International Journal of Pharmaceutics (v.392, #1-2)

Pulmonary administration of drugs presents several advantages in the treatment of many diseases. Considering local and systemic delivery, drug inhalation enables a rapid and predictable onset of action and induces fewer side effects than other routes of administration. Three main inhalation systems have been developed for the aerosolization of drugs; namely, nebulizers, pressurized metered-dose inhalers (MDIs) and dry powder inhalers (DPIs). The latter are currently the most convenient alternative as they are breath-actuated and do not require the use of any propellants. The deposition site in the respiratory tract and the efficiency of inhaled aerosols are critically influenced by the aerodynamic diameter, size distribution, shape and density of particles. In the case of DPIs, since micronized particles are generally very cohesive and exhibit poor flow properties, drug particles are usually blended with coarse and fine carrier particles. This increases particle aerodynamic behavior and flow properties of the drugs and ensures accurate dosage of active ingredients. At present, particles with controlled properties are obtained by milling, spray drying or supercritical fluid techniques. Several excipients such as sugars, lipids, amino acids, surfactants, polymers and absorption enhancers have been tested for their efficacy in improving drug pulmonary administration. The purpose of this article is to describe various observations that have been made in the field of inhalation product development, especially for the dry powder inhalation formulation, and to review the use of various additives, their effectiveness and their potential toxicity for pulmonary administration.
Keywords: Inhalation; Dry powder inhalation; Excipients; Sugars; Lipids; Toxicology;

Active and passive tumor targeting of a novel poorly soluble cyclin dependent kinase inhibitor, JNJ-7706621 by Fabienne Danhier; Bernard Ucakar; Nicolas Magotteaux; Marcus E. Brewster; Véronique Préat (20-28).
The anti-cancer cyclin dependent kinase (CDK) inhibitors are poorly soluble drugs. The aims of this work were (i) to formulate a novel CDK inhibitor, JNJ-7706621, in polymeric micelles and nanoparticles, (ii) to compare passive and active targeting on tumor growth and (iii) to evaluate the potential synergy of JNJ-7706621 with Paclitaxel. Therefore, JNJ-7706621 was encapsulated in self-assembling diblock copolymers made up of ɛ-caprolactone (CL) and trimethylene carbonate (TMC) (PEG-p-(CL-co-TMC)) polymeric micelles and in (poly(lactide-co-glycolide)) (PLGA)-based PEGylated nanoparticles (passive targeting) as well as in RGD-grafted nanoparticles (active targeting). In vivo, the transplantable liver tumor growth was more decreased by active targeting with RGD-grafted nanoparticles than by passive targeting with micelles or ungrafted nanoparticles. Moreover, a synergy between JNJ-7706621 and Paclitaxel was demonstrated. Therefore, active targeting of JNJ-7706621-loaded nanocarriers may be considered as an effective anti-cancer drug delivery system for cancer chemotherapy, particularly in combination with Paclitaxel.
Keywords: Polymeric micelles; PLGA nanoparticles; RGD; CDK inhibitor; Tumor targeting;

Taste-masked quinine pamoate tablets for treatment of children with uncomplicated Plasmodium falciparum malaria by E. Kayitare; C. Vervaet; E. Mehuys; P.C. Kayumba; J.D. Ntawukulilyayo; C. Karema; Van Bortel; J.P. Remon (29-34).
Children with uncomplicated malaria are generally treated with oral medication, except those unable to take oral drugs. Even though quinine has shown to be effective in treatment of African children with uncomplicated malaria its high bitterness limited the paediatric use. This study aimed to develop taste-masked quinine tablets suitable for children and offering dosing flexibility to adjust the quinine dose in function of body weight.Insoluble quinine pamoate was used to formulate fast-disintegrating tablets, using a specific tablet design (rectangular tablet which can be divided into 8 subunits) to allow dosing flexibility. The physical properties of tablets were evaluated in vitro, as well as the quinine bioavailability in healthy adults (n  = 18) and the efficacy for treatment of children with uncomplicated Plasmodium falciparum malaria (n  = 56) using a 7-day regimen of 8 mg quinine/kg.Quinine pamoate tablets complied with the pharmacopoeial requirements for mass uniformity, friability, content uniformity, breakability, disintegration and dissolution. The quinine pharmacokinetic parameters after single administration of a quinine pamoate tablet were similar to a commercially available quinine sulfate tablet. The fast decline in parasitemia (28.6%/24 h), the reduction rate of fever (all children were apyretic after 72 h) and the steady state quinine plasma concentration (5.7–15.8 μg/ml) proved the efficacy of the quinine pamoate tablets against P. falciparum.Fast-dispersible and taste-masked quinine pamoate tablets improved dosing accuracy, allowed easy administration and resulted in a high efficacy during the treatment of children with uncomplicated malaria.
Keywords: Malaria; Quinine; Taste; Tablets; Bioavailability; Efficacy; Paediatrics;

Effect of lipopolysaccharide on P-glycoprotein-mediated intestinal and biliary excretion of rhodamine123 in rats by Mikio Tomita; Atsushi Kanbayashi; Hiroyuki Murata; Ayako Tanaka; Mariko Nakaike; Megumi Hatanaka; Masahiro Hayashi (35-41).
The effects of lipopolysaccharide (LPS) on the ileal and biliary excretion of rhodamine123 were investigated in rats at different times after intraperitoneal (i.p.) injection (1 mg/kg and 5 mg/kg of body weight). P-gp protein decreased 8 h after injection of LPS and returned to the control level 24 h after i.p. injection of LPS in the ileum. There was a marked decrease in the expression level of mdr1a mRNA in the ileum and liver 8 h after i.p. injection of LPS when compared with the control condition. Also, the ileal and biliary clearance of rhodamine123 significantly decreased 8 h after i.p. injection of LPS, but returned to the control levels 24 h after i.p. injection of LPS. These results suggest that LPS-induced decreases in P-gp-mediated ileal and biliary excretion of rhodamine123 were probably due to impaired P-gp-mediated transport ability. The levels of iNOS and IL-1β mRNA in the ileum and liver increased 2 and 8 h after i.p. injection of LPS, respectively, and returned to the control levels 24 h after injection of LPS. These findings suggest that LPS markedly decreases P-gp-mediated ileal and biliary excretion of rhodamine123, probably by partly decreasing the expression of P-gp protein levels, likely due to increased lipid peroxidation levels through iNOS mRNA and inflammatory mediators such as IL-1β.
Keywords: Lipopolysaccharide; Intestine; Liver; P-glycoprotein-mediated transport; Inducible nitric oxide synthesis (iNOS); Interleukin 1β (IL-1β);

Preparation and evaluation of biodegradable microspheres containing a new potent osteogenic compound and new synthetic polymers for sustained release by Nobuo Umeki; Takayuki Sato; Masahiro Harada; Junko Takeda; Shuji Saito; Yasunori Iwao; Shigeru Itai (42-50).
In order to achieve the sustained release of 3-ethyl-4-(4-methylisoxazol-5-yl)-5-(methylthio) thiophene-2-carboxamide (BFB0261), a new potent osteogenic compound for the treatment of bone disorders, we prepared microspheres containing BFB0261 and newly synthesized three poly (d, l-lactic acid) (PLA), four poly (d, l-lactic acid–co-glycolic acid) (PLGA), and eight poly (d, l-lactic acid)-block-poly(ethylene glycol) (PLA-PEG) biodegradable polymers or copolymers, and evaluated the release pattern of BFB0261 from the microspheres in vitro and in vivo. The mean particle size of the microspheres, except for the microspheres constructed from PLA-PEG with a greater than 20% PEG component, was in the range of approximately 10–50 μm, and the preparations showed a spherical shape with a smooth surface. In an in vitro release study, the release of BFB0261 from PLA-1 (Mw: 36 kDa), PLAPEG9604H (PLA/PEG ratio: 96:4, Mw: 181 kDa), or PLAPEG8317 (PLA/PEG ratio: 83:17, Mw: 106 kDa) microspheres occurred in a zero-order manner with a slow release, and more than 50% of BFB0261 remained in each type of microsphere at 12 weeks after incubation. When the BFB0261 microspheres constructed from various polymers were intramuscularly administered to the rat femur, the microspheres constructed from PLA-1 or PLAPEG9604H were able to achieve a sustained release of BFB0261 at the injection site for 6 weeks. The present information indicates that microspheres constructed from PLA-1 or PLAPEG9604H may be feasible for bone engineering.
Keywords: Bone tissue engineering; Localized drug delivery; Microspheres; Poly (d, l-lactic acid) polymer; Poly (d, l-lactic acid-co-glycolic acid) polymer; Poly (d, l-lactic acid)-block-poly (ethylene glycol) polymer;

In vivo efficacy of paclitaxel-loaded injectable in situ-forming gel against subcutaneous tumor growth by Ju Young Lee; Kyung Sook Kim; Yun Mi Kang; E. Sle Kim; Sung-Joo Hwang; Hai Bang Lee; Byoung Hyun Min; Jae Ho Kim; Moon Suk Kim (51-56).
Injectable in situ-forming gels have received considerable attention as localized drug delivery systems. Here, we examined a poly(ethylene glycol)–b-polycaprolactone (MPEG–PCL) diblock copolymer gel as an injectable drug depot for paclitaxel (Ptx). The copolymer solution remained liquid at room temperature and rapidly gelled in vivo at body temperature. In vitro experiments showed that Ptx was released from MPEG–PCL copolymer gels over the course of more than 14 days. Experiments employing intratumoral injection of saline (control), gel-only, Taxol, or Ptx-loaded gel into mice bearing B16F10 tumor xenografts showed that Ptx-loaded gel inhibited the growth of B16F10 tumors more effectively than did saline or gel alone. Further, intratumoral injection of Ptx-loaded gel was more efficacious in inhibiting the growth of B16F10 tumor over 10 days than was injection of Taxol. A histological analysis demonstrated an increase in necrotic tissue in tumors treated with Ptx-loaded gel. In conclusion, our data show that intratumoral injection of Ptx-loaded MPEG–PCL diblock copolymer yielded an in situ-forming gel that exhibited controlled Ptx release profile, and that was effective in treating localized solid tumors.
Keywords: Injectable in situ gel; Tumors; Paclitaxel; Intratumoral injection;

The advantages of a novel CoQ10 delivery system in skin photo-protection by Yang Yue; Huafeng Zhou; Guanlan Liu; Yan Li; Zemin Yan; Mingxing Duan (57-63).
Skin photo-ageing induced by ultraviolet (UV) radiation is mainly ascribed to oxidative stress and reactive oxygen species (ROS). Coenzyme Q10 (CoQ10) has been reported as a powerful antioxidant in plasma. However, CoQ10 was barely satisfactory in topical drug delivery because of its lipid solubility. To improve the anti-oxidative efficiency of CoQ10 in skin photo-ageing, the present research prepared a novel CoQ10 nano-structured lipid carrier (CoQ10-NLC) and characterised it by size and freeze-fracture transmission electron microscopy (FF-TEM). In UVA-irradiated fibroblasts, the protection of CoQ10-NLC was more effective than the CoQ10-emulsion as demonstrated by cell viability and morphological changes of the cell body and nucleus. In addition, malondialdehyde (MDA, the product of lipid peroxidation) concentration decreased by 61.5% in the group treated with CoQ10-NLC compared to the group subjected to general CoQ10-emulsion. In the presence of CoQ10-NLC, the activities of the anti-oxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were reinstated to 81% and 75%, respectively, of the control group. In vivo, the CoQ10-NLC displayed a stronger capability to penetrate the stratum corneum and permeate the dermis after a topical skin application. These results reveal that CoQ10-NLC has greater antioxidant properties and topical skin penetration than the CoQ10-emulsion.
Keywords: Coenzyme Q10; Nano-structured lipid carriers; Ultraviolet; Antioxidant; Skin penetration;

Preparation and cytotoxic activity of hydroxycamptothecin nanosuspensions by Yong-Xing Zhao; Hai-Ying Hua; Min Chang; Wei-Jing Liu; Yang Zhao; Hong-Min Liu (64-71).
Hydroxycamptothecin is a promising anticancer agent that possesses the ability to inhibit the growth of a wide range of human tumors. Owing to its poor solubility and instability, the pharmaceutical development and clinical utilization of hydroxycamptothecin have been limited. In the present study, a novel precipitation-combined high-pressure homogenization (PCH) technique was used to prepare hydroxycamptothecin nanosuspensions. Based on the homogenization pressure and number of cycles, the process with 10 cycles at 18,000 psi of homogenization pressure was found to be the most efficient method to achieve consistent particle size reduction. It was used to prepare nanosuspensions for characterization and evaluation of the formulation performance. Lyophilization of hydroxycamptothecin nanosuspensions, the shape and crystal form of the drug, and antiproliferative activity were also studied. The mean particle size (z-ave) of the reconstituted freeze-dried powder was small and uniform. The freeze-dried powder might be a good choice for intravenously administrating poorly soluble hydroxycamptothecin, which proved to have higher cytotoxicity against the cancer cells than hydroxycamptothecin injections (p  < 0.001). Overall, these studies have demonstrated that the PCH technique can be used successfully to prepare hydroxycamptothecin nanosuspensions.
Keywords: Hydroxycamptothecin; Nanosuspensions; High-pressure homogenization; Precipitation; Cytotoxicity;

Transdermal permeation of geniposide in the herbal complex liniment in vivo and in vitro by Yugang Wang; Lele Li; Huiying Li; Zhaoyun Zhu; Lei Hua; Fan Lei; Michael M. Kheir; Lijun Du (72-77).
Zhongtong Caji, a kind of liniment, is a traditional Chinese medicinal formula that is widely used for clinical treatment of inflammation and sprains. In this study, the principal effective compound of this formula, geniposide, was used as a criterion to represent the transdermal permeability of the whole formula. A passive diffusion of Zhongtong Caji through the stratum corneum was discovered by an in vitro experiment. The dosage–content relationship detected in subcutaneous tissue after in vivo drug administration was further evidence of its permeation. Blood analysis after different dosages showed that the geniposide could be absorbed and accumulated by subcutaneous tissue within 1 h after drug administration, and it would be eliminated by blood circulation 1 h after drug treatment.
Keywords: Transdermal penetration; Liniment; Geniposide; Mice;

A charge-switched nano-sized polymeric carrier for protein delivery by Bo Reum Lee; Kyung Taek Oh; Hye Jung Baik; Yu Seok Youn; Eun Seong Lee (78-82).
A novel synthetic nanocomplex was constructed from glycol chitosan (GCS) grafted with 2,3-dimethylmaleic anhydride (DMA) (denoted as ‘GCD’ hereafter) and lysozyme (isoelectric point = 10.9) as a model protein. This is a core-shell supramolecular assemble formed through electrostatic interactions between anionic GCD and cationic lysozyme at a pH 7.4. The pH-sensitivity of the nanocomplexes originates from the dissociation of DMA block from GCD at a slightly acidic pH (i.e., pH 6.8), resulting in an increased electrostatic repulsion between cationic GCS and cationic lysozyme. This pH-induced charge switching of GCD provides a mechanism for triggered protein drug release from the nanocomplexes triggered by the small change in pH (pH 7.4–6.8).
Keywords: Charge-switched nanocomplex; Protein delivery; Acidosis;

Potential use of γ-cyclodextrin polypseudorotaxane hydrogels as an injectable sustained release system for insulin by Irhan Ibrahim Abu Hashim; Taishi Higashi; Takayuki Anno; Keiichi Motoyama; Abd-ElGawad Helmy Abd-ElGawad; Mohamed H. El-Shabouri; Thanaa Mohamed Borg; Hidetoshi Arima (83-91).
The development of injectable hydrogels for protein delivery is a major challenge. In this study, insulin/α-cyclodextrin (α-CyD) and γ-CyD polypseudorotaxane (PPRX) hydrogels were prepared through inclusion complexation between high molecular weight poly(ethylene glycol) (PEG) and CyDs. The α-CyD and γ-CyD PPRX hydrogels were formed by inserting one PEG chain in the α-CyD cavity and two PEG chains in the γ-CyD cavity. Insulin/CyD PPRX hydrogel formation was based on physical crosslinking induced by self-assembling without chemical crosslinking reagent. The supramolecular structures of insulin/CyD PPRX hydrogels were confirmed with 1H nuclear magnetic resonance (1H NMR), X-ray diffraction, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The in vitro release study showed that the release rate of insulin from the CyDs PPRX hydrogels decreased in the order of γ-CyD PPRX hydrogel > α-CyD PPRX hydrogel. This decrease was controlled by the addition of CyDs to the medium. The serum insulin level after subcutaneous administration of γ-CyD PPRX hydrogel to rats was significantly prolonged, accompanying with an increase in the area under serum concentration–time curve, which was clearly reflected in the prolonged hypoglycemic effect. In conclusion, these results suggest the potential use of γ-CyD PPRX hydrogel as an injectable sustained release system for insulin.
Keywords: Insulin; Cyclodextrin; Polypseudorotaxane; Hydrogel; Sustained release system;

Chitosomes as drug delivery systems for C-phycocyanin: Preparation and characterization by M. Manconi; S. Mura; M.L. Manca; A.M. Fadda; M. Dolz; M.J. Hernandez; A. Casanovas; O. Díez-Sales (92-100).
The aim of this work was to investigate chitosomes, i.e. liposomes coated by a polyelectrolyte complex between chitosan (CH) and xantan gum (XG), as potential delivery system for oral administration of the protein C-phycocyanin. To this purpose several CH–XG-microcomplexes were prepared in aqueous lactic acid at different chitosan–xanthan gum percent ratios and rheological properties of the microcomplexes were studied to analyse the contribution of chitosan and xanthan gum in the reaction of microcomplexation. After establishing the best microcomplexes, chitosomes were prepared by coating C-phycocyanin loaded liposomes with the CH–XG hydrogels using spray-drying or freeze-drying. The chitosomes were characterized in terms of morphology, size distribution, zeta potential, swelling properties, drug release, and mucoadhesive properties. Rheological studies showed the influence of xanthan gum in the microcomplex properties. Moreover, obtained results demonstrated the effects of formulation and process variables on particle size, drug content, swelling, drug release, and especially on the mucoadhesiveness of C-PC chitosomes of CH–XG. In particular, chitosomes prepared by spray-drying technique using CH–XG in 0.5/8.0 (w/w) ratio showed a regular surface and a drug release characteristic for a Fickian diffusion of the active ingredient. The in vitro mucoadhesive study revealed that the spray-drying method is advantageous to prepare C-phycocyanin loaded chitosomes with excellent mucoadhesive properties for colonic drug delivery.
Keywords: Liposomes coated; Drug delivery; Release study; Diffusion coefficient; Swelling; Mucoadhesive properties;

The feasibility of α-glucosyl hesperidin (Hsp-G) to improve the dissolution and bioavailability of poorly water-soluble drug was investigated. A spray-dried powder (SDP) of Hsp-G and flurbiprofen (FP), an acidic drug (pK a  = 3.78) with low water solubility, was prepared by a spray-drying method. Powder X-ray diffraction analysis revealed the conversion of FP from the crystal to the amorphous form when dispersed in Hsp-G. The SDPs of FP/Hsp-G resulted in pronounced improvement in both the dissolution rate and solubility of FP. The apparent solubility of FP in hydrochloric acid solution (pH 1.2) was improved by 10-fold more than untreated FP crystals when prepared as SDPs in Hsp-G. The bioavailability of FP from the prepared SDPs was evaluated in vivo after oral administration to rats, in comparison with the untreated FP crystals. The results revealed 2.5- and 2.8-fold improvement in the C max and AUC values, respectively, after oral administration of the SDPs of FP/Hsp-G. In conclusion, Hsp-G is a potentially safe material to enhance the dissolution and absorption of poorly water-soluble drugs.
Keywords: α-Glucosyl hesperidin; Flurbiprofen; Solubility enhancement; Spray-dried powder; Absorption enhancement;

The coefficient of rolling resistance (CoRR) of some pharmaceutical tablets by William R. Ketterhagen; Rahul Bharadwaj; Bruno C. Hancock (107-110).
Experiments have been conducted to measure the coefficient of rolling resistance (CoRR) of some pharmaceutical tablets and several common materials, such as glass beads and steel ball bearings. CoRR values are required as inputs for discrete element method (DEM) models which can be used to model particulate flows and solid dosage form manufacturing processes. Until now there have been no CoRR data reported for pharmaceutical materials, and thus these new data will help to facilitate more accurate modeling of pharmaceutical systems.
Keywords: Tablet; Coefficient of rolling resistance; Discrete element method;

Biochemical and biophysical characterization of lysozyme modified by PEGylation by Débora da Silva Freitas; José Abrahão-Neto (111-117).
PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH2 modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FTIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 °C, and demonstrated resistance to proteolytic degradation.
Keywords: Antimicrobial activity; Lysozyme; PEGylation; Physical–chemical stability; Proteolysis resistance;

Brush-shaped polycation with poly(ethylenimine)-b-poly(ethylene glycol) side chains as highly efficient gene delivery vector by Xi-Qiu Liu; Jin-Zhi Du; Chu-Pei Zhang; Fang Zhao; Xian-Zhu Yang; Jun Wang (118-126).
A brush-shaped polymer PHEMA-g-(PEI-b-PEG) with poly(2-hydroxyethyl methacrylate) (PHEMA) backbone and linear poly(ethylenimine)-b-poly(ethylene glycol) (PEI-b-PEG) side chains was synthesized and evaluated as a vector for potential cancer gene therapy. The characterizations by 1H NMR and laser light scattering demonstrated the brush structure of the polymer. PHEMA-g-(PEI-b-PEG) was much less cytotoxic when compared with branched poly(ethylenimine) with M w of 25 kDa. The capacity of plasmid DNA condensation by PHEMA-g-(PEI-b-PEG) was demonstrated by gel retardation assay, and they formed nanosized complexes with surface zeta potential around 20 mV at N/P ratios higher than 5:1. The complexes of PHEMA-g-(PEI-b-PEG) with plasmid DNA were more efficiently internalized by BT474 cells in comparison with the complexes of PEI25K, leading to higher gene transfection in cells. Further investigation using complexes of PHEMA-g-(PEI-b-PEG) with plasmid DNA encoding wild-type p53 gene showed its potential as a carrier for cancer gene therapy. The complexes of PHEMA-g-(PEI-b-PEG) successfully induced elevated wild-type p53 expression in BT474 cells and led to enhanced apoptosis of BT474 cells. Transfection of wild-type p53 using the complexes also significantly increased the sensitivity of BT474 cells to doxorubicin chemotherapy, suggesting the potential of this carrier in cancer gene therapy.
Keywords: Non-viral vector; Poly(ethylenimine); Brush polymer; Gene therapy; p53 gene; Doxorubicin;

Transdermal delivery of insulin using microneedle rollers in vivo by Cui-Ping Zhou; Yu-Ling Liu; Hong-Liang Wang; Peng-Xiao Zhang; Jin-Lan Zhang (127-133).
This study characterizes skin perforation by commercially available microneedle rollers and evaluates the efficacy of transdermal delivery of insulin to diabetic rats. Three different needle lengths, 250, 500 and 1000 μm, were used in this work. Creation and resealing of the skin holes that were produced by the needles were observed by Evan's blue (EB) staining and transepidermal water loss (TEWL) measurements. EB clearly showed that microchannels were formed in the skin and that the pores created by the longest microneedle (1000 μm) persisted no longer than 8 h, while the hypodermic injury was still observed 24 h later. TEWL significantly increased after the application of the needles and then decreased with time, which explains the recovery of skin barrier function and agrees well with EB results. The extent of permeation was demonstrated by insulin delivery in vivo. The rapid reduction of blood glucose levels in 1 h was caused by the increased permeability of the skin to insulin after applying microneedle rollers. The reduced decrease after 1 h is closely associated with hole recovery. In conclusion, microneedle rollers with 500-μm or shorter lengths are safe and useful in transdermal delivery of insulin in vivo.
Keywords: Transdermal drug delivery; Microneedle roller; Transepidermal water loss; Insulin; Diabetes;

Online monitoring of transepithelial electrical resistance (TEER) in an apparatus for combined dissolution and permeation testing by Marco Muendoerfer; Ulrich F. Schaefer; Petra Koenig; Jutta S. Walk; Petra Loos; Stefan Balbach; Thomas Eichinger; Claus-Michael Lehr (134-140).
The aim of this study was to evaluate a newly implemented feature for the online monitoring of transepithelial electrical resistance (TEER) in an apparatus for combined in vitro dissolution and permeation testing. In a first step, the course of TEER was analyzed simultaneously to the permeability of sodium fluorescein, and a time frame of cell monolayer integrity inside the apparatus of approximately 3 h was found. In successive experiments cell monolayer integrity was challenged by application of EDTA (8, 6, 3 and 2 mM) in the apical compartment. After application of high EDTA concentrations of 8 and 6 mM for 45 min TEER did not recover, and permeability of the monolayer was steadily increasing. For the lower concentrations TEER recovered again while permeability of sodium fluorescein remained at an elevated level. This suggests that the EDTA induced opening of the tight junctions was preserved during the period of TEER recovery and did not change within the lifespan of the cell monolayer inside the apparatus. Online monitoring of TEER appeared to be a suitable method for real-time control of barrier integrity throughout each experiment. Moreover, this feature is intended to be used to analyze formulation approaches aiming for an improved oral drug bioavailability by application of excipients that increase the paracellular permeability of the intestinal epithelial barrier.
Keywords: Caco-2 cells; TEER; Tight junctions; EDTA; Sequential Injection Analysis; In vitro drug absorption;

Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid by Hyun-Ki Kim; Enkhzaya Davaa; Chang-Seon Myung; Jeong-Sook Park (141-147).
Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of cationic liposomes that contain a new PEG-lipid. The new lipid, poly-l-arginine-conjugated polyethylene glycol (PLR–PEG), was synthesized. To confirm the synthesis of the amino acid-conjugated PEG-lipid, 1H NMR and gel permeation chromatography (GPC) were performed. Cationic liposomes as non-viral vectors were formulated using the cationic lipids 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolaminepropane (DOPE), cholesterol (Chol) and PLR–PEG. Physicochemical properties of cationic liposomes were investigated. A GFP siRNA was used as a model siRNA to test the efficiency of cationic liposome-mediated siRNA delivery. The liposomes could enhance delivery efficiency and decrease cytotoxicity at an optimized lipid composition. The new cationic liposome formulation using a new PEG-lipid (PLR–PEG) showed not only enhanced intracellular delivery of siRNA but also decreased cytotoxicity in H4II-E and HepG2 cell lines. The GFP siRNA delivered by new cationic liposomes using PLR–PEG was effective in reducing the GFP protein expression levels of the gene. These results suggest that the new cationic liposomes could be used for efficient delivery of siRNA therapeutics.
Keywords: siRNA delivery; Poly-l-arginine; Polyethylene glycol; Cationic liposomes;

A dry-coated tablet is a solid dosage form with a controlled drug-release system, which consists of a core and an outer layer. The accuracy of its geometric (e.g. the outer layer wall and core thicknesses) and mechanical properties (e.g. Young's moduli and mass densities of associated materials) could be crucial to its therapeutic and structural functions. The objective of current study is to develop a non-destructive technique for determining the geometric and mechanical properties of dry-coated tablets. Two contact ultrasonic techniques (i.e. pitch-catch and pulse-echo measurement modes) are employed and the properties of all the structural components of a set of experimental tablets are measured and reported. The thicknesses of the outer layers of the sample tablets are used to obtain the eccentricity of the core tablets. The two approaches are compared for their effectiveness in obtaining these properties of the sample dry-coated tablets. The thicknesses of the outer layers obtained with the proposed approach and with the direct (destructive) measurements are also compared. A good agreement is found; there is an approximately 2% difference. The eccentricity and concentricity of a set of tablets are determined and it is concluded that the observed consistent anomaly in eccentricity can be attributed to the same root cause and its correction can be achieved by control input based on monitoring data. Potential of the approach for in-die real-time monitoring of compaction presses and its PAT (Process Analytical Technology) applications for the pharmaceutical manufacturing are also discussed.
Keywords: Dry-coated tablets; Core eccentricity; Real-time compaction monitoring; Mechanical properties; Acoustic monitoring techniques; Process Analytical Technology (PAT);

Pulmonary delivery of cisplatin–hyaluronan conjugates via endotracheal instillation for the treatment of lung cancer by Yumei Xie; Kristin L. Aillon; Shuang Cai; Jason M. Christian; Neal M. Davies; Cory J. Berkland; M. Laird Forrest (156-163).
Cisplatin (CDDP) intravenous treatments suffer several dose-limiting toxicity issues. Hyaluronan (HA), a naturally occurring biopolymer in the interstitium, is primarily cleared by the lymphatic system. An alteration in input rate and administration route through pulmonary delivery of hyaluronan–cisplatin (HA–Pt) conjugate may increase local lung CDDP concentrations and decrease systemic toxicity.Sprague–Dawley rats were split into four groups: i.v. CDDP (3.5 mg/kg), i.v. HA–Pt conjugate (3.5 mg/kg equivalent CDDP), lung instillation CDDP and lung instillation HA–Pt conjugate. Total platinum level in the lungs of the HA–Pt lung instillation group was 5.7-fold and 1.2-fold higher than the CDDP intravenous group at 24 and 96 h, respectively. A 1.1-fold increase of Pt accumulation in lung draining nodes for the HA–Pt lung instillation group was achieved at 24 h relative to the CDDP i.v. group. In the brain and kidneys, the CDDP i.v. group had higher tissue/plasma ratios compared to the HA–Pt lung instillation group. Augmented tissue distribution from CDDP i.v. could translate into enhanced tissue toxicity compared to the altered input rate and distribution of the intrapulmonary nanoformulation.In conclusion, a local pulmonary CDDP delivery system was developed with increased platinum concentration in the lungs and draining nodes compared to i.v. therapy.
Keywords: Cisplatin; Hyaluronan; Pharmacokinetics; Pulmonary delivery; Lung chemotherapeutics;

Sustained release of ATP encapsulated in chitosan oligosaccharide nanoparticles by Yong-Zhong Du; Xiao-Ying Ying; Ling Wang; You Zhai; Hong Yuan; Ri-Sheng Yu; Fu-Qiang Hu (164-169).
The chemical cross-linked chitosan oligosaccharide (CSO) nanoparticles containing ATP/CSO ionic complex nano-components were prepared using combination techniques of W/O miniemulsion, chemical cross-linking and ionic complexation. The resulted nanoparticles had about 110 nm diameter and 20 mV surface zeta potential. The ATP loading efficiencies in nanoparticles could reach up to 40.6–69.5%. It was found that the ATP loading efficiency increased with increasing the amount and the molecular weight of chitosan oligosaccharide, and decreased with increasing molar ratio of glutaraldehyde to chitosan oligosaccharide. In vitro ATP release from chemical cross-linked CSO nanoparticles could continue for 24 h, and could also be adjusted by the amount and molecular weight of CSO, and the molar ratio of glutaraldehyde to CSO. The higher molecular weight and smaller amount of CSO, and the lower molar ratio of glutaraldehyde to CSO led the slower ATP release rate. Furthermore, it was also found that the CSO nanoparticles could be uptaken by HepG-2 tumor cells, and could be applied for intracellular drug delivery.
Keywords: Chitosan oligosaccharide; ATP; Nanoparticles; Ionic complex; Controlled release;

Design and evaluation of micellar nanocarriers for 17-allyamino-17-demethoxygeldanamycin (17-AAG) by Thripthy Chandran; Usha Katragadda; Quincy Teng; Chalet Tan (170-177).
17-Allyamino-17-demethoxygeldanamycin (17-AAG) is a potent anticancer agent currently undergoing phases I and II clinical trials. However, the clinical development of 17-AAG has been hindered by its poor aqueous solubility and hepatotoxicity. This study aimed to devise novel micellar nanocarriers for 17-AAG that improve its solubility and retain the incorporated drug for a prolonged period of time. We have found that 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]/d-α-tocopheryl polyethylene glycol 1000 (PEG-DSPE/TPGS) mixed micelles (at a 1:2 molar ratio) can deliver 17-AAG at clinically relevant doses. By modulating the concentrations of micelle-forming copolymers, the burst release of 17-AAG from PEG-DSPE/TPGS mixed micelles was substantially reduced with a release half-life up to about 8 h. Our 1H NMR spectroscopy results revealed that the incorporation of TPGS into PEG-DSPE micelles restricted internal molecular motions of copolymers in both the corona and core regions of the micelles, leading to the delayed drug release. Cytotoxicity of 17-AAG formulated in PEG-DSPE/TPGS mixed micelles against human ovarian cancer SKOV-3 cells was comparable to that of free 17-AAG. 17-AAG-loaded PEG-DSPE/TPGS mixed micelles may offer a promising alternative to the current 17-AAG formulations for the treatment of solid tumors.
Keywords: Polymeric micelles; PEG-DSPE; TPGS; 17-AAG; Drug release;

The enhanced aerosol performance of salbutamol from dry powders containing engineered mannitol as excipient by Waseem Kaialy; Gary P. Martin; Martyn D. Ticehurst; Mohammed N. Momin; Ali Nokhodchi (178-188).
The aim of the present study was to investigate the effect of crystallising mannitol from different binary mixtures of acetone/water on the resultant physical properties and to determine the effects of any changes on in vitro aerosolisation performance, when the different mannitol crystals were used as a carrier in dry powder inhaler formulations containing salbutamol sulphate. Mannitol particles were crystallised under controlled conditions by dissolving the sugar in water and precipitating the sugar using binary mixtures of acetone/water in different percentages as anti-solvent media. For comparison purposes the physical properties and deposition behaviour of commercially available mannitol were also studied. SEM showed that all crystallised mannitol particles were more elongated than the commercial mannitol. Solid state studies revealed that commercial mannitol and mannitol crystallised using acetone in the presence of 10–25% v/v water as anti-solvent was β-polymorphic form whereas mannitol crystallised in the presence of a small amount of water (0–7.5%) was the α-form. All the crystallised mannitol samples showed poor flowability. Nevertheless, the powdered crystallised mannitol and commercial samples were blended with salbutamol in the ratio 67.5:1. The aerosolisation performance of the formulations containing the engineered mannitol (evaluated using Multi Stage Liquid Impinger) was considerably better than that of the commercial mannitol formulation (the fine particle fraction was increased from 15.42% to 33.07–43.99%, for the formulations containing crystallised mannitol). Generally, carriers having a high tapped density and high fraction of fine carrier particles produced a high FPF. The improvement in the DPI performance could be attributed to the presence of elongated carrier particles with smooth surfaces since these are believed to have less adhesive forces between carrier and the drug resulting in easier detachment of the drug during the inhalation.
Keywords: Dry powder inhaler; Mannitol; Micromeritic; Deposition study; Crystallisation; Ratio of acetone/water;

Correlation between the FT-IR characteristics and metoprolol tartrate release of methylcellulose-based patches by József Papp; József Horgos; Virág Szente; Romána Zelkó (189-191).
The aim of the present study was to investigate how the drug release and FT-IR characteristics of metolose patches were influenced by the changes of Metolose SM 4000 (methylcellulose) and Metolose 90SH 100.000SR (hypromellose) proportions.FT-IR spectroscopy measurements were performed in parallel with the metoprolol tartrate release study to track the effect of the composition on the drug release. The metoprolol tartrate release profile of the patches was evaluated by Weibull distribution. Linear relationship was found with good correlation between the logarithm of time interval necessary to release 63.2% of metoprolol tartrate (τ d values) and the peak area measured within the characteristic FT-IR wavenumbers of patches. The application of FT-IR measurements can be recommended as a rapid, non-destructive screening method during the in-process control of patches.
Keywords: Methylcellulose; Hypromellose; Metoprolol tartrate release; Transdermal patch; FT-IR spectroscopy;

Gel formulations for treatment of the ophthalmic complications in cystinosis by Barbara Buchan; Graeme Kay; Anne Heneghan; Kerr H. Matthews; Donald Cairns (192-197).
Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised lysosomal levels of cystine in the cells of all organs. It is treated by regular administration of the aminothiol, cysteamine. Corneal crystal deposition is one of the most troublesome complications affecting patients and requires the hourly administration of cysteamine eye drops. In an attempt to reduce this frequency and improve the treatment, the preparation and evaluation of cysteamine containing Carbomer gel is reported. The results demonstrated that a weak gel network was formed at low shear–stress, the bioadhesion of the gel was increased with inclusion of a cysteamine derivative (e.g. mean force of 0.067 N compared to 0.107 N with compound included) and first-order release from the gel was observed. In conclusion these results offer the possibility to formulate cysteamine in an ocular applicable gel formulation.
Keywords: Cystinosis; Ophthalmic delivery; Gel; Modified release;

Comment to “The pharmacopeial evolution of Intralipid injectable emulsion in plastic containers: From a coarse to a fine emulsion” by Anders Ellborg; Denise Ferreira; Javad Mohammadnejad; Torbjörn Wärnheim (198-200).
The droplet size distribution of 50 batches of multi-chamber bags containing the parenteral nutrition emulsions Intralipid (Kabiven and Kabiven Peripheral) or Structolipid (StructoKabiven and StructoKabiven Peripheral), respectively, has been investigated. The results show that the non-compounded lipid emulsions analysed are in compliance with the United States Pharmacopeia (USP) chapter 729, Method II limit for the droplet size distribution, PFAT5  < 0.05%.
Keywords: PFAT5; Lipid emulsion; Droplet size; Parenteral nutrition; Multi-chamber bag;

Lipid-coated nano-calcium-phosphate (LNCP) for gene delivery by Chenguang Zhou; Bo Yu; Xiaojuan Yang; Tianyao Huo; L. James Lee; Rolf F. Barth; Robert J. Lee (201-208).
While calcium-phosphate has been used to deliver plasmid DNA (pDNA) for decades, the method is typically characterized by low and irreproducible transfection efficiency relative to the other non-viral approaches, such as liposomes and polymers. Here we report a novel gene transfer vector comprising lipid-coated nano-calcium-phosphate (LNCP) that provides consistently efficient and satisfactory pDNA delivery. It is based on core-shell nanoparticles comprising a calcium-phosphate core and a cationic lipid shell. This method, in contrast to the solution precipitation methods used in the past, yields colloidally stable calcium-phosphate nanoparticles inside the cationic liposomes. Our results indicate that the particle size and the size distribution of the LNCP remain virtually unchanged even after 21 days of storage. Atomic force microscopy measurements reveal that the LNCP have a 5-fold higher rigidity than common cationic liposomes. The LNCP transfected pDNA 24 times greater than the naked pDNA and 10-fold greater relative to the standard calcium-phosphate precipitation preparations, suggesting that the LNCP may have potential as a novel transfection agent for gene therapy.
Keywords: Nanoparticles; Cationic liposome; Calcium-phosphate; Gene therapy;

Optimizing partition-controlled drug release from electrospun core–shell fibers by Sandeep Kumar Tiwari; Roey Tzezana; Eyal Zussman; Subbu S. Venkatraman (209-217).
Controlled release of hydrophilic entities, such as peptides, proteins and even pDNA, is difficult to accomplish with conventional approaches. This work suggests one possible approach for controlled release of such actives using electrospun core–shell fiber structures. In particular, we propose strategies for partition control of the release. The fibers consist of two layers, with the outer polymer sleeve serving containing the inner core, in which the drug is encapsulated. By varying the physical and chemical properties of the core and shell solutions, we have shown that the release rate of a hydrophilic drug, metoclopramide hydrochloride, is controllable. Experimental results show a clear difference in the release pattern between monolithic fibers made of hydrophilic and hydrophobic polymers and various core–shell fibers with PCL, PLLA and PLGA 80/20 as shell polymers. The study yields insight into when partition control of release can be achieved in core–shell fibers, and with that, options for controlled release systems for hydrophilic drugs, peptides and pDNA.
Keywords: Co-electrospinning; Core–shell fibers; Controlled drug release; Hydrophilic drug; Metoclopramide hydrochloride; Partitioning;

T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes by Hiroyuki Koide; Tomohiro Asai; Kentaro Hatanaka; Shuji Akai; Takayuki Ishii; Eriya Kenjo; Tatsuhiro Ishida; Hiroshi Kiwada; Hideo Tsukada; Naoto Oku (218-223).
Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [3H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [3H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.
Keywords: Polyethylene glycol; Liposome; Accelerated blood clearance; Thymus-independent type 2 antigen; Nanocarriers;

Ternary nanoparticles of anionic lipid nanoparticles/protamine/DNA for gene delivery by Hong Yuan; Wei Zhang; Yong-Zhong Du; Fu-Qiang Hu (224-231).
In this study, the lipid nanoparticles with excellent cellular uptake capacity were utilized to prepare lipid nanoparticles/protamine/DNA ternary nanoparticles for gene delivery. Anionic lipid nanoparticles consisting of monostearin (MS) and different content of oleic acid (OA) were prepared. The lipid nanoparticles had an average size ranging from 23.6 to 71.3 nm and a zeta potential about −30 mV. The protamine/DNA complex was prepared by mixing the DNA and protamine solution with 1:2 mass ratio. The average size of protamine/DNA complex was 128.5 nm and with 19.4 mV zeta potential. Lipid nanoparticles/protamine/DNA ternary nanoparticles were then prepared by combining the protamine/DNA binary complex with lipid nanoparticles. The ternary nanoparticles had an average size ranging from 192.7 to 260.6 nm and were negatively charged. Gene transfection efficiency was improved by increasing OA content in lipid nanoparticles. Results of cellular uptake showed that the uptake ability of lipid nanoparticles was enhanced by increasing the OA content. Lipid nanoparticles with 20 wt% OA/protamine/DNA ternary nanoparticles (w/w/w, 65:6:3) showed the best and the most durable gene transfection even in the presence of serum.
Keywords: Lipid nanoparticles; Protamine; Plasmid DNA; Ternary nanoparticles; Gene transfection;

Gel characterisation and in vivo evaluation of minocycline-loaded wound dressing with enhanced wound healing using polyvinyl alcohol and chitosan by Jung Hoon Sung; Ma-Ro Hwang; Jong Oh Kim; Jeong Hoon Lee; Yong Il Kim; Jeong Hoon Kim; Sun Woo Chang; Sung Giu Jin; Jung Ae Kim; Won Seok Lyoo; Sung Soo Han; Sae Kwang Ku; Chul Soon Yong; Han-Gon Choi (232-240).
The purpose of this study was to develop a minocycline-loaded wound dressing with an enhanced healing effect. The cross-linked hydrogel films were prepared with polyvinyl alcohol (PVA) and chitosan using the freeze-thawing method. Their gel properties, in vitro protein adsorption, release, in vivo wound healing effect and histopathology were then evaluated. Chitosan decreased the gel fraction, maximum strength and thermal stability of PVA hydrogel, while it increased the swelling ability, water vapour transmission rate, elasticity and porosity of PVA hydrogel. Incorporation of minocycline (0.25%) did not affect the gel properties, and chitosan hardly affected drug release and protein adsorption. Furthermore, the minocycline-loaded wound dressing composed of 5% PVA, 0.75% chitosan and 0.25% drug was more swellable, flexible and elastic than PVA alone because of relatively weak cross-linking interaction of chitosan with PVA. In wound healing test, this minocycline-loaded PVA-chitosan hydrogel showed faster healing of the wound made in rat dorsum than the conventional product or the control (sterile gauze) due to antifungal activity of chitosan. In particular, from the histological examination, the healing effect of minocycline-loaded hydrogel was greater than that of the drug-loaded hydrogel, indicating the potential healing effect of minocycline. Thus, the minocycline-loaded wound dressing composed of 5% PVA, 0.75% chitosan and 0.25% drug is a potential wound dressing with excellent forming and enhanced wound healing.
Keywords: Minocycline; Chitosan; Polyvinyl alcohol; Wound dressing; Wound healing effect; Histology;

Target-specific technique can significantly enhance the efficacy of gene delivery system which was limited by many cellular barriers. In this work, a new folate-PEG modified poly(2-(2-aminoethoxy)ethoxy)phosphazene (PAEP), namely, folate-PEG-PAEP was synthesized as a folate receptor (FR) targeted carrier, and the cytotoxicity, transfection efficiency, cellular uptake and intracellular trafficking of folate-PEG-PAEP/DNA nanoparticles (FPPN) were investigated. Compared with the PAEP/DNA nanoparticles (PN), the cytotoxicity of FPPN decreased significantly at high dose. FPPN showed much higher transfection efficiency (15.85 ± 1.23%) compared with PN (6.71 ± 0.42%) in FR overexpressing Hela cells, but no significant difference was observed in CHO-k1 cells lacking FR. The transfection activity of FPPN could be reversed in the presence of 1.0 mM free folic acid in Hela cells. The cellular uptake of FPPN was 37.38% higher than that of PN in Hela cells. These results indicated that FPPN could be a potential targeted gene delivery system.
Keywords: DNA; Poly(phosphazene); Folic acid; Nanoparticles; Gene;

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3 by Hiroyuki Kamiya; Hitomi Goto; Genki Kanda; Yuma Yamada; Hideyoshi Harashima (249-253).
The intranuclear disposition of plasmid DNA is extremely important for transgene expression. Exogenous histones have been used as carriers of plasmid DNA in histone-mediated gene delivery. In this study, the effects of exogenous histone H3 complexed with plasmid DNA on transgene expression efficiency were examined. The plasmid–histone complexes in various ratios were transfected into HeLa cells by osmotic pressure. Histone H3 suppressed transgene expression in the nucleus in a dose-dependent manner. Our results suggest that the histone-mediated gene delivery is unlikely to be useful, from the viewpoint of the intranuclear disposition.
Keywords: Exogenous DNA; Histone; H3; Intranuclear disposition; Expression efficiency;

Deoxycholic acid modified-carboxymethyl curdlan conjugate as a novel carrier of epirubicin: In vitro and in vivo studies by Fuping Gao; Lei Li; Huizhu Zhang; Wenzhi Yang; Hongli Chen; Jing Zhou; Zhimin Zhou; Yinsong Wang; Yuanyuan Cai; Xuemin Li; Lingrong Liu; Qiqing Zhang (254-260).
Deoxycholic acid hydrophobically modified-carboxymethylated-curdlan (DCMC) conjugate was developed as a novel carrier for the anticancer drugs. Epirubicin (EPB), as a model drug, was physically loaded into DCMC self-assembled nanoparticles. EPB-loaded DCMC nanoparticles were almost spherical in shape and their size, in the range of 327.4–511.5 nm, increased with the EPB-loading content increasing. In vitro release of EPB from DCMC self-assembled nanoparticles showed sustained drug release pattern and the release rate was related to pH of release media and drug loading content. The cytotoxic activity of EPB-loaded DCMC nanoparticles was assayed by the MTT colorimetric assay. Compared with free drug, EPB-loaded DCMC nanoparticles showed the higher cytotoxicity, which may be attributed to the enhanced cellular uptake. In vivo toxicity study indicated that DCMC conjugate did not induce unexpected side effects. Tissue biodistribution study was performed in tumor-bearing mice. The result showed that DCMC increased the uptake of EPB in the tumor and decreased the uptake of EPB in kidney and heart, compared to free drug. Moreover, tumor volume reductions induced by DCMC conjugate, free EPB and EDNs were 24.3%, 58.9% and 70%, respectively, which suggested that EDNs could effectively retard the growth of the tumor.
Keywords: Carboxymethyl curdlan; Deoxycholic acid; Epirubicin; Nanomedicine; Anti-tumor effect;

Rational development of a stable liquid formulation for nanomedicine products by Enikő R. Tőke; Orsolya Lőrincz; Eszter Somogyi; Julianna Lisziewicz (261-267).
DermaVir vaccine is a novel “pathogen-like” nanomedicine containing a plasmid DNA complexed with a polyethylenimine that is mannobiosylated to target antigen-presenting cells and to induce immune responses (pDNA/PEIm). To develop a commercially viable vaccine product we have systematically investigated the variability of raw materials and their relationship with the product's biological activity. We demonstrated that the cGMP quality requirements are not sufficient to reproducible formulate the nanomedicine with optimal biological activity. Unexpectedly, we found that the high cationic concentration of the pDNA favored the biological activity, but did not support the stability of the nanomedicine. Similarly, the presence of EDTA in the pDNA increased the size of the nanoparticle to microparticles causing the drop of its biological activity. A new parameter, the Cl/N ratio of the PEIm, also influenced the biological activity together with the chemical properties of the solvent. Based on these findings we have developed a pDNA/PEIm formulation capable to maintain the physical stability and the biological activity of the nanomedicine. This work illustrates some of the key steps that must be taken for the implementation of “Quality by Design” (QbD) approach for a biotech product.
Keywords: DermaVir; Polyethylenimine; Plasmid DNA; pDNA/PEIm nanoparticle; Liquid formulation;

Monoolein (MO) cubic phases entrapping hydroxypropyl β-cyclodextrin (HPβCD)/minoxidil (MXD) complex were prepared by hydrating molten MO with the complex solution, where the concentrations of HPβCD/MXD were 1.0%/0.32%–19.4%/1.98%. Without HPβCD, the maximum content of MXD loaded in the cubic phase was only 0.071%, but with aid of HPβCD, the content in the cubic phase increased up to 5.72%. The nanoparticles of the cubic phase were prepared by a bath type sonication using a Pluronic F127 as a dispersant. HPβCD/MXD complex had little effect on the size and the structure of cubic phase nanoparticles. In vitro skin permeation of MXD loaded in the cubic phase nanoparticles (2.44 mg/cm2 for 18 h), were higher than that of MXD dissolved in propylene glycol/water/ethanol (20/30/50, v/v/v) (1.91 mg/cm2 for 18 h), but the amount of MXD remained within skin was higher with the MXD solution (0.068 mg/cm2 for 18 h) than with the nanoparticles (0.023 mg/cm2 for 18 h).
Keywords: Cubic phase; Nanoparticle; Minoxidil; Hydroxypropyl β-cyclodextrin; Skin permeation;

Effect of surfactant concentration on transdermal lidocaine delivery with linker microemulsions by Jessica S. Yuan; Alice Yip; Nga Nguyen; Jacquelene Chu; Xiao-Yan Wen; Edgar J. Acosta (274-284).
A limited number of studies have been conducted to investigate the effect of surfactant concentration on microemulsion-mediated transdermal transport. Some studies suggest that increasing surfactant concentration reduces the partition of the active in the skin, and the overall transport. Other studies suggest that increasing surfactant concentration improves mass transport across membranes by increasing the number of “carriers” available for transport. To decouple these partition and mass transport effects, a three-compartment (donor, skin, receiver) mass balance model was introduced. The model has three permeation parameters, the skin-donor partition coefficient (K sd ), the donor-skin mass transfer coefficient (k ds ) and the skin-receiver mass transfer coefficient (k sr ), also known as skin permeability. The model was used to fit the permeation profile of lidocaine formulated in oil-in-water (Type I) and water-in-oil (Type II) lecithin–linker microemulsions. The results show that surfactant concentration has a relatively minor effect on the mass transfer coefficients, suggesting that permeation enhancement via disruption of the structure of the skin is not a relevant mechanism in these lecithin–linker microemulsions. The most significant effect was the increase in the concentration of lidocaine in the skin with increasing surfactant concentration. For Type I systems such increase in lidocaine concentration in the skin was linked to the increase in lidocaine solubilization in the microemulsion with increasing surfactant concentration. For Type II systems, the increase in lidocaine concentration in the skin was linked to the increase in skin-donor partition. A surfactant-mediated absorption/permeation mechanism was proposed to explain the increase in lidocaine concentration in skin with increasing surfactant concentration. The penetration profiles of hydrophobic and amphiphilic fluorescence probes are consistent with the proposed mechanism.
Keywords: Partition; Transdermal; Mechanism; Mass balance; Model; Lidocaine;

Silica nanoparticle coated liposomes: A new type of hybrid nanocapsule for proteins by Vellore J. Mohanraj; Timothy J. Barnes; Clive A. Prestidge (285-293).
A hybrid silica–liposome nanocapsule system containing insulin has been developed and the encapsulation, protection and release properties are evaluated. The formulation strategy is based on using insulin-loaded 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol liposomes as a template for the deposition of inert silica nanoparticles. The influence of formulation and process variables on particle size, zeta potential and liposome entrapment of insulin is reported. The ability to protect against lipolytic degradation and sustain insulin release in vitro in simulated GI conditions is also reported. Depending on the concentration and charge ratio of liposomes and silica nanoparticles, nanoparticle coated liposomes with varied size and zeta potential were obtained with an insulin entrapment efficiency of 70%. The silica nanoparticle coating protected liposomes against degradation by digestive enzymes in vitro; the release rate of insulin from silica coated liposomes was reduced in comparison to uncoated liposomes. Thus the liposomal release kinetics and stability can be controlled by including a specifically engineered nanoparticle layer. Silica nanoparticle–liposomes hybrid nanocapsules show promise as a delivery vehicle for proteins and peptides.
Keywords: Liposomes; Silica nanoparticles; Insulin release; Encapsulation; Lipolysis;

Enhanced gene transfection efficiency by polyamidoamine (PAMAM) dendrimers modified with ornithine residues by Ajay Kumar; Venkata K. Yellepeddi; Gareth E. Davies; Kevin B. Strychar; Srinath Palakurthi (294-303).
Aim of the study was to prepare and to evaluate gene transfection efficiency and cytotoxicity of the ornithine-conjugated PAMAMG4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative gene transfection study between PAMAMG4 dendrimers and the surface modified dendrimers was conducted in HEK 293T, GM7373 and NCI H157G cell lines. Effect of excess of ornithine (100 μM) on transfection efficiency of the ornithine-conjugated PAMAMG4 dendrimers was investigated in separate experiment. Cytotoxicity of the dendriplexes was tested in HEK 293T cells by MTT assay. 1H NMR and MALDI-TOF spectral analysis showed that about 60 molecules of ornithine (PAMAMG4-ORN60) were conjugated to a PAMAMG4 dendrimer. Preliminary studies indicated that dendriplexes at charge ratio (N/P 10) show higher transfection efficiency and presence of serum does not affect the transfection efficiency of the dendriplexes. Transfection efficiency of PAMAMG4-ORN60 dendriplexes was slightly higher in cancer cells (NCI H157G) as compared to HEK 293T cells. Transfection efficiency of the PAMAMG4-ORN60 dendrimers decreased in presence of excess of ornithine while there was no effect on the parent PAMAMG4 dendrimers. Cytotoxicity assay has shown that PAMAMG4-ORN60 dendriplexes at N/P 10 were safe at concentrations ≤50 μg/mL. It may be concluded that the ornithine-conjugated dendrimers possess the potential to be novel gene carrier.
Keywords: PAMAMG4; Ornithine-conjugated PAMAMG4 dendrimers (PAMAMG4-ORN60); Dendriplexes; Transfection efficiency; Endocytosis;

Transdermal absorption enhancement through rat skin of gallidermin loaded in niosomes by Aranya Manosroi; Penpan Khanrin; Warangkana Lohcharoenkal; Rolf G. Werner; Friedrich Götz; Worapaka Manosroi; Jiradej Manosroi (304-310).
Gallidermin (Gdm) loaded in anionic niosomes composed of Tween 61/CHL/DP (1:1:0.05 molar ratio) gave the highest entrapment efficiency (45.06%). This formulation gave antibacterial activity against Propionibacterium acnes and Staphylococcus aureus with the MIC and MBC of 3.75 and 7.5; 7.5 and 15 μg/μl, respectively. Gdm loaded in niosomes was more chemically stable than Gdm in aqueous solution of about 1.5 times. Gdm loaded and unloaded in niosomes were not found in the receiver solution investigated by vertical Franz diffusion cells at 37 °C for 6 h. Gdm loaded in niosomes showed higher cumulative amounts in viable epidermis and dermis (VED) of rat skin of about 2 times more than unloaded Gdm. Gdm loaded in niosomes and incorporated in gel exhibited the highest cumulative amounts (82.42 ± 9.28 μg cm−2) and fluxes (13.74 ± 1.55 μg cm−2  h−1) in stratum corneum (SC) and comparative cumulative amounts (183.16 ± 30.32 μg cm−2) and fluxes (25.74 ± 5.05 μg cm−2  h−1) in VED to the unloaded Gdm incorporated in gel. This study has suggested that Gdm loaded in anionic niosomes and incorporated in gel is the superior topical antibacterial formulation because of the high accumulation in the skin with no risk of systemic effect.
Keywords: Niosomes; Gallidermin; Antibacterial; Transdermal absorption;

Hybrid-modified poly(d,l-lactide-co-glycolide) nanospheres for a novel cellular drug delivery system by Kohei Tahara; Sahori Furukawa; Hiromitsu Yamamoto; Yoshiaki Kawashima (311-313).
We prepared surface-modified poly(d,l-lactide-co-glycolide) (PLGA) nanospheres (NS) for use as cellular drug and gene delivery systems using an emulsion solvent diffusion method. In this study, we screened for an appropriate surface modifier to improve NS cellular uptake. Poly-l-lysine (PLL)-modified PLGA NS were taken up by A549 cells in significantly higher amounts (17-fold) than unmodified NS. In order to provide a novel function, PLGA NS were hybrid-modified using both; a cationic polymer, PLL, and a nonionic surfactant, polysorbate 80, to improve cellular uptake in serum-containing medium (SCM). Hybrid modification abrogated the decreased PLGA NS cellular uptake in SCM as a result of better dispersion in serum compared to PLL-PLGA NS, which aggregated in SCM. Luciferase activity of Hybrid-NS/pCMV-Luc complexes in A549 cells in SCM was 122-fold higher than PLL-NS. Hybrid-PLGA NS were not cytotoxic for A549 cells. In conclusion, Hybrid-PLGA NS have great potential as effective cellular drug delivery carriers.
Keywords: Poly(d,l-lactide-co-glycolide); Nanospheres; Polysorbate 80; Poly-l-lysine; Hybrid modification;