poly (methyl vinyl ether co maleic acid) (PMVE/MA) MN arrays. It was found that the choice of in vitro skin model had a significant effect on the permeation of a wide range of small hydrophilic molecules released from dissolving MNs. For example, when Silescol® was used as the model membrane, the cumulative percentage permeation of methylene blue 24 h after the application of dissolvable MNs was found to be only approximately 3.7% of the total methylene blue loaded into the MN device. In comparison, when dermatomed and full thickness neonatal porcine skin were used as a skin model, approximately 67.4% and 47.5% of methylene blue loaded into the MN device was delivered across the skin 24 h after the application of MN arrays, respectively. The application of methylene blue loaded MN arrays in a rat model in vivo revealed that the extent of MN-mediated percutaneous delivery achieved was most similar to that predicted from the in vitro investigations employing dermatomed neonatal porcine skin (300–350 μm) as the model skin membrane. On the basis of these results, a wider discussion within the MN community will be necessary to standardise the experimental protocols used for the evaluation and comparison of MN devices.
Keywords: Microneedles; Transdermal drug delivery; Skin model; In vitro release;

In transdermal drug delivery systems (TDDS), it is a challenge to achieve stable and prolonged high permeation rates across the skin since the concentrations of the drug dissolved in the matrix have to be high in order to maintain zero order release kinetics. Several attempts have been reported to improve the permeability of poorly soluble drug compounds using supersaturated systems, however, due to thermodynamic challenges, there was a high tendency for the drug to nucleate immediately after formulating or even during storage. The present study focuses on the efficiency of drug crystals at the submicron/nano range in presence of different solubilizers to improve the permeation rate. Effect of several solubilizers, e.g. Pluronic F-127, Vitamin E TPGS, propylene glycol were studied on the submicron suspension systems of ibuprofen as a model drug. Various stabilizers such as hydroxylpropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) were examined to evaluate their crystal inhibitory effects on particle growth of the drug compound at submicron range. The overall permeation enhancement process through the skin seems to be influenced by the presence of solubilizers and also the presence of submicron drug crystal. The most promising stable formulation was developed with Vitamin E TPGS + HPMC submicron suspension, which produced higher permeation rate compared to other vehicles.
Keywords: Transdermal drug delivery; Wet media milling; Permeation rate; Vitamin E TPGS; Nanoparticles; Crystal growth; Porcine skin;

Hyperbranched PEGmethacrylate linear pDMAEMA block copolymer as an efficient non-viral gene delivery vector by Asha Mathew; Hongliang Cao; Estelle Collin; Wenxin Wang; Abhay Pandit (99-105).
A unique hyperbranched polymeric system with a linear poly-2-dimethylaminoethyl methacrylate (pDMAEMA) block and a hyperbranched polyethylene glycol methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EGDMA) block was designed and synthesized via deactivation enhanced atom transfer radical polymerisation (DE-ATRP) for efficient gene delivery. Using this unique structure, with a linear pDMAEMA block, which efficiently binds to plasmid DNA (pDNA) and hyperbranched polyethylene glycol (PEG) based block as a protective shell, we were able to maintain high transfection levels without sacrificing cellular viability even at high doses. The transfection capability and cytotoxicity of the polymers over a range of pDNA concentration were analysed and the results were compared to commercially available transfection vectors such as polyethylene imine (branched PEI, 25 kDa), partially degraded poly(amido amine)dendrimer (dPAMAM; commercial name: SuperFect®) in fibroblasts and adipose tissue derived stem cells (ADSCs).
Keywords: 2-(Dimethylamino) ethyl methacrylate; Deactivation enhanced atom transfer radical polymerisation; Gene therapy;

The objective of this work was to modulate transdermal drug delivery by iontophoresis though skin microchannels created by microneedles. Calcein and human growth hormone were used as a model small and large molecule, respectively. In vitro permeation studies were performed on porcine ear skin under three different settings: (a) modulated iontophoresis alone, (b) pretreatment with microneedles and (c) combination of microneedles pretreatment and modulated iontophoresis. For modulated iontophoresis, 0.5 mA/cm2 current was applied for 1 h each at 2nd and 6th hour of the study. Methylene blue staining, calcein imaging and pore permeability index suggested maltose microneedles created uniform microchannels in skin. Application of iontophoresis provided two peaks in flux of 1.04 μg/(cm2  h) at 4th hour and 2.09 μg/(cm2  h) at 8th hour of study for calcein. These peaks in flux were significant higher when skin was pretreated with microneedles (p  < 0.05). Similarly, for human growth hormone, modulation in transdermal flux was achieved with combination of microneedles and iontophoresis. This combination also provided significant increase in cumulative amount of calcein and human growth hormone delivered as compared to microneedles or iontophoresis alone (p  < 0.05). Therefore, iontophoresis can be used to modulate drug delivery across skin microchannels created by microneedles.
Keywords: Calcein; Human growth hormone; Microneedle; Iontophoresis; Programmable drug delivery; Transdermal;

Comparison of in vitroin vivo release of Risperdal® Consta® microspheres by Archana Rawat; Upkar Bhardwaj; Diane J. Burgess (115-121).
In vitroin vivo relationship between accelerated in vitro release and in vivo absorption profiles (time scaled) of Risperdal® Consta® 25 mg long acting injection (risperidone microspheres).The objective was to investigate the relationship between in vitro and in vivo release of commercial Risperdal® Consta® microspheres. A modified USP apparatus 4 method was used for accelerated and real-time in vitro release testing. The in vivo plasma profile (clinical data) reported for the product was deconvoluted for comparison with the in vitro release profiles. The in vivo profile differed from the real-time in vitro profile and was faster initially and then slower after approximately 30 days. This effect is considered to be due to differences in the in vivo conditions such as small interstitial volume, low pH and immune response. Accelerated in vitro release profiles obtained at temperatures (50 °C and 54.5 °C) above the microsphere glass transition temperature (Tg ∼ 48 °C) overlapped with the in vivo profile after time scaling. A linear in vitroin vivo relationship was observed with correlation coefficients of 0.97 and 0.99 at 50 °C and 54.5 °C, respectively. The accelerated test performed below the Tg had a similar release profile to that of the real-time in vitro test. The accelerated tests performed above the Tg of the microspheres showed the potential to be used for in vivo performance prediction as well as for quality control purposes.
Keywords: In vitroin vivo relationship (IVIVR); PLGA microspheres; Accelerated in vitro release; USP apparatus 4;

Liquisolid technique to enhance and to sustain griseofulvin dissolution: Effect of choice of non-volatile liquid vehicles by Amal Ali Elkordy; Ebtessam Ahmed Essa; Shreyas Dhuppad; Poojitha Jammigumpula (122-132).
Liquisolid systems were originally designed to enhance dissolution of hydrophobic drugs. Recently, the same technique was explored to control drug release via hydrophobic carriers. This work aimed to study the effects of different liquid vehicles on release characteristics of griseofulvin as a model hydrophobic drug. Fast dissolution tablets were prepared using three different non-ionic surfactants namely Cremophor®EL, Synperonic®PE/L61 and Capryol™ 90, on the contrary Kollicoat®SR 30D was used for production of grieseofulvin sustained release formulations. Avicel® PH102 and Cab-O-Sil® M5 were used as carrier and coat materials, respectively. The effect of formulation parameters, such as drug concentration and carrier to coat ratio, on enhancing drug dissolution was explored. Drug concentrations of 20% and 40% (w/w), and R-values (carrier to coat ratio) of 10 and 20 were used. The mathematical model was utilized to formulate liquisolid powder systems. All fast release liquisolid formulations showed higher percentage drug dissolution efficiency (%DE) than conventional directly compacted tablets. Cremophor®EL showed the best dissolution enhancement with %DE of about 90%, compared to only 23% for conventional tablets; DSC data suggested loss of griseofulvin crystallinity and thermal behavior. Kollicoat® SR 30D retarded the drug release even in the presence of hydrophilic carrier; DSC data suggested that only small fraction of the drug was present in the molecular state within the system. The used liquisolid vehicles showed promise to enhance and to control (depend on the choice of the liquid vehicle) the release of griseofulvin from liquisolid compacts.
Keywords: Liquisolid formulations; Griseofulvin; Cremophor® EL; Synperonic® PE/L61; Capryol™ 90; Kollicoat® SR 30 D;

Dissolution: It is not a dot nor a plot but the story contained in the movie shotFor efficient and effective drug development it is desirable to acquire a deep understanding of the dissolution behaviour of potential candidate drugs and their physical forms as early as possible and with the limited amounts of material that are available at that time. Using 3–10 mg sample quantities, the ability of a UV imaging system is investigated to provide deep mechanistic insight into the intrinsic dissolution profiling of a range of compounds and physical forms assessed under flow conditions. Physical forms of indomethacin, theophylline and ibuprofen were compressed and their solid-state form confirmed before and after compression with X-ray methods and/or Raman spectroscopy. Intrinsic dissolution rates (IDRs) were determined using the compact's UV-imaging profile. The ratio in the IDRs for theophylline anhydrate over hydrate was 2.1 and the ratio for the alpha form of indomethacin over the gamma form was approximately 1.7. The discriminatory power of the novel UV area visualisation approach was shown to be high in that process-induced solid-state dissolution differences post-micronisation could be detected. Additionally, the scale-down system was able to visualise a previously observed increase in ibuprofen IDR with an increase in concentration of sodium dodecyl sulphate. The mechanistic dissolution insights from the visualisation approach are evident.
Keywords: Biorelevant discriminatory dissolution; Intrinsic dissolution; Polymorphism; Salt forms; UV area imaging analysis; Poorly soluble drugs;

This article has been retracted at the request of the Investigation Committee of the University of Colorado, Denver, USA, and the Editor.The Committee has come to the conclusion that author Kadam has knowingly and intentionally falsified and/or fabricated results by manipulating LC-MS/MS peak area data to smooth kinetics and/or alter statistical significance for Figure 3.Data on sustained delivery of triamcinolone acetate were extensively falsified to reduce variability in the results. This appears to falsely support the conclusions regarding the influences on drug delivery reported in the paper.The Committee found no evidence that any of the other authors was aware of and/or participated in any activity amounting to Scientific Misconduct.

Novel formulations of dipyridamole with microenvironmental pH-modifiers for improved dissolution and bioavailability under hypochlorhydria by Chika Taniguchi; Ryo Inoue; Yohei Kawabata; Kazuhiro Yamashita; Koichi Wada; Yukinori Yamauchi; Shizuo Yamada; Satomi Onoue (148-154).
Pharmacokinetic behavior of dipyridamole granule (DPG) with microenvironment pH modifier.This study was undertaken to develop new dipyridamole (DP) formulations with acidic microenvironmental pH-modifiers for improving dissolution and absorption under hypochlorhydric conditions. Dipyridamole granules (DPG) with ten acidic pH-modifiers were prepared with conventional wet granulation, and their manufacturability, stability and dissolution behavior were characterized. Pharmacokinetic profiling of the optimized DPG with acid was carried out in omeprazole-treated rats as a hypochlorhydric model. On the basis of the manufacturability, stability and dissolution behavior of new DPG formulations, p-toluenesulfonic acid (TS) was found to be a suitable acidic pH-modifier for DPG formulation. Although DPG showed pH-dependent dissolution behavior, DPG with TS exhibited a high rate and extent of dissolution in both acidic and neutral media. After oral administration of DPG (10 mg DP/kg) in omeprazole-treated hypochlorhydric rats, there was ca. 40% reduction of the area under the curve of plasma concentration vs. time from zero to 3 h (AUC0–3) for DPG compared with that in normal rats. However, AUC0–3 for DPG/TS under hypochlorhydria was almost identical to that of DPG in normal rats. From these findings, the addition of TS as a microenvironmental pH-modifier in DP formulation might be beneficial in expanding the therapeutic potential of DP in hypochlorhydric patients.
Keywords: Dipyridamole; pH-modifier; Dissolution; Bioavailability; Hypochlorhydric;

Three most commonly used preparative methods, dry-film, reverse phase evaporation and ethanol injection were employed to prepare cationic liposomes composed of DC-Chol and DOPE, respectively. The resulting samples were contrasted through morphology observation, particle size and zeta potential analysis. Sephadex filtration method with high selectivity was developed to determine the encapsulation efficiency of plasmid DNA-loaded cationic vectors, on this basis, cationic liposomes formulation was further optimized by applying Box Behnken design with encapsulation efficiency as evaluation index. The results showed that liposomes prepared by dry-film method were of best quality and stability, moreover, the optimum formulation of cationic liposomes and optimal value of each influencing factors were quantitatively obtained, measured value was highly consistent with predicted results. These findings preliminarily clarified the effect of preparative methods on performance of cationic liposome, as well as formulation factors on encapsulation efficiency, and will provide important methodological reference for further study of liposomes carriers for gene delivery.
Keywords: Cationic liposomes; Preparative methods; Encapsulation efficiency; Box Behnken design; Formulation optimization;

Treatments of paclitaxel with poly(vinyl pyrrolidone) to improve drug release from poly(ɛ-caprolactone) matrix for film-based stent by Fei Lu; Yuan-Yuan Shen; Yan-Qing Shen; Jing-Wen Hou; Zhong-Min Wang; Sheng-Rong Guo (161-168).
Drug-loaded biodegradable films as a principal part of film-based stent were investigated for controlled drug delivery systems. In this study, solid dispersion technique, a pretreatment method of paclitaxel (PTX), was applied to prepare the PTX-loaded poly(ɛ-caprolactone) (PCL) films. Drug dissolution rates and characteristics of the poly(vinyl pyrrolidone) (PVP)/PTX solid dispersions (SDs) and physical mixtures (PMs) were investigated to show that the PVP/PTX SDs were successfully prepared before being incorporated in biodegradable films. Afterwards, the effect of the application of SDs on improving drug release behavior, weightlessness, crystalline states, and surface and internal morphologies of the films were studied. It was found that, the films with SDs showed a higher drug release rate than the films with PMs or pure PTX. In addition, the content of PVP in the SDs also had impact on drug release behavior: the more PVP in SDs, the faster the drug was released. According to the drug release test and weightlessness study, the possible drug release mechanism was put forward for the films with SDs. The application of solid dispersion technique showed a remarkable effect on improving drug release behavior for film-based biodegradable stent drug delivery systems.
Keywords: Solid dispersion; Paclitaxel (PTX); Poly(vinyl pyrrolidone) (PVP); Film; Drug delivery systems;

Idebenone-loaded solid lipid nanoparticles for drug delivery to the skin: In vitro evaluation by Lucia Montenegro; Chiara Sinico; Ines Castangia; Claudia Carbone; Giovanni Puglisi (169-174).
Idebenone (IDE), a synthetic derivative of ubiquinone, shows a potent antioxidant activity that could be beneficial in the treatment of skin oxidative damages. In this work, the feasibility of targeting IDE into the upper layers of the skin by topical application of IDE-loaded solid lipid nanoparticles (SLN) was evaluated. SLN loading different amounts of IDE were prepared by the phase inversion temperature method using cetyl palmitate as solid lipid and three different non-ionic surfactants: ceteth-20, isoceteth-20 and oleth-20. All IDE loaded SLN showed a mean particle size in the range of 30–49 nm and a single peak in size distribution. In vitro permeation/penetration experiments were performed on pig skin using Franz-type diffusion cells. IDE penetration into the different skin layers depended on the type of SLN used while no IDE permeation occurred from all the SLN under investigation. The highest IDE content was found in the epidermis when SLN contained ceteth-20 or isoceteth-20 as surfactant while IDE distribution into the upper skin layers depended on the amount of IDE loaded when oleth-20 was used as surfactant. These results suggest that the SLN tested could be an interesting carrier for IDE targeting to the upper skin layers.
Keywords: Idebenone; Skin permeation; Skin penetration; Solid lipid nanoparticles; Skin delivery;

The influence of plate design on the properties of pellets produced by extrusion and spheronization by Helen Michie; Fridrun Podczeck; John Michael Newton (175-182).
The aim of this work was to produce pellets using a standard formulation by means of extrusion and spheronization. Three different spheroniser friction plate patterns (i.e. cross-hatch, radial, striated edge pattern) have been used in order to investigate whether the plate pattern affects physical properties of the pellets such as pellet size distribution, yield, shape, mechanical strength, density and drug dissolution. Extrusion was performed with a screen extruder and the screen size was varied to determine whether the extrudate produced could affect the physical properties of pellets. The plate load was also varied. Diclofenac sodium was chosen as a model drug. The pattern of the friction plate used in the spheronization of extrudates affected the properties of the pellets. Yield values varied by up to 20%, and for an otherwise optimised formulation the use of a striated edge plate appeared advantageous in this respect. However, these pellets had a reduced mechanical strength despite their lower porosity, which might be disadvantageous. In addition, other factors such as the amount of extrudate loaded into the spheroniser, the maintenance of a constant moisture content within the spheroniser and the size of the extruder screen influenced these findings significantly. The only physical property of the pellets that did not respond to the various changes in the manufacturing process of the pellets is the pellet shape, which remained spherical. The dissolution of the drug appeared to be related to the median pellet size and was only marginally affected by changes in the spheronization process.
Keywords: Cross-hatch plate; Extrusion; Friction plate; Radial plate; Spheronization; Striated edge plate;

Considering that the skin barrier properties are closely linked to the ceramides composition and conformation within the SC, our work focused on developing a new evaluation criterion in complement of the Log Pow and MW: lipids retentive role within the SC. We developed an in vitro model to study exogenous molecules (Mol) and SC lipids interaction by fluorescence spectroscopy. As ceramides do not fluoresce, fluorescence probes that emit a fluorescence signal in contact with lipidic chains were selected for the study. A protocol was developed based on the exogenous molecule (cosmetic actives) affinity for the SC lipids. A fluorescence criterion (ΔI) was calculated from our results and compared to ex vivo skin penetration measurements realized with a Franz cell device. Our results indicated that polarity seems to be very representative of the ceramide and exogenous molecule interaction for most of the molecules tested. However, the ΔI calculated highlighted the particular interaction of some exogenous molecules with ceramides and their skin distribution. This particular behavior was not initially possible to estimate with the Log Pow and MW. This work aimed to develop a new alternative method to enhance the percutaneous penetration estimation of exogenous molecules for the risk analysis.
Keywords: Percutaneous absorption; Ceramides; Fluorescence spectroscopy; Predictive model;

In vitro and in vivo evaluation of N,N,N-trimethylphytosphingosine-iodide (TMP) in liposomes for the treatment of angiogenesis and metastasis by Chung Kil Song; Ju-Hee Lee; Alexander Jahn; Myeong Jun Choi; Sung Keon Namgoong; Soon-Sun Hong; Saeho Chong; Chang-Koo Shim; Suk-Jae Chung; Dae-Duk Kim (191-198).
Phytosphingosine and methyl derivatives are important mediators on cellular processes, and are associated with cell growth and death. The antitumor activity of N,N,N-trimethylphytosphingosine-iodide (TMP) as a novel potent inhibitor of angiogenesis and metastasis was evaluated in B16F10 murine melanoma cells. The results indicated that TMP itself effectively inhibited in vitro cell migration, tube formation, and the expression of angiogenic factors as well as in vivo lung metastasis. However, TMP slightly suppressed in vivo experimental tumor metastasis in its free form and induced side effects including hemolysis and local side effects. Therefore, in an attempt to reduce the toxicity and the undesirable side effects of TMP, a liposomal formulation was prepared and tested for its effectiveness. TMP liposomes retained the effectiveness of TMP in vitro while side effects were reduced, and both in vivo experimental and spontaneous tumor metastasis were significantly suppressed. These results support the conclusion that TMP effectively inhibits in vitro angiogenesis as well as in vivo metastasis, and a liposomal formulation is more efficient delivery system for TMP treatment than solution.
Keywords: N,N,N-Trimethylphytosphingosine-iodide; Liposomes; Angiogenesis; Metastasis;

Calcium phosphate-mediated gene delivery using simulated body fluid (SBF) by Alireza Nouri; Rita Castro; José L. Santos; César Fernandes; João Rodrigues; Helena Tomás (199-208).
The present study aimed at developing a new approach in gene delivery of calcium phosphate nanoparticles through simulated body fluid (CaP-SBF). The physicochemical and biological characteristics of the CaP-SBF nanoparticles were compared with those made in pure water (CaP-water) via a similar procedure. The CaP-SBF and CaP-water solutions were then adjusted to two different pH values of 7.4 and 8.0, mixed with plasmid DNA (pDNA), and added in varying amounts to human embryonic kidney (HEK 293T) cells. The transfection efficiency and cell viability were studied in vitro by reporter gene (luciferase and Enhanced Green Fluorescent Protein) expression and the resazurin reduction assay, respectively, 24 and 48 h after the incubation with the nanoparticles. Our results indicated considerably high in vitro transfection efficiency for CaP-SBF/DNA complexes at physiological pH (7.4) with high amounts of CaP. Additionally, the SBF solution exhibited the ability to reduce the rapid growth of CaP particles over time, leading to higher transfection efficiency of CaP-SBF/DNA complexes than those made in water (CaP-water/DNA).
Keywords: Simulated body fluid (SBF); Calcium phosphate; Gene delivery; Transfection;

Self-assembled peptide (CADY-1) improved the clinical application of doxorubicin by Ying Li; Xuemin Zheng; Zhenghong Cao; Weiren Xu; Jianning Zhang; Min Gong (209-214).
Predicted interactions between doxorubicin and CADY-1. Simulation was carried out at pH 7.0 using the GROMACS program and the all-atom force field GROMACS software. The CADY-1 peptide was solvated in a 3 × 3 matrix containing a single doxorubicin molecule in the middle. The pitch interval of CADY-1 was at 1.2 nm. MD simulations were performed with periodic boundary conditions at constant temperatures at 298 K. Bond lengths were constrained with the SHAKE algorithm and the time step for dynamics was 5 ns. Doxorubicin is shown in sphere model, peptides are shown in ribbon representation and the nearby residues are shown in stick model.CADY-1 is an amphipathic peptide that possesses cell-penetrating activity. As an amphipathic peptide, CADY-1 is capable of forming complexes by self-assembly, and they are these complexes that possess cell-penetrating activity. This distinct characteristic of CADY-1 makes it a potent cell-penetrating drug delivery system. Doxorubicin is a widely used cytotoxic anti-cancer drug but is limited by its toxicity. Although the liposomal formulation of doxorubicin ameliorates its toxicity, its complicated synthesis remains an obstacle to its wide clinical use. In this study, our findings revealed that CADY-1 and doxorubicin form a stable complex at optimised molar ratios in a self-assembling manner. Formation of the complex extended the blood residence time of doxorubicin in a similar fashion to that of liposomal doxorubicin. In addition, the complex was capable of carrying doxorubicin across the cell membrane, which increased the therapeutic index of doxorubicin. Experimental animals treated with a CADY-1/doxorubicin complex exhibited better tolerance and anti-tumour activity than animals treated with either liposomal doxorubicin or the free form of doxorubicin. Collectively, the findings in this study support the advantages of using complexes formed by the self-assembled peptide CADY-1 and suggest that CADY-1 is a potent drug delivery system.
Keywords: Self-assembled peptide; Doxorubicin; Cell penetrating peptide; Blood residence time;

Theophylline radial release from cellulose derivatives with different substitution type (HPMC K4M, HPC H, MC A4M) matrix tablets has been modulated by the introduction of a new inert polymeric excipient, at different proportions (75, 50, 25%). The new polymer was hydroxypropylcellulose-methyl methacrylate (HCMMA), which was dried either in a vacuum oven (OD-HCMMA) or freeze-dried (FD-HCMMA). MC A4M and its mixtures presented the best compaction properties results, especially mixed with FD-HCMMA, according to 100% mixtures. Only high levels of HCMMA (75%) in the matrices showed interesting differences to drug release modulation. Also, at this proportion (75:25), the HPC H mixtures presented the highest differences in relation with OD or FD HCMMA respect to the other cellulose polymers. HPMC K4M and HPC H mixtures showed a combination of diffusion and erosion release mechanisms. The last one was nearly negligible in MC A4M mixtures, according with its highest diffusion rate constant values, and the absence of hydroxypropyl substituents. Only HPMC K4M mixtures presented a diffusion front that moves outwards, while HPC H and MC A4M moves inwards. The modulation of theophylline radial release was obtained using a high percentage of HCMMA, and the use of two cellulosic ethers, one of them with just one type of substituent (MC A4M or HPC H) and the other with two types of substituent (HPMC K4M). Another possibility is changing the HCMMA copolymer (OD or FD) in the 75/25 mixture with HPC.
Keywords: Hydroxypropyl methylcellulose; Hydroxypropyl cellulose; Methyl cellulose; Hydroxypropylcellulose-methyl methacrylate; Substitution type; Release modulation; Drug delivery system; Theophylline;

A nipple shield delivery system for oral drug delivery to breastfeeding infants: Microbicide delivery to inactivate HIV by Stephen E. Gerrard; Mary Lynn Baniecki; David C. Sokal; Mary K. Morris; Sandra Urdaneta-Hartmann; Fred C. Krebs; Brian Wigdahl; Barbara F. Abrams; Carl V. Hanson; Nigel K.H. Slater; Alexander D. Edwards (224-234).
Cross sectional diagram of milk leaving breast passing through nipple shield delivery system insert.A new drug delivery method for infants is presented which incorporates an active pharmaceutical ingredient (API)-loaded insert into a nipple shield delivery system (NSDS). The API is released directly into milk during breastfeeding. This study investigates the feasibility of using the NSDS to deliver the microbicide sodium dodecyl sulfate (SDS), with the goal of preventing mother-to-child transmission (MTCT) of HIV during breastfeeding in low-resource settings, when there is no safer alternative for the infant but to breastfeed. SDS has been previously shown to effectively inactivate HIV in human milk. An apparatus was developed to simulate milk flow through and drug release from a NSDS. Using this apparatus milk was pulsed through a prototype device containing a non-woven fiber insert impregnated with SDS and the microbicide was rapidly released. The total SDS release from inserts ranged from 70 to 100% of the average 0.07 g load within 50 ml (the volume of a typical breastfeed). Human milk spiked with H9/HIVIIIB cells was also passed through the same set-up. Greater than 99% reduction of cell-associated HIV infectivity was achieved in the first 10 ml of milk. This proof of concept study demonstrates efficient drug delivery to breastfeeding infants is achievable using the NSDS.
Keywords: Breastfeeding; Microbicide; Pediatric drug delivery; HIV; Mother-to-child transmission; MTCT; Sodium dodecyl sulfate; SDS; Breast milk;

Development and evaluation of taste-masked drug for paediatric medicines – Application to acetaminophen by Thanh Huong Hoang Thi; Sylvain Morel; Fatma Ayouni; Marie-Pierre Flament (235-242).
The aim of this work was to produce and characterize taste-masked powders of a model drug (acetaminophen) prepared using potentially tolerable and safe excipients for paediatric use, i.e. sodium caseinate and lecithin. The powders were produced by spray-drying aqueous dispersions. The characteristics of taste-masked drug particles were determined by scanning electron microscopy, differential scanning calorimetry and X-ray photoelectron spectroscopy to analyse the surface composition of particles. Taste assessment was approached by an indirect method through drug release studies. We developed a method with a syringe pump using small volumes of aqueous medium and low flow rates, to mimic the behaviour in the mouth. This method was compared to the electronic tongue analysis. SEM, DSC and XPS analysis indicated differences in surface composition of spray-dried particles according to the caseinate/lecithin ratio and to relate it with taste-masking. The “coating” consisting of caseinate and lecithin had a significant role in decreasing the release of drug during the first 2 min and so in taste-masking. Higher content in lecithin results in higher taste-masking efficiency. The association of sodium caseinate and lecithin seems to be promising to mask the bitterness of acetaminophen. A good agreement between release study and electronic tongue analysis was established.
Keywords: Taste-masked drug; Spray-drying; Electronic tongue; X-ray photoelectron spectroscopy (XPS); Drug release studies;

Fully biodegradable and cationic poly(amino oxalate) particles for the treatment of acetaminophen-induced acute liver failure by Hyungmin Kim; Yerang Kim; Kyeonghye Guk; Donghyuck Yoo; Hyungsuk Lim; Gilson Kang; Dongwon Lee (243-250).
Acute inflammatory diseases are one of major causes of death in the world and there is great need for developing drug delivery systems that can target drugs to macrophages and enhance their therapeutic efficacy. Poly(amino oxalate) (PAOX) is a new family of fully biodegradable polymer that possesses tertiary amine groups in its backbone and has rapid hydrolytic degradation. In this study, we developed PAOX particles as drug delivery systems for treating acute liver failure (ALF) by taking the advantages of the natural propensity of particulate drug delivery systems to localize to the mononuclear phagocyte system, particularly to liver macrophages. PAOX particles showed a fast drug release kinetics and excellent biocompatibility in vitro and in vivo. A majority of PAOX particles were accumulated in liver, providing a rational strategy for effective treatment of ALF. A mouse model of acetaminophen (APAP)-induced ALF was used to evaluate the potential of PAOX particles using pentoxifylline (PTX) as a model drug. Treatment of PTX-loaded PAOX particles significantly reduced the activity of alanine transaminase (ALT) and inhibited hepatic cell damages in APAP-intoxicated mice. The high therapeutic efficacy of PTX-loaded PAOX particles for ALF treatment may be attributed to the unique properties of PAOX particles, which can target passively liver, stimulate cellular uptake and trigger a colloid osmotic disruption of the phagosome to release encapsulated PTX into the cytosol. Taken together, we believe that PAOX particles are a promising drug delivery candidate for the treatment of acute inflammatory diseases.
Keywords: Acute liver failure; Inflammation; Poly(amino oxalate); Drug delivery; Microparticles;

The goal of this study was to obtain physically stable non-aqueous in situ forming microparticle (ISM) emulsions capable of forming biodegradable microparticles upon injection. ISM emulsions consist of a biocompatible organic PLGA solution dispersed in a continuous oil phase prepared in a two-syringe/connector system prior to administration. A variety of parenteral approved excipients were tested for a stability-enhancing effect and possible stabilization mechanisms evaluated. Glycerol monostearate (GMS) showed superior stabilizing potential prolonging the emulsion stability from a few minutes to more than 12 h. Flow behavior analysis, differential scanning calorimetry, polarized light- and Cryo-electron microscopy revealed, that the stabilization was caused by an immediate, more than 5-fold viscosity increase in the continuous phase after emulsification and by a stabilized interface through a liquid crystalline GMS layer around the polymer solution droplets. Despite the viscosity increase the injectability of the stabilized ISM emulsion was improved by about 30% compared to the corresponding highly viscous PLGA solution (in situ implant) due to a pronounced shear thinning of the GMS containing oil phase. The injectability improvement allows a faster administration or enables the use of thinner needles and hence reduced patient discomfort.
Keywords: Biodegradable microparticles; Emulsion stability; Injectability; In situ forming; Non-aqueous emulsion; Poly(lactide-co-glycolide);

Multifunctional poly (lactide-co-glycolide) nanoparticles for luminescence/magnetic resonance imaging and photodynamic therapy by Dong Jin Lee; Ga Young Park; Kyung Taek Oh; Nam Muk Oh; Dong Sup Kwag; Yu Seok Youn; Young Taik Oh; Jin woo park; Eun Seong Lee (257-263).
Poly (lactide-co-glycolide) (PLGA) coupled with methoxy poly (ethylene glycol) (mPEG) or chlorin e6 (Ce6) was synthesized using the Steglich esterification method. PLGA-linked mPEG (PLGA-mPEG), PLGA-linked Ce6 (PLGA-Ce6), and Fe3O4 were utilized to constitute multifunctional PLGA nanoparticles (∼160 nm) via the multi-emulsion W1/O/W2 (water-in-oil-in-water) method. The photo-sensitizing properties of Ce6 molecules anchored to PLGA nanoparticles enabled in vivo luminescence imaging and photodynamic therapy for the tumor site. The encapsulation of Fe3O4 allowed high contrast magnetic resonance (MR) imaging of the tumor in vivo. Overall, PLGA nanoparticles resulted in a significant tumor volume regression for the light-illuminated KB tumor in vivo and enhanced the contrast at the tumor region, compared to that of Feridex® (commercial contrast agent).
Keywords: Poly (lactide-co-glycolide); Fe3O4; Photodynamic therapy (PDT); Magnetic resonance imaging (MRI);

Enhanced solubility and oral bioavailability of itraconazole by combining membrane emulsification and spray drying technique by Young Keun Choi; Bijay K. Poudel; Nirmal Marasini; Kwan Yeol Yang; Jeong Whan Kim; Jong Oh Kim; Han-Gon Choi; Chul Soon Yong (264-271).
The objective of the present study was to enhance solubility and bioavailability of itraconazole by a combined use of membrane emulsification and spray drying solidification technique. A shirasu-porous-glass (SPG) membrane with a mean pore size of 2.5 μm was used to produce monodispersed microemulsions of itraconazole consisting of methylene chloride as the dispersed phase, a mixture of Transcutol HP and Span 20 as a stabilizer, and dextran as solid carrier dissolved in water as the continuous phase. The dispersed phase permeated through the SPG membrane into the continuous phase at an agitator speed of 150 rpm, a feed pressure of 15 kPa and a continuous phase temperature of 25 °C and the resultant emulsion was solidified using spray-drying technique. Solid state characterizations of the solid emulsion showed that the crystal state of itraconazole in solid emulsion was converted from crystalline to amorphous form. The solid emulsion of itraconazole displayed a significant increase in the dissolution rate than that of pure itraconazole. Furthermore, the solid emulsion after oral administration gave about eight-fold higher AUC and about ten-fold higher C max in rats than pure itraconazole powder (p  < 0.05), indicating this formulation greatly improved the oral bioavailability of drug in rats. Thus, these results demonstrated that the SPG membrane emulsification system combined with spray-drying technique could be used as a promising technique to develop solid formulation of itraconazole with enhanced solubility and bioavailability.
Keywords: Membrane emulsification; Spray-drying; Itraconazole; Solubility; Bioavailability;

Exploring the effects of high shear blending on lactose and drug using fluidised bed elutriation by J.P. Willetts; P.T. Robbins; T.C. Roche; M. Bowley; R.H. Bridson (272-279).
Powder formulations comprising inhalation grade lactose and a mimic drug (cholesterol) were prepared using a high shear blending process for which the total energy input could be quantified. The formulations were fluidised in a classic fluidised bed system, to determine whether blending-induced changes could be determined through either bulk fluidisation behaviour or the characteristics of elutriated fractions from the powder beds. The evolution of the fluidisation regime within the powder beds (Δ pressure vs. superficial gas velocity) and total mass of elutriated material were not sensitive measures to differentiate between blended and unblended samples. However, blended and unblended material could be distinguished by the size distributions of the elutriated fractions. The study also showed that there were no further changes in the size distribution of the elutriated fractions once a chemically homogenous mixture of lactose and drug had been produced. However, further blending beyond this ‘point of homogeneity’ continued to change the lactose particle size distribution of the bulk powder; this may have implications for blend end point determination for these types of formulation.
Keywords: Dry powder inhaler; High shear blending; Lactose; Fluidisation; Elutriation;

In vitro methods are commonly used in order to estimate the extent of systemic absorption of chemicals through skin. Due to the wide variability of experimental procedures, types of skin and data analytical methods, the resulting permeation measures varies significantly between laboratories and individuals. Inter-laboratory and inter-individual variations with the in vitro measures of skin permeation lead to unreliable extrapolations to in vivo situations. This investigation aimed at a comprehensive assessment of the available data and development of validated models for in vitro skin flux of chemicals under various experimental and vehicle conditions.Following an exhaustive literature review, the human skin flux data were collated and combined with those from EDETOX database resulting in a dataset of a total of 536 flux reports. Quantitative structure–activity relationship techniques combined with data mining tools were used to develop models incorporating the effects of permeant molecular structure, properties of the vehicle, and the experimental conditions including the membrane thickness, finite/infinite exposure, skin pre-hydration and occlusion.The work resulted in statistically valid models for estimation of the skin flux from varying experimental conditions, including relevant real-world mixture exposure scenarios. The models indicated that the most prominent factors influencing flux values were the donor concentration, lipophilicity, size and polarity of the penetrant, and the melting and boiling points of the vehicle, with skin occlusion playing significant role in a non-linear way. The models will aid assessment of the utility of dermal absorption data collected under different conditions with broad implications on transdermal delivery research.
Keywords: QSAR; Skin; Dermal; Permeation; Absorption; In vitro; Decision tree; Finite dosing; Occlusion; Hydration;

Synthesis, characterization of chitosan–tripolyphosphate conjugated chloroquine nanoparticle and its in vivo anti-malarial efficacy against rodent parasite: A dose and duration dependent approach by Satyajit Tripathy; Sabyasachi Das; Subhankari Prasad Chakraborty; Sumanta Kumar Sahu; Panchanan Pramanik; Somenath Roy (292-305).
Various strategies to deliver antimalarials using nanocarriers have been evaluated. However, taking into account the peculiarities of malaria parasites, the focus is placed mainly polymer-based chitosan nanocarriers. Our purpose of the study is to develop chitosan–tripolyphosphate (CS–TPP) nanoparticles (NPs) conjugated chloroquine in application for attenuation of Plasmodium berghei infection in Swiss mice. NPs were prepared by ionotropic gelation between CS and sodium TPP. In the study, the interaction of CS and TPP and the presence of chloroquine at the surface of chitosan–TPP NPs have been investigated by means of different methods like FTIR, DLS, and zeta potential. After drug preparation, effective dose of the nanoconjugated chloroquine (Nch) among 100, 250, and 500 mg/kg bw/day, was studied against P. berghei infection in Swiss mice by blood smear staining and biochemical assay of different inflammatory markers, and antioxidant enzyme levels also performed. After evaluating the effective dose, dose-dependent duration study was performed for 5, 10, 15 days. From the present study the maximum effect of Nch was found at 250 mg/kg bw concentration for 15 days treatment. So, this Nch might have potential of application as therapeutic anti-malarial and antioxidant agent.
Keywords: Malaria; Chitosan; Tripolyphosphate; Nanochloroquine; Anti-malarial drug; Plasmodium berghei;

The objectives of this study were (i) to characterize the interaction of vandetanib with P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) in vitro and in vivo (ii) to study the modulation of P-gp and BCRP mediated efflux of vandetanib with specific transport inhibitors and m-TOR inhibitors, everolimus and temsirolimus. Cellular accumulation and bi-directional transport studies in MDCKII cell monolayers were conducted to delineate the role of efflux transporters on disposition of vandetanib. Brain distribution studies were conducted in male FVB wild-type mice with vandetanib administered intravenously either alone or in the presence of specific inhibitors and m-TOR inhibitors. In vitro studies suggested that vandetanib is a high affinity substrate of Bcrp1 but is not transported by P-gp. Interestingly, in vivo brain distribution studies in FVB wild type mice indicated that vandetanib penetration into the brain is restricted by both Bcrp1 and P-gp mediated active efflux at the blood brain barrier (BBB). Co-administration of elacridar, a dual P-gp/BCRP inhibitor increased the brain to plasma concentration ratio of vandetanib upto 5 fold. Of the two m-TOR pathway inhibitors examined; everolimus showed potent effect on modulating vandetanib brain penetration whereas no significant affect on vandetanib brain uptake was observed following temsirolimus co-administration. This finding could be clinically relevant as everolimus can provide synergistic pharmacological effect in addition to primary role of vandetanib efflux modulation at BBB for the treatment of brain tumors.
Keywords: P-glycoprotein; Breast cancer resistant protein; Vandetanib; Brain; Efflux; Pharmacokinetics;

Targeted lipid based drug conjugates: A novel strategy for drug delivery by Aswani Dutt Vadlapudi; Ramya Krishna Vadlapatla; Deep Kwatra; Ravinder Earla; Swapan K. Samanta; Dhananjay Pal; Ashim K. Mitra (315-324).
A majority of studies involving prodrugs are directed to overcome low bioavailability of the parent drug. The aim of this study is to increase the bioavailability of acyclovir (ACV) by designing a novel prodrug delivery system which is more lipophilic, and at the same time site specific. In this study, a lipid raft has been conjugated to the parent drug molecule to impart lipophilicity. Simultaneously a targeting moiety that can be recognized by a specific transporter/receptor in the cell membrane has also been tethered to the other terminal of lipid raft. Targeted lipid prodrugs i.e., biotin-ricinoleicacid-acyclovir (B-R-ACV) and biotin-12hydroxystearicacid-acyclovir (B-12HS-ACV) were synthesized with ricinoleicacid and 12hydroxystearicacid as the lipophilic rafts and biotin as the targeting moiety. Biotin-ACV (B-ACV), ricinoleicacid-ACV (R-ACV) and 12hydroxystearicacid-ACV (12HS-ACV) were also synthesized to delineate the individual effects of the targeting and the lipid moieties. Cellular accumulation studies were performed in confluent MDCK-MDR1 and Caco-2 cells. The targeted lipid prodrugs B-R-ACV and B-12HS-ACV exhibited much higher cellular accumulation than B-ACV, R-ACV and 12HS-ACV in both cell lines. This result indicates that both the targeting and the lipid moiety act synergistically toward cellular uptake. The biotin conjugated prodrugs caused a decrease in the uptake of [3H] biotin suggesting the role of sodium dependent multivitamin transporter (SMVT) in uptake. The affinity of these targeted lipid prodrugs toward SMVT was studied in MDCK-MDR1 cells. Both the targeted lipid prodrugs B-R-ACV (20.25 ± 1.74 μM) and B-12HS-ACV (23.99 ± 3.20 μM) demonstrated higher affinity towards SMVT than B-ACV (30.90 ± 4.19 μM). Further, dose dependent studies revealed a concentration dependent inhibitory effect on [3H] biotin uptake in the presence of biotinylated prodrugs. Transepithelial transport studies showed lowering of [3H] biotin permeability in the presence of biotin and biotinylated prodrugs, further indicating a carrier mediated translocation by SMVT. Overall, results from these studies clearly suggest that these biotinylated lipid prodrugs of ACV possess enhanced affinity towards SMVT. These prodrugs appear to be potential candidates for the treatment of oral and ocular herpes virus infections, because of higher expression of SMVT on intestinal and corneal epithelial cells. In conclusion we hypothesize that our novel prodrug design strategy may help in higher absorption of hydrophilic parent drug. Moreover, this novel prodrug design can result in higher cell permeability of hydrophilic therapeutics such as genes, siRNA, antisense RNA, DNA, oligonucleotides, peptides and proteins.
Keywords: Acyclovir; Targeted lipid prodrugs; Biotin; SMVT; Affinity; Drug delivery;

Enhanced oral bioavailability of novel mucoadhesive pellets containing valsartan prepared by a dry powder-coating technique by Qing-Ri Cao; Yan Liu; Wei-Juan Xu; Beom-Jin Lee; Mingshi Yang; Jing-Hao Cui (325-333).
The aim of this study was to develop novel mucoadhesive pellets containing valsartan (VAL) with enhanced oral bioavailability. Two types of VAL loaded core pellets were prepared by an extrusion/spheronization method, and further dry-coated with a mixture of hydroxypropylmethylcellulose (HPMC) and carbomer (CB) at different ratios. The effects of the pellet core composition, HPMC:CB ratio and coating level on the drug release from the coated pellets were investigated. The physicochemical properties of the core and coated pellets were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In addition, the in vitro and in vivo mucoadhesion properties as well as the bioavailability of the coated pellets in rats were evaluated by using VAL suspension and core pellets as control preparations. The results of the release study demonstrated that the two types of core pellets, especially the pellets formulated with a solubilizer and a pH modulator gave considerably faster drug release than the VAL powder. However, the core and coated pellets exhibited similar release profiles indicating that the dry powder-coating did not retard the drug release. Strong molecular interactions were observed between the drug and the carriers in FT-IR analysis. The coated pellets displayed distinct mucoadhesive property in vitro and delayed gastrointestinal (GI) transit in vivo. Furthermore, the coated pellets exhibit significantly higher AUC0–12 h and C max, as compared to the core pellets and drug suspension. It was concluded that the mucoadhesive pellets could render poorly water soluble drugs like VAL with a rapid drug release, delayed GI transit and enhanced oral bioavailability.
Keywords: Valsartan; Mucoadhesive pellet; Rapid drug release; Delayed GI transit; Dry powder-coating; Bioavailability;

Contact drying: A review of experimental and mechanistic modeling approaches by Ekneet Kaur Sahni; Bodhisattwa Chaudhuri (334-348).
Drying is one of the most complex unit operations with simultaneous heat and mass transfer. The contact drying process is also not well understood as several physical phenomena occur concurrently. This paper reviews current experimental and modeling approaches employed towards a better understanding of the contact drying operation. Additionally, an overview of some fundamental aspects relating to contact drying is provided. A brief discussion of some model extensions such as incorporation of noncontact forces, interstitial fluids and attrition rate is also presented.
Keywords: Contact drying; PAT approaches; Mechanistic modeling;

Application of quality by design to formulation and processing of protein liposomes by Xiaoming Xu; Antonio P. Costa; Mansoor A. Khan; Diane J. Burgess (349-359).
Quality by design (QbD) principles were explored in the current study to gain a comprehensive understanding of the preparation of superoxide dismutase (SOD) containing liposome formulations prepared using freeze-and-thaw unilamellar vesicles (FAT-ULV). Risk analysis and D-optimal statistical design were performed. Of all the variables investigated, lipid concentration, cholesterol mol%, main lipid type and protein concentration were identified as critical parameters affecting SOD encapsulation efficiency, while the main lipid type was the only factor influencing liposome particle size. Using a model generated by the D-optimal design, a series of three-dimensional response spaces for SOD liposome encapsulation efficiency were established. The maximum values observed in the response surfaces indirectly confirmed the existence of a specific SOD–lipid interaction, which took place in the lipid bilayer under the following optimal conditions: (1) appropriate membrane thickness and curvature (DPPC liposomes); and (2) optimal “pocket size” generated by cholesterol content. With respect to storage stability, the prepared SOD liposomes remained stable for at least 6 months in aqueous dispersion state at 4 °C. This research highlights the level of understanding that can be accomplished through a well-designed study based on the philosophy of QbD.
Keywords: Liposome; QbD; Superoxide dismutase; Freeze-and-thaw unilamellar vesicles (FAT-ULV); Encapsulation efficiency; D-optimal design; Design space; Risk analysis;

Thymol nanospheres as an effective anti-bacterial agent by Anna Wattanasatcha; Sirirat Rengpipat; Supason Wanichwecharungruang (360-365).
Among thymol, carvacrol, citronellal, eugenol and terpinen-4-ol, thymol showed the highest antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Thymol was then encapsulated into water dispersible submicron sized ethylcellulose/methylcellulose spheres, attaining the relatively high thymol loading level of 43.53% (weight of encapsulated thymol to weight of the thymol-loaded spheres). When tested against the same three bacterial strains, the encapsulated thymol gave comparable minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) values to the unencapsulated compound while mostly showing lower MIC and MBC values than the conventionally used preservative, methyl-p-hydroxybenzoate (methylparaben). The use of encapsulated thymol at 0.078, 0.156 and 0.625 mg ml−1 (0.52, 1.04 and 4.16 mmol−1, respectively) in cosmetic lotion formulations provided total suppression of viable E. coli, S. aureus and P. aeruginosa growth (all initially seeded at 105  cfu ml−1), respectively, over the three month test period, whereas unencapsulated thymol showed effective suppression for only 2–4 weeks. Effective bacterial suppression by encapsulated thymol was also observed when used in cream and aqueous gel cosmetic formulations.
Keywords: Encapsulation; Thymol; Antibacterial activity; Nanoparticle; Preservative;

Sulfated β-cyclodextrin was synthesized from sulfonation of β-cyclodextrin and sulfated polymer was crosslinked with cotton fabric using ethylenediaminetetraacetic acid as crosslinker. ZnO, TiO2 and Ag nanoparticles were prepared and characterized by XRD, UV, DLS, SEM and PSA. The prepared nanoparticles were coated on crosslinked cotton fabric. The crosslinking and nanoparticles coating effects of cotton fabrics were studied by FTIR and SEM analysis. The antibacterial test was done against gram positive Staphylococcus aureus and gram negative Escherichia coli bacterium.
Keywords: Sulfonation; Pad-dry-cure; β-Cyclodextrin; Crosslinking; Antibacterial activity; Nanoparticles;

Alginate based bilayer hydrocolloid films as potential slow-release modern wound dressing by Hnin-Ei Thu; Mohd Hanif Zulfakar; Shiow-Fern Ng (375-383).
The aims of this research were to develop a novel bilayer hydrocolloid film based on alginate and to investigate its potential as slow-release wound healing vehicle. The bilayer is composed of an upper layer impregnated with model drug (ibuprofen) and a drug-free lower layer, which acted as a rate-controlling membrane. The thickness uniformity, solvent loss, moisture vapour transmission rate (MVTR), hydration rate, morphology, rheology, mechanical properties, in vitro drug release and in vivo wound healing profiles were investigated. A smooth bilayer film with two homogenous distinct layers was produced. The characterisation results showed that bilayer has superior mechanical and rheological properties than the single layer films. The bilayers also showed low MVTR, slower hydration rate and lower drug flux in vitro compared to single layer inferring that bilayer may be useful for treating low suppurating wounds and suitable for slow release application on wound surfaces. The bilayers also provided a significant higher healing rate in vivo, with well-formed epidermis with faster granulation tissue formation when compared to the controls. In conclusions, a novel alginate-based bilayer hydrocolloid film was developed and results suggested that they can be exploited as slow-release wound dressings.
Keywords: Bilayer film; Alginate; Slow-release; Hydrocolloid dressing; Wound dressing; Wound healing;

Ultrasound-triggered thrombolysis using urokinase-loaded nanogels by Haiqiang Jin; Hui Tan; Lingling Zhao; Weiping Sun; Lijun Zhu; Yongan Sun; Hongjun Hao; Haiying Xing; Linlin Liu; Xiaozhong Qu; Yining Huang; Zhenzhong Yang (384-390).
To find a way to modulate the effect of thrombolytic proteins by increasing their specificity, minimizing their adverse effect as well as lengthening their circulation time for the treatment of ischemic vascular disease holds great promise. In this work, urokinase-type plasminogen activator (uPA) was encapsulated into hollow nanogels which are generated by the reaction of glycol chitosan and aldehyde capped poly(ethylene glycol) (OHC-PEG-CHO) through a one-step approach of ultrasonic spray. The uPA-loaded nanogels, with size of 200–300 nm, have longer circulation time than that of the nude urokinase in vivo, besides the protein can be triggered to release in faster rate under diagnostic ultrasonic condition of 2 MHz, which significantly enhanced the thrombolysis of clots. The results are promising for increasing the specificity and positive effects of thrombolytic agents like recombinant tissue plasminogen activator (rt-PA) for the current treatment of ischemic vascular disease.
Keywords: Hollow nanogel; Ultrasound responsive; Urokinase-type plasminogen activator; Thrombolysis;

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles by Lais Alonso; Sebastião Antônio Mendanha; Cássia Alessandra Marquezin; Marina Berardi; Amando Siuiti Ito; A. Ulises Acuña; Antonio Alonso (391-398).
Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50 °C. This effect becomes negligible at temperatures higher that ca. 60 °C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis.
Keywords: EPR; Fluorescence; Spin label; Miltefosine; Stratum corneum; Phospholipid bilayer;

The effects of betamethasone dipropionate and fish oil on HaCaT proliferation and apoptosis by Mohd Hanif Zulfakar; Charlene M.Y. Ong; Charles M. Heard (399-405).
The current work examined the effect of fish oil (FO) and betamethasone dipropionate (BD) on the growth of immortalized HaCaT keratinocytes. HaCaT cells were grown and treated with FO and/or BD, and proliferation determined using the MTT method. The cells were further probed by immunocytochemistry (ICC) techniques for apoptosis using Cleaved Caspase-3 Asp175, and inflammatory processes using cyclooxygenase-2 (COX-2). The addition of FO increased the inhibition of HaCaT cells by 27.2%, from 43.15% to 70.35% compared to BD alone (p 0.034). FO alone appeared to induce expression of Asp175 and the effect was greater in combination with BD. The net effect, however, were less than BD alone. Similar observations were seen with regards to COX-2 inhibition. The added benefits of FO to the effect of BD on the inhibition of cell growth, induction of apoptosis and inhibition of inflammation have now been demonstrated on a cellular level. Each of these activities supports beneficial effects in hyperproliferative skin disorders, such as psoriasis.
Keywords: Betamethasone dipropionate; Fish oil; EPA; Psoriasis; HaCaT; Cell culture; Apoptosis;

Utility of in situ sodium alginate/karaya gum gels to facilitate gastric retention in rodents by Kimberly A. Foster; Mike Morgen; Brice Murri; Ian Yates; R. Marcus Fancher; Jon Ehrmann; Olafur S. Gudmundsson; Michael J. Hageman (406-412).
Barium sulfate suspension (left) and in situ gel containing barium sulfate (right) 1 h post-dose.Target validation or demonstration of efficacy requires adequate in vivo exposure of tool molecules to determine their activity in order to validate the model or show the potential usefulness of the pharmacophore. Early discovery work is often carried out with compounds which possess undesirable PK properties in small rodents where the discovery formulation scientist is often forced to dose 2–4 times per day. Gastric retentive formulations in small rodents (rats/mice) could enable increased duration of exposure for compounds with narrow absorption windows or increased residence time for compounds with targets located in the GI tract. The aim of this work is to establish an easily administered gastric retentive gel for rodents in situ using a mixture of sodium alginate and karaya gum. Feasibility studies were conducted in Sprague-Dawley rats using barium sulfate as a radio-opaque tracer. The results show that gastric retention of barium was achieved for rats dosed with the gel formulation relative to a barium suspension. The gastric residence time of the gel varied from 1 h to >8 h (n  = 3). The data suggest that sodium alginate/karaya gum gels may be a useful tool to achieve gastric retention in rodent studies.
Keywords: Alginate; Formulation; Controlled release; Hydrogels; Digital imaging; Gastroretention;

To overcome the poor aqueous solubility of docetaxel (DTX) and the side effects of the emulsifier in the marketed formulation, we have developed a DTX-loaded micelle using a nontoxic and biodegradable amphiphilic diblock copolymer, methoxy polyethylene glycol-distearoylphosphatidylethanolamine (mPEG2000-DSPE). The prepared micelles exhibited a core–shell structure, and DTX was successfully encapsulated in the core with an encapsulation efficiency of 97.31 ± 2.95% and a drug loading efficiency of 3.14 ± 0.13%. The micelles were spherical with a hydrodynamic diameter of approximately 20 nm, which could meet the requirement for in vivo administration, and were expected to enhance the drug's antitumor efficacy and to decrease its toxicity. To evaluate the DTX-loaded micelles, we chose a well marketed formulation, Taxotere®, as the control. The prepared DTX micelle had a similar antiproliferative effect to Taxotere® in vitro but a significantly better antitumor efficacy than Taxotere® in vivo, which may be caused by passive targeting of the tumor by the micelles. In addition, the safety evaluation revealed that the DTX micelle was a qualified drug for use in vivo. Based on the experimental results, we propose that mPEG2000-DSPE micelle is a potent carrier for DTX.
Keywords: Docetaxel (DTX); Micelle; Antitumor efficacy; Safety evaluation; Solubilization;

Decylglucoside-based microemulsions for cutaneous localization of lycopene and ascorbic acid by Dominique Pepe; Jessica Phelps; Kevin Lewis; Jared DuJack; Katrina Scarlett; Sumaya Jahan; Emilee Bonnier; Tatjana Milic-Pasetto; Martha A. Hass; Luciana B. Lopes (420-428).
Cutaneous delivery of combinations of antioxidants offers the possibility of enhanced protection against UV-radiation. In this study, we investigated the potential of sugar-based microemulsions containing monoglycerides to promote simultaneous cutaneous delivery of lycopene and ascorbic acid, and increase tissue antioxidant activity. Lycopene and ascorbic acid were incorporated (0.04% and 0.2% (w/w), respectively) in decylglucoside-based microemulsions containing isopropyl myristate mixed with monocaprylin (ME-MC), monolaurin (ME-ML) or monoolein (ME-MO) as oil phase. The microemulsions increased lycopene delivery into porcine ear skin by 3.3- to 8-fold compared to a drug solution. The effect of microemulsions on ascorbic acid cutaneous delivery was more modest (1.5–3-fold), and associated with an approximately 2-fold increase in transdermal delivery. According to their penetration-enhancing ability, the microemulsions were ranked ME-MC > ME-MO > ME-ML. This superiority of ME-MC coincided with a stronger effect in decreasing skin electrical resistance. After 18 h of treatment, the viability of bioengineered skin treated with ME-MC was 2.2-times higher compared to Triton-X100 (moderate irritant), demonstrating that ME-MC is less cytotoxic. Skin treatment with ME-MC containing both antioxidants increased the tissue antioxidant activity by 10.2-fold, but no synergism between the antioxidants was observed.
Keywords: Lycopene; Ascorbic acid; Skin; Microemulsion; Topical delivery; Monoglycerides;

PEGylation of an osteoclast inhibitory peptide: Suitable candidate for the treatment of osteoporosis by Jabed Akhtar; Vandana Mallaredy; Jagneshwar Dandapat; Prasanta Maiti; Sanjeeb K. Sahoo; Sujay Singh (429-436).
Osteoporosis is a condition of bone loss due to excessive osteoclastic activity. Several protein factors, such as receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), have been identified that are important in the pathogenesis of osteoporosis. RANKL binds to RANK and activates the NF-κB pathway by interaction of its cytoplasmic domain with an intracellular adapter protein, TNF receptor associated factors 6 (TRAF 6). This interaction can be inhibited by cell-permeable peptides that prevent RANK-TRAF 6 interaction. However, similar to the peptides/proteins used in clinical setting, the effective application of this TRAF 6 Inhibitory peptide as a therapeutic agent is marred by several limitations for instance short half-life, rapid renal clearance and immunogenicity. In the present study, we have developed PEGylated TRAF 6 Inhibitory peptide by conjugating TRAF 6 Inhibitory peptide to linear PEG backbone that exhibits longer bioavailability in plasma in the animal model. Besides, it has an enhanced uptake at its site of action, i.e., bone marrow.
Keywords: TRAF 6 Inhibitory peptide; RANK; PEGylated peptide; Bioavailability;

Polymerized spermine as a novel polycationic nucleic acid carrier system by Zixiu Du; Moying Chen; Qianqian He; Yi Zhou; Tuo Jin (437-443).
Spermine, an endogenous amino-group bearing monomer that condenses DNA in sperm, was used as the basic building block to form polycationic nucleic acid carriers via condensation with one of three linker molecules – bischloroformate, succinyl chloride, and glyoxal. The three cationic polymers, polyspermine carbamate (PSP-Carb), polyspermine amide (PSP-Amide) and polyspermine imine (PSP-Imine) were examined for their degradability, cytotoxicity, ability to condense nucleic acids to nanoparticles, and ability to transfect genes or siRNA to cells. PSP-Carb and PSP-Amide exhibited a half-life of more than 2 months when incubated in aqueous buffers at 37 °C, while the half-life of PSP-Imine was 11 h. Relative cytotoxicity of the polymers, as measured by COS-7 and HepG2 cell viability, was in the order of PSP-Carb > PSP-Amide > PSP-Imine. Each cationic polymer condensed the luciferase plasmid to nanoparticles of 150–200 nm diameters and with a zeta potential of +15–30 mV when the mass ratio of polymer-to-DNA was over 8/1. The three polycationic carriers showed similar luciferase transfection activity in COS-7 cells, while the transfection efficiency of PSP-Carb was significantly higher than that of the other two in HepG2 cells. PSP-Amide exhibited significantly higher gene silencing activity in COS-7 cells, suggesting the linkage structures play an important role in the activity of the polyspermine-based nucleic acid carriers.
Keywords: Polyspermine; Gene delivery; Cytotoxicity; Transfection; Gene silencing;

Chains of magnetosomes isolated from Magnetospirillum magneticum strain AMB-1 magnetotactic bacteria by sonication at 30 W during 2 h are tested for magnetic hyperthermia treatment of tumors. These chains are composed of magnetosomes, which are bound to each other by a filament made of proteins. When they are incubated in the presence of cancer cells and exposed to an alternating magnetic field of frequency 198 kHz and average magnetic field strength of 20 or 30 mT, they produce efficient inhibition of cancer cell proliferation. This behavior is explained by a high cellular internalization, a good stability in solution and a homogenous distribution of the magnetosome chains, which enables efficient heating. When the chains are heated during 5 h at 90 °C in the presence of 1% SDS, the filament binding the magnetosomes together is denatured and individual magnetosomes are obtained. By contrast to the chains of magnetosomes, the individual magnetosomes are prone to aggregation, are not stable in solution and do not produce efficient inhibition of cancer cell proliferation under application of an alternating magnetic field.
Keywords: Magnetosomes; Magnetotactic bacteria; Cancer; Tumor; Chains of magnetosomes; Individual magnetomes; Alternating magnetic field; Magnetic hyperthermia;

Reformulation of etoposide with solubility-enhancing rubusoside by Fang Zhang; Gar Yee Koh; Javoris Hollingsworth; Paul S. Russo; Rhett W. Stout; Zhijun Liu (453-459).
Etoposide (ETO), a widely used anti-cancer drug, is constrained by its low aqueous solubility and by side effects from both the drug and its solubilizing excipients. In this study, a recently discovered natural solubilizer rubusoside (RUB) was used to achieve the solubilization of ETO. Dynamic light scattering and freeze-fracture transmission electron microscopy studies showed that ETO and RUB formed ETO–RUB nanoparticles (∼6 nm in diameter). The powder of ETO–RUB nanoparticles was completely reconstitutable in water and remained stable in this solution at 25 and 37 °C for at least 24 h. Under other physiologic conditions, ETO solution was clear and free of precipitation at 25 °C, but underwent various structural transformations. In PBS and simulated intestinal fluid, RUB-solubilized ETO underwent epimerization and equilibrated to cis-ETO. In simulated gastric fluid, RUB-solubilized ETO degraded to 4′-demethylepipodophyllotoxin-beta-d-glucoside and 4′-demethylepipodophyllotoxin. Higher temperatures favored epimerization or degradation. Furthermore, a side-by-side comparison with DMSO-solubilized ETO confirmed that the RUB-solubilized ETO showed no significant differences in cytotoxicity in colon, breast and prostate cancer cell lines. RUB effectively solubilized and stabilized etoposide, which sets the stage for further toxicology, bioavailability, and efficacy investigations.
Keywords: Etoposide; Formulation; Nanoparticles; Rubusoside; Stability; Anti-cancer;

Deformability of nanoparticles might affect their behaviour at biological interfaces. Lipid nanocapsules (LNCs) are semi-solid particles resembling a hybrid of polymer nanoparticles and liposomes. Deformability of LNCs of different sizes was modelled by drop tensiometer technique. Two purification methods, dialysis and tangential flow filtration (TFF), were applied to study experimental behaviour and deformability of LNCs in order to evaluate if these properties contributed to membrane passing. Rheological parameters obtained from the drop tensiometer analysis suggested decreasing surface deformability of LNCs with increase in diameter. Dialysis results showed that up to 10% of LNCs can be lost during the process (e.g. membrane accumulation) but no clear evidence of the membrane passing was observed. Instead, LNCs with initial size and size distribution could be found in the TFF filtrate although molecular weight cut-off (MWCO) of the membrane used was smaller than the LNC diameter.
Keywords: Lipid nanocapsules; Drop tensiometer; Dialysis; Tangential flow filtration; Rheology;

Epinephrine (EPI) and norepinephrine (NE) play an important role in emergency medicine and acute treatment of hypotension and shocks in the intensive care unit. Injectable solutions can either be provided as proprietary medicinal products or as individually prepared dilutions. Due to the chemical structure of EPI and NE, the stability of the corresponding solutions is limited. Thus, most manufacturers of EPI and NE injectable solutions use sulfites and nitrogen for stabilization, Nevertheless, storage conditions such as temperature and light have to be considered, but are often neglected in the daily hospital routine. In addition, hospital pharmacies prepare EPI and NE solutions and dilute commercially available solutions for individual therapy, especially on ICUs. Since the influence of dilution and the presence of excipients and other preservatives are not systematically explored, we collected published data and investigations on stability on the potency of EPI and NE injectable solutions in order to deduce storage recommendations for diluted EPI and NE solutions of different concentration.
Keywords: Epinephrine; Norepinephrine; Dilution; Stability; Stability-indicating methods; Hospital pharmacy;

Pulmonary delivery of dry powders to rats: Tolerability limits of an intra-tracheal administration model by A. Guillon; J. Montharu; L. Vecellio; V. Schubnel; G. Roseau; J. Guillemain; P. Diot; M. de Monte (481-487).
The inhaled route is increasingly developed to deliver locally acting or systemic therapies, and rodent models are used to assess tolerance before clinical studies. Endotracheal intubation of rats with a probe which generates powder aerosols enables controlled administration of drug directly into the respiratory tract. However, preliminary observations of intratracheal powder administration procedures have raised concerns with regard to pulmonary safety. The aim of the present work was to evaluate the safety of intra-tracheal administration of dry powder in a rat model.Sixty animals were administered various volumes of air alone, lactose or magnesium stearate through a Microsprayer® (Pencentury, USA). The mass of powder actually delivered to each animal was calculated. Rats were sacrificed immediately after administration, and the lungs, trachea and larynx were removed and examined for gross pathology.The mass of powder delivered varied, the full dose being rarely delivered. About one third of the administration procedures resulted in respiratory failure, and macroscopic pulmonary lesions were observed in about 55% of animals. Lung damages were observed with air alone, lactose and magnesium stearate. In conclusion, artifacts observed with this technique may limit the relevance of the model. These observations are particularly important in the context of regulatory toxicity studies.
Keywords: Lung tolerance; Powder; Intratracheal; Inhalation; Microsprayer®;

Cell-penetrating peptide mimicking polymer-based combined delivery of paclitaxel and siRNA for enhanced tumor growth suppression by Yeon Lim Jang; Ui Jeong Yun; Min Sang Lee; Myung Goo Kim; Sohee Son; Kyuri Lee; Su Young Chae; Dong Woo Lim; Hong Tae Kim; Sun Hwa Kim; Ji Hoon Jeong (488-493).
Cancer chemotherapy is often limited, since more than one molecule is usually involved with the cancer pathogenesis. A combination of therapeutic drugs would be a promising approach to overcome the complexity of tumors. In this study, a conjugate (DA3) of deoxycholic acid and low molecular weight polyethylenimine (PEI 1.8 kDa), which has a property that mimics that of cell penetrating peptides (CPPs), was used for simultaneous delivery of an anticancer drug and siRNA. When complexed with siRNA, DA3 showed significantly higher target gene silencing efficiency than PEI 25 kDa. The gene silencing efficiency of DA3 was maintained even in the presence of endocytosis inhibitors, suggesting that the polymeric carrier can mediate an endocytosis-independent macromolecular transduction similar to CPPs. The capability of forming a micelle-like core–shell structure enables the conjugates to encapsulate and dissolve paclitaxel (PTX), a water-insoluble drug. The drug-loaded cationic micelles can then interact with siRNA to form stable complexes (PTX/DA3/siRNA). The PTX/DA3/siRNA showed significantly enhanced inhibition of cancer cell growth. When administered into tumor-bearing animals, the PTX/DA3/siRNA demonstrated significant suppression of tumor growth, providing potential usefulness in clinical settings.
Keywords: Biomimetic polymer; Combined delivery; siRNA; Anticancer drug; Combination cancer therapy;

Direct measurement of the time-dependent mechanical response of HPMC and PEO compacts during swelling by Kathryn Otim Hewlett; Jennifer L’Hote-Gaston; Michael Radler; Kenneth R. Shull (494-501).
Mechanical indentation is used to measure the time dependent mechanical properties of three model compact formulations during swelling in aqueous media. The formulations are based on polyethylene oxide (PEO), hydroxypropyl methylcellulose (HPMC) and a PEO/HPMC blend. The technique is sensitive to changes in compact thickness and mechanical response and is used to characterize changes in the mechanical properties of the model compacts during the swelling process. The gel thickness and the effective elastic modulus of the gel layer are obtained from the load/displacement relationship during initial indentation. The HPMC and hybrid compacts showed significantly more swelling (110%) than the PEO compact (67%). Viscoelastic properties of the gel layer are determined throughout the swelling process by an oscillatory indentation method. Results show the complex modulus of all three compacts decreasing by approximately an order of magnitude over the course of swelling for 6 h. The measurement techniques presented here can easily be extended to more complex systems.
Keywords: Mechanical properties; Drug release compacts; Indentation;

On the performance qualification of hypromellose capsules by Brian E. Jones; Fridrun Podczeck (503-506).