International Journal of Pharmaceutics (v.454, #1)

Contribution of hydrophobicity, DNA and proteins to the cytotoxicity of cationic PAMAM dendrimers by Inessa Halets; Dzmitry Shcharbin; Barbara Klajnert; Maria Bryszewska (1-3).
In most articles, cytotoxicity of cationic polyamidoamine (PAMAM) dendrimers toward red blood cells has been exclusively explained by their surface charge. We have focused on dendrimer hydrophobicity as a second possible factor that determines this cytotoxicity. Using PAMAM-NH2 dendrimers from the 3rd to the 6th generations and PAMAM-NH2-C12(25%) dendrimer of the 4th generation bearing 25% acyl groups, these induced hemolysis that increased with their surface charge and hydrophobicity. Interaction of PAMAM-NH2-C12(25%) G4 dendrimer with blood proteins (γ-globulin, α-thrombin, human serum albumin) and calf thymus DNA (ctDNA) significantly reduced their cytotoxicity toward red blood cells.
Keywords: Dendrimer; Hemolysis; Hydrophobicity; DNA; Protein;

Eluting combination drugs from stents by Rajesh Thipparaboina; Wahid Khan; Abraham J. Domb (4-10).
Cardiovascular diseases (CVD) are one of the leading causes of death across the globe. Pathogenesis of coronary artery disease (CAD) is lead by the progression of atherosclerotic lacerations in coronary arteries. Percutaneous coronary intervention (PCI) using balloon angioplasty was introduced in 1979 and was majorly used in the treatment of these lesions. Introduction of bare metal stents (BMS) has revolutionized stenting procedures overcoming elastic recoil and reducing restenosis commonly associated with balloon angioplasty, but follow up studies have shown 20–30% prevalence of in-stent restenosis (ISR), this led to the development of drug eluting stents (DES). But long-term follow up studies have shown increased liability of stent thrombosis. Boosting the development of safer and effective DES expounding for therapies like biodegradable polymer based DES, polymer free DES, bioresorbable DES and combination DES to collectively reduce neointimal hyperplasia and promote endothelial healing. In dual-DES development, a combination employing an anti-restenotic agent (for preventing VSMC's proliferation), which is released for the first few weeks, and then the second drug a pro-healing agent (promoting re-endothelialization) released after a month would be ideal. Growing understanding in the areas of polymer therapeutics, nanoscale surface modifications and gene therapy would assist in the delivery of multiple drugs, which would further help in the design of promising therapeutic strategies for the treatment of CAD using stent based therapies.
Keywords: Coronary artery disease; Dual-drug eluting stents; Anti-restenotic agent; Pro-healing agents; Gene delivery;

T7 peptide-functionalized nanoparticles utilizing RNA interference for glioma dual targeting by Yuyang Kuang; Sai An; Yubo Guo; Shixian Huang; Kun Shao; Yang Liu; Jianfeng Li; Haojun Ma; Chen Jiang (11-20).
Among all the malignant brain tumors, glioma is the deadliest and most common form with poor prognosis. Gene therapy is regarded as a promising way to halt the progress of the disease or even cure the tumor and RNA interference (RNAi) stands out. However, the existence of the blood–brain barrier (BBB) and blood tumor barrier (BTB) limits the delivery of these therapeutic genes. In this work, the delivery system targeting to the transferrin (Tf) receptor highly expressed on both BBB and glioma was successfully synthesized and would not compete with endogenous Tf. U87 cells stably express luciferase were employed here to simulate tumor and the RNAi experiments in vitro and in vivo validated that the gene silencing activity was 2.17-fold higher with the targeting ligand modification. The dual-targeting gene delivery system exhibits a series of advantages, such as high efficiency, low toxicity, stability and high transaction efficiency, which may provide new opportunities in RNAi therapeutics and nanomedicine of brain tumors.
Keywords: RNA interference; T7 peptide; Glioma dual targeting; Dendrigraft poly-l-lysines; Transferrin;

Synergistic inhibition of breast cancer metastasis by silibinin-loaded lipid nanoparticles containing TPGS by Pengfei Xu; Qi Yin; Jianan Shen; Lingli Chen; Haijun Yu; Zhiwen Zhang; Yaping Li (21-30).
Tumor metastasis is the leading cause of breast cancer-related mortality and remains to be the principal obstacle for the successful chemotherapy of breast cancer. To block metastasis of breast cancer, silibinin-loaded lipid nanoparticles (SLNs) containing TPGS and phosphatidylcholine were designed and prepared by a thin-film hydration method. The optimized SLNs were approximately 45 nm in particle size with high stability in serum, which were further demonstrated to be efficiently uptaken by MDA-MB-231 breast cancer cells. Importantly, the SLNs could accumulate within tumor tissues with high efficiency and amounts. Compared with free silibinin, SLNs exhibited much stronger inhibitory effects on the invasion and migration of MDA-MB-231 cells through the downregulation of MMP-9 and Snail. More importantly, systematic in vivo evaluations demonstrated that SLNs treatment group resulted in 67% and 39% less pulmonary metastases formation than saline treatment group in the spontaneous and blood vessel metastasis models, respectively. Interestingly, the blank lipid nanoparticles without silibinin were also found, for the first time, to possess the efficient anti-metastatic capabilities to some extent. The biocompatibility assay reveals that SLNs treatment did not exhibit obvious systemic toxicity in two mouse models. Therefore, SLNs are the promising delivery systems against metastasis of breast cancer cells.
Keywords: Metastasis; Silibinin; TPGS; Nanoparticles; Breast cancer;

Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified with cholesterol anchored transferrin and TAT by Jie Tang; Li Zhang; Yayuan Liu; Qianyu Zhang; Yao Qin; Yujia Yin; Wenmin Yuan; Yuting Yang; Yafei Xie; Zhirong Zhang; Qin He (31-40).
This study was mainly focused on developing a dual-ligand liposomal delivery system to enhance both targeting specificity and cellular uptake. The specific ligand transferrin (TF) and the cationic cell-penetrating peptide TAT were connected with cholesterol via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (TAT/TF-PEG-LP). Then the in vitro cellular uptake by three kinds of cells that possessed different expressing levels of transferrin receptor (TFR) and the in vivo delivery efficiency were evaluated. Compared to the single-ligand TAT or TF modified liposomes (TAT-PEG-LP or TF-PEG-LP), TAT/TF-PEG-LP exhibited the enhanced cellular uptake and selectivity via the synergistic effect of both ligands in vitro. The ex vivo fluorescence imaging of tumors, the qualitative observation of tumor frozen section and the quantitative determination of cellular uptake in tumor tissues altogether showed the in vivo delivery efficiency of TAT/TF-PEG-LP was higher than that of other liposomes. In conclusion, the dual-ligand liposomes co-modified with TF and TAT possessed a strong capability for synergistic targeted delivery of payload into tumor cells both in vitro and in vivo.
Keywords: Dual-ligand; Synergistic effect; Transferrin; Cell penetrating peptide; Liposomes;

Pharmacoscintigraphy confirms consistent tamsulosin release from a novel triple-layered tablet by L.A. Hodges; K.A. Sime; L.A. Creech; S.M. Connolly; S.T. Barclay; Min Chang Kwon; Byeong Jin Jeon; Sang Min Shim; Hun Sik Wang; H.N.E. Stevens; Jun Sang Park (41-46).
Conventional modified release preparations of tamsulosin HCl have been linked to increased incidence of cardiovascular adverse events, possibly due to rapid drug peaks soon after ingestion. A ‘flattened’ absorption profile has been shown to reduce the occurrence of these unwanted effects while improving symptom control.The potential of a novel triple-layered tablet to effect prolonged release and continuous absorption of tamsulosin HCl in the gastrointestinal tract was investigated in this clinical study. Gastrointestinal (GI) transit behaviour was monitored by scintigraphic imaging of technetium-labelled tablets. Drug absorption levels were simultaneously determined through pharmacokinetic analysis of blood samples.A mean C max of 6 ± 3 ng/nL was achieved after 324 ± 184 min (mean t max). The mean AUC0-24 was noted as 4359 ± 1880 ng/mL min. The mean gastric emptying and colon arrival times of the tablets were 105.2 ± 68.9 and 270.1 ± 32.0 min post-dose; giving a mean small intestine transit time of 164.9 ± 83.6 min. Variations in gastrointestinal transit did not appear to influence drug absorption.Correlation of scintigraphic and PK data indicated that tamsulosin HCl is released steadily throughout the entire GI tract, suggesting that the mechanism of drug release is independent of GI site allowing drug release even in the low moisture environment of the colon.
Keywords: Tamsulosin hydrochloride; GLARS tablet; Gamma scintigraphy; Gastrointestinal transit;

Hydrogel blends with adjustable properties as patches for transdermal delivery by Stefania Mazzitelli; Cinzia Pagano; Danilo Giusepponi; Claudio Nastruzzi; Luana Perioli (47-57).
The effect of different preparation parameters were analyzed with respect to the rheological and pharmaceutical characteristics of hydrogel blend patches, as transdermal delivery formulations.The effect of different preparation parameters were analyzed with respect to the rheological and pharmaceutical characteristics of hydrogel blend patches, as transdermal delivery formulation. Mixtures of pectin and gelatin were employed for the production of patches, with adjustable properties, following a two-step gelation procedure. The first gelation, a thermal one, is trigged by the presence of gelatin, whereas, the second gelation, an ionic one, is due to the formation of the typical egg box structure of pectin. In particular, the patch structural properties were assessed by oscillation stress sweep measurements which provided information concerning their viscolelastic properties. In addition, different modalities for drug loading were analyzed with respect to drug homogeneous distribution; testosterone was employed as model drug for transdermal administration. Finally, the performances of the produced transdermal patches were studied, in term of reproducibility and reliability, by determination of in vitro drug release profiles.
Keywords: Gelatin; Pectin; Hydrogels; Transdermal delivery formulation;

Comparison of ionic and non-ionic drug release from multi-membrane spherical aerogels by Anja Veronovski; Željko Knez; Zoran Novak (58-66).
Alginate multi-membrane aerogels, prepared using Ca2+ and Ba2+ ions for alginate cross-linking, were used as ionic and non-ionic drug carriers for oral drug delivery. After optimising parameters during sol–gel synthesis, prolonged release of both drugs was obtained.The presented research was oriented towards the preparation of dry biodegradable alginate aerogels with multi-membranes using a multi-step sol–gel process with potential applications as carriers during oral drug delivery. First alginate spherical hydrogels were formed in CaCl2 or BaCl2 solutions by ionic cross-linking. These cores were further immersed into alginate sodium solution, filtered through a sieve, and dropped into the salt solution again. Multi-membrane hydrogels were obtained by repeating the above process. They were further converted into aerogels by supercritical drying. The effect of the number of membranes was investigated regarding the loading and release of the model drugs nicotinic acid and theophylline. Moreover, the efficiencies of Ba2+ and Ca2+ metal ions for forming tridimensional networks that retain and extend drug release were also investigated. Nicotinic acid release was prolonged by adding membranes around the core and using Ca2+ for cross-linking. However, retarded theophylline release was only obtained by using Ba2+ for cross-linking. Namely, by increasing the number of membranes and BaCl2 concentration drug release became linear versus time in all studied cases. In the case of nicotinic acid loading increased by adding membranes around the core, however, for theophylline the opposite results were obtained due to the different nature of the model drugs.
Keywords: Natural polysaccharide; Alginate gel; Controlled drug release; Drug carrier; Multi-membrane gel;

Effect of surface chemistry of porous silicon microparticles on glucagon-like peptide-1 (GLP-1) loading, release and biological activity by Anne Huotari; Wujun Xu; Juha Mönkäre; Miia Kovalainen; Karl-Heinz Herzig; Vesa-Pekka Lehto; Kristiina Järvinen (67-73).
Recently, mesoporous silicon (PSi) microparticles have been shown to extend the duration of action of peptides, reducing the need for frequent injections. Glucagon-like peptide 1 (GLP-1) is a potential novel treatment for type 2 diabetes.The aim of this study was to evaluate whether GLP-1 loading into PSi microparticles reduce blood glucose levels over an extended period. GLP-1 (pI 5.4) was loaded and released from the negatively charged thermally oxidized (TOPSi, pI 1.8) and thermally carbonized (TCPSi, pI 2.6) PSi microparticles and from the novel positively charged amine modified microparticles, designated as TOPSi-NH2-D (pI 8.8) and TCPSi-NH2-D (pI 8.8), respectively.The adsorption of GLP-1 onto the PSi microparticles could be increased 3–4-fold by changing the PSi surface charge from negative to positive, indicating that the positive surface charge of PSi promoted an electrostatic interaction between the negatively charged peptide. All the GLP-1 loaded PSi microparticles lowered the blood glucose levels after a single s.c. injection but surprisingly, TOPSi-NH2-D and TCPSi-NH2-D were not able to prolong the effect when compared to TOPSi, TCPSi or GLP-1 solution. However, TOPSi-NH2-D and TCPSi-NH2-D microparticles were able to carry improved payloads of active GLP-1 encouraging continuing further attempts to achieve sustained release.
Keywords: In vivo; Mesoporous silicon; Peptide delivery; GLP-1; Blood glucose levels;

Ursodeoxycholic acid-conjugated chitosan for photodynamic treatment of HuCC-T1 human cholangiocarcinoma cells by Hye Myeong Lee; Young-Il Jeong; Do Hyung Kim; Tae Won Kwak; Chung-Wook Chung; Cy Hyun Kim; Dae Hwan Kang (74-81).
Chitosan was hydrophobically modified with ursodeoxycholic acid (UDCA) to fabricate nano-photosensitizer for photodynamic therapy (PDT) of HuCC-T1 cholangiocarcinoma cells. Synthesis of UDCA-conjugated chitosan (ChitoUDCA) was confirmed using 1H NMR spectra. Chlorin E6 (Ce6) was used as a photosensitizer and incorporated into ChitoUDCA nanoparticles through formation of ion complexes. Morphology of Ce6-incorporated ChitoUDCA nanoparticles was observed using TEM and their shapes were spherical with sizes around 200–400 nm. The PDT potential of Ce6-incorporated ChitoUDCA nanoparticles were studied with HuCC-T1 human cholangiocarcinoma cells. The results showed that ChitoUDCA nanoparticles enhances of Ce6 uptake into tumor cells, phototoxicity, and ROS generation compared to Ce6 itself. Furthermore, Ce6-incorporated ChitoUDCA nanoparticles showed quenching in aqueous solution and sensing at tumor cells. We suggest that Ce6-incorporated ChitoUDCA nanoparticles are promising candidates for PDT of cholangiocarcinoma cells.
Keywords: Ursodeoxycholic acid; Cholangiocarcinoma; Chlorin e6; Chitosan; Photodynamic therapy;

Effect of iron liposomes on anemia of inflammation by Li Yuan; Lina Geng; Lan Ge; Peng Yu; Xianglin Duan; Jun Chen; Yanzhong Chang (82-89).
Supplementation with iron-fortified foods is an effective method for treating iron deficiency diseases. However, traditional iron agents used to treat anemia of inflammation (AI) have little effect. In this study, two types of iron liposomes, heme liposomes (HEME-LIP) and ferric citrate liposomes (FAC-LIP), were prepared by the rotary-evaporated film-ultrasonication method, and the encapsulation efficiencies, microstructures, size distributions and zeta potentials were assessed. Both types of iron liposomes showed stable physical characteristics. When used to treat rat models of AI, FAC-LIP and HEME-LIP could increase serum iron levels by 119% and 54% higher than did ferric citrate (FAC) and heme, respectively. Furthermore, the hepcidin, a key regulator of iron homeostasis was up-regulated by these iron liposomes, especially by HEME-LIP. These results indicate that the absorption of iron liposomes was improved over that of unencapsulated iron agents. Thus, iron liposomes may be used to fortify food in treating iron deficiency diseases, especially AI.
Keywords: Iron; Liposome; Anemia; Inflammation; Hepcidin;

Extrudates with laminar shape for delivery of drugs were manufactured at room temperature and without solvents. Different lipidic materials, formulations and extrusion conditions were studied, as well as the influence of the size of particles of coumarin on the release from the extrudates.Materials were mixed prior to feeding an in-house built ram extruder with a rectangular shaped die. The process of extrusion was characterized for the force at steady state whereas extrudates were characterized immediately after production and over storage for density, porosity, bending strength, elasticity, stiffness, deformation, thermal behavior and release performance of coumarin, the model drug.The variety of lipid excipients and their proportions in the formulations directly influenced the properties of the extrudates, namely the bending strength, stiffness, deformation and elasticity and, the density and porosity, which changed over storage time: in general, the bending strength, stiffness and porosity increased over time, whereas deformation, elasticy and density decreased. The thermal analysis supported these observations as there was an increase in the enthalpies of fusion of the extrudates over time. The release of coumarin from the extrudates occured both by diffusion within the extrudates and by structural alterations of the extrudates and an increase of the coumarin particle size corresponded to a decrease of the release rate.The study has proved the ability to manufacture extrudates in a continuous fashion, with laminar shape using a green technology.
Keywords: Extrusion; Green technology; Laminar extrusion; Mechanical properties; Room-temperature extrusion; Solvent-free extrusion;

In our previous study, a novel alginate-based bilayer film for slow-release wound dressings was successfully developed. We found that alginate alone yielded poor films; however, the addition of gelatine had significantly enhanced the drug dispersion as well as the physical properties. Here, an investigation of the drug–polymer interactions in the bilayer films was carried out. Drug content uniformity test and microscopy observation revealed that the addition of gelatine generated bilayer films with a homogenous drug distribution within the matrix. The FTIR and XRD data showed an increase in film crystallinity which might infer the presence of drug–polymer crystalline microaggregates in the films. DSC confirmed the drug–polymer interaction and indicated that the gelatine has no effect on the thermal behaviour of the microaggregates, suggesting the compatibility of the drug and excipients in the bilayer films. In conclusion, the addition of gelatine can promote homogenous dispersion of hydrophobic drugs in alginate films possibly through the formation of crystalline microaggregates.
Keywords: Sodium alginate; Gelatine; Hydrophobic drug dispersion; Crystalline microaggregates; Bilayer films;

Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments by Salome-Juliette Koussoroplis; Sam Heywood; Catherine Uyttenhove; Céline Barilly; Jacques Van Snick; Rita Vanbever (107-115).
The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab′)2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.
Keywords: Antibody fragment; PEGylation; Anti-interleukin-17A; Bronchial hyperreactivity;

Modulation of stability and mucoadhesive properties of chitosan microspheres for therapeutic gastric application by Mariana Fernandes; Inês C. Gonçalves; Stefania Nardecchia; Isabel F. Amaral; Mário A. Barbosa; M. Cristina L. Martins (116-124).
Chitosan microspheres have been explored for pharmaceutical applications, namely as a drug delivery systems for Helicobacter pylori gastric infection treatment, due to their mucoadhesive capacity. In this study, a different application of chitosan microspheres is proposed aiming the creation of an H. pylori-binding system where, after oral administration, microspheres will capture and remove these bacteria from infected patients, taking advantage of their muco/bacterial adhesive process. However, mucoadhesion is influenced by the degree of crosslinking necessary to avoid microspheres dissolution in the acidic gastric environment.During this work, the effect of genipin crosslinking on the stability, size, charge and mucoadhesive properties of chitosan microspheres under acidic pH was studied.Chitosan microspheres with ∼170 μm were produced by ionotropic gelation and subsequently covalently crosslinked with genipin in different degrees. The crosslinking reaction was followed by infrared spectroscopy and time-lapse fluorescence microscopy, since we have demonstrated that the fluorescence intensity of chitosan microspheres increases with genipin chemical bonding to chitosan.Results showed that both the zeta potential and the swelling capacity of chitosan microspheres decrease with increasing crosslinking. When immersed in simulated gastric fluid (SGF) with pepsin for 7 days, chitosan microspheres crosslinked with 10 mM of genipin for 1 h did not dissolve and doubled their size to approximately 345 μm. Furthermore, they maintained their in vitro mucoadhesion to soluble gastric mucins at both pH tested (3.6 and 6.5) and presented an in vivo retention time of around 2 h in the stomach of C57BL/6 mice.
Keywords: Microspheres; Chitosan; Genipin; Mucoadhesion; Biomaterials; Helicobacter pylori;

Baicalein and hydroxypropyl-γ-cyclodextrin complex in poloxamer thermal sensitive hydrogel for vaginal administration by Qiuna Zhou; Lin Zhong; Xiaohui Wei; Wei Dou; Guixin Chou; Zhengtao Wang (125-134).
The present study aimed to prepare a chemically and physically stable formulation of baicalein (Ba) in an in situ thermally sensitive hydrogel for vaginal administration. An inclusion complex of Ba and hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was first developed to increase the stability and solubility of Ba in an aqueous solution. The formation of the Ba-HP-γ-CD complex was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray powder diffractometry (XRPD). Poloxamers 407, 188 (P407, P188), sodium alginate (SA), hydroxypropylmethylcellulose (HPMC), and benzalkonium bromide were mixed to obtain a hydrogel with an appropriate gelation temperature for vaginal use. Among the formulations examined, P407/Ba-HP-γ-CD/HPMC/P188/benzalkonium bromide (18/0.96/0.5/4.0/0.02%) represented the appropriate gelation temperature and acceptable drug release rate at the administration site. Release of Ba-HP-γ-CD from the poloxamer hydrogel followed Peppas equation, which suggests that it occurred through coupled corrosion-diffusion mechanism, and the corrosion-release curve showed that corrosion was the primary mode of release. In animal study, treatment by using Ba-HP-γ-CD thermosensitive hydrogel could produce a restoration of damaged tissues. Thermosensitive hydrogel formulation of the Ba-HP-γ-CD complex appears to be a promising treatment for cervicitis.
Keywords: Baicalein; Hydroxypropyl-γ-cyclodextrin; Thermosensitive; Vaginal hydrogel; Cervicitis;

A novel strategy to design sustained-release poorly water-soluble drug mesoporous silica microparticles based on supercritical fluid technique by Wang Li-hong; Che Xin; Xu Hui; Zhou Li-li; Han Jing; Zou Mei-juan; Liu Jie; Liu Yi; Liu Jin-wen; Zhang Wei; Cheng Gang (135-142).
The organic solvent solution immersion method was often used to achieve the loading of the drugs into mesoporous silica, but the drugs that have loaded into the pores of the mesoporous silica would inevitable migrate from the inside to the external surface or near the outside surface during the process of drying. Hence, it often leads to the pores of mesoporous materials not be fully utilized, and results in a low drug loading efficiency and a fast releasing rate.The purpose of this study was to develop a novel drug loading strategy to avoid soluble component migration during the process of drying, then, to prepare poorly water-soluble drug mesoporous silica microparticles with higher drug loading efficiency and longer sustained-release time.Ibuprofen was used as model drug. The microparticles were prepared by a novel method based on mesoporous silica and supercritical fluid (SCF) technique. The drug-loaded mesoporous silica microparticles prepared by SCF technique were analyzed by thermogravimetric analysis (TGA), N2 adsorption/desorption, scanning electron microscopy (SEM), powder X-ray diffraction (XRD) and differential scanning calorimetry (DSC). In vitro releasing study was used to evaluate the sustained-release effect of the drug-loaded microparticles.By virtue of the high diffusibility and the high dissolving capacity of the supercritical carbon dioxide (SCF-CO2), the poorly water-soluble drugs, ibuprofen, entered the pores of the mesoporous silica. The amount and the depth of ibuprofen entered the pores of the mesoporous silica by SCF technique were both larger than those by the solution immersion method. It was found that ibuprofen loaded into the mesoporous silica by SCF technique was amorphous and the largest amount of the ibuprofen loaded into the mesoporous silica by SCF technique could reach 386 mg/g (w/w, ibuprofen/SiO2), it was more than that by the solution immersion method. In vitro releasing study showed that the sustained-release effect of ibuprofen in the samples prepared by SCF technique was 50% in 15 min and 90% in 60 min. It was longer than that prepared by the solution immersion method.Present study showed that sustained-release poorly water-soluble drug mesoporous silica microparticle based on SCF technique has twofold advantages. One is the larger drug loading amount in internal pores of the mesoporous silica, the other is the longer drug releasing time.
Keywords: Mesoporous silica; Sustained-release; Loading capacity; Ibuprofen; Supercritical CO2 fluid;

Do iodinated nano-emulsions designed for preclinical vascular imaging alter the vascular reactivity in rat aorta? by Nicolas Anton; Marina Atzenhoffer; François Daubeuf; Xiang Li; Valérie B. Schini-Kerth; Barbara Delmotte; Thierry F. Vandamme; Thierry Chataigneau (143-148).
Display OmittedThis study proposes a new methodology to evaluate the putative consequences of the long-lasting circulation in the blood pool of nanoparticulate systems widely used in nanomedicine, Indeed, the blood pool contrast agent for micro-computed tomography, i.e. iodinated nano-emulsions, have recently been developed, for their great potential in medical applications such as advanced diagnosis, image-guided surgery, personalized medicine or theragnostics. Stealth nanoparticles exhibit a low recognition by the reticuloendothelial system, resulting in a prolonged circulation in the bloodstream and long-lasting contact with the endothelium. Therefore, the aim of the present study is to determine whether this prolonged interaction could induce an alteration of the vascular reactivity in rat aorta. The Iodinated nano-emulsions were intravenously injected in anesthetized rats. After 1 h of contrast agent circulation in the blood pool, the thoracic aorta was removed for the study of vascular reactivity. These animals were compared with control (untreated) rats and a third group of rats receiving an injection of phosphate buffered saline (i.e. dispersing phase of the nano-emulsions). Phenylephrine-induced concentration-dependent contractions of the isolated rat thoracic aorta were not modified whatever the group. Sodium nitroprusside (a nitric oxide (NO) donor)-induced relaxations of endothelium-denuded aorta were also unaltered in response to the different administrations. In contrast, in comparison with control animals, endothelium-dependent NO-mediated relaxations to acetylcholine were significantly impaired in thoracic aorta from PBS-treated rats, but not in animals receiving the iodinated nano-emulsion. In addition, neither isoprenaline-induced nor levcromakalim-induced relaxations were modified in the aorta from the three groups of animals. These findings indicate that even with a long-lasting residence time of the iodinated nano-emulsion in the blood flow, these iodinated nano-emulsions do not alter the vascular reactivity and thus can be used as contrast agent for preclinical vascular imaging on small laboratory animals.
Keywords: Nano-emulsion; Aorta; Nitric oxide; Contraction; Relaxation;

Preparation, characterization, and transport of dexamethasone-loaded polymeric nanoparticles across a human placental in vitro model by Hazem Ali; Irina Kalashnikova; Mark Andrew White; Michael Sherman; Erik Rytting (149-157).
The purpose of this study was to prepare dexamethasone-loaded polymeric nanoparticles and evaluate their potential for transport across human placenta. Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics: particle size, polydispersity index, zeta potential, and drug encapsulation efficiency. Dexamethasone and nanoparticle transport was subsequently investigated using the BeWo b30 cell line, an in vitro model of human placental trophoblast cells, which represent the rate-limiting barrier for maternal–fetal transfer. Encapsulation efficiency and drug transport were determined using a validated high performance liquid chromatography method. Nanoparticle morphology and drug encapsulation were further characterized by cryo-transmission electron microscopy and X-ray diffraction, respectively. Nanoparticles prepared from poly(lactic-co-glycolic acid) were spherical, with particle sizes ranging from 140 to 298 nm, and encapsulation efficiency ranging from 52 to 89%. Nanoencapsulation enhanced the apparent permeability of dexamethasone from the maternal compartment to the fetal compartment more than 10-fold in this model. Particle size was shown to be inversely correlated with drug and nanoparticle permeability, as confirmed with fluorescently labeled nanoparticles. These results highlight the feasibility of designing nanoparticles capable of delivering medication to the fetus, in particular, potential dexamethasone therapy for the prenatal treatment of congenital adrenal hyperplasia.
Keywords: Dexamethasone; Nanoparticles; Congenital adrenal hyperplasia; BeWo cells; Placenta; Pregnancy;

This paper reports synthesis and evaluation of Janus type generation G-1 and G-2 dendrimers. The dendrimers have been constructed by linking two building blocks, dendrons and oleic acid, through ester and amide bonds and were well characterized by Fourier-transform infrared (FT-IR), 1H NMR, 13C NMR and electrospray ionization mass spectrometry (ESI-MS). The dendrimers have been evaluated for in vitro cytotoxicity using sulforhodamine B assay (SRB assay) and in vivo skin irritation potential. The ester linked dendrimers did not exhibit any cytotoxicity even up to 80 μg/ml while G-1 and G-2 generations dendrimers with amide linkage exhibited toxicity above 70 μg/ml and 21 μg/ml, respectively, none of the dendrimers showed any skin irritation. All the dendrimers, tested for their skin permeation enhancement potential using diclofenac sodium (DS) as a model drug at a concentration of 1% in gels, showed significant increase in steady-state flux (ER flux ) of the drug as compared to control (without enhancer), and oleic acid. Amongst the dendrimers, the ester linked G-1 and G-2 dendrimers showed highest ER flux , 3.33 ± 0.31 and 3.39 ± 0.21, respectively.
Keywords: Dendrimers; Transdermal delivery; Penetration enhancers; Diclofenac sodium; Oleic acid; Anti-inflammatory; Oleodenrimers;

Detecting phase separation of freeze-dried binary amorphous systems using pair-wise distribution function and multivariate data analysis by Norman Chieng; Hjalte Trnka; Johan Boetker; Michael Pikal; Jukka Rantanen; Holger Grohganz (167-173).
The purpose of this study is to investigate the use of multivariate data analysis for powder X-ray diffraction-pair-wise distribution function (PXRD-PDF) data to detect phase separation in freeze-dried binary amorphous systems. Polymer–polymer and polymer–sugar binary systems at various ratios were freeze-dried. All samples were analyzed by PXRD, transformed to PDF and analyzed by principal component analysis (PCA). These results were validated by differential scanning calorimetry (DSC) through characterization of glass transition of the maximally freeze-concentrate solute (T g ’). Analysis of PXRD-PDF data using PCA provides a more clear ‘miscible’ or ‘phase separated’ interpretation through the distribution pattern of samples on a score plot presentation compared to residual plot method. In a phase separated system, samples were found to be evenly distributed around the theoretical PDF profile. For systems that were miscible, a clear deviation of samples away from the theoretical PDF profile was observed. Moreover, PCA analysis allows simultaneous analysis of replicate samples. Comparatively, the phase behavior analysis from PXRD-PDF-PCA method was in agreement with the DSC results. Overall, the combined PXRD-PDF-PCA approach improves the clarity of the PXRD-PDF results and can be used as an alternative explorative data analytical tool in detecting phase separation in freeze-dried binary amorphous systems.
Keywords: Freeze-drying; Phase separation; Powder X-ray diffraction; Pair-wise distribution function; Principal component analysis;

Bioactive insulin microparticles produced by supercritical fluid assisted atomization with an enhanced mixer by Zhe Du; Chuan Tang; Yi-Xin Guan; Shan-Jing Yao; Zi-Qiang Zhu (174-182).
Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA–HCM) was used to micronize insulin from aqueous solution without use of any organic solvents. Insulin microparticles produced under different operating conditions including solution type, solution concentration and precipitator temperature presented distinct morphologies such as highly folded, partly deflated, corrugated or smooth hollow spherical shape. Solution concentration had a striking influence on particle size, and insulin microparticles produced from acidic solution had mean diameters increasing from 1.4 μm to 2.7 μm when protein concentration increased from 3 g/L to 50 g/L. HPLC chromatograms showed no degradation of insulin after SAA–HCM processing and FTIR, CD and fluorescence data further confirmed the structural stability. TGA analysis revealed that insulin microparticles remained moderate moisture content compared with raw material. In vivo study showed that insulin processed by SAA–HCM from acidic solution retained identical bioactivity. SAA–HCM is demonstrated to be a very promising process for insulin inhaled formulation development.
Keywords: Supercritical fluid assisted atomization; Hydrodynamic cavitation mixer; Insulin; Micronization; Bioactivity;

Injectable extracellular matrix hydrogel developed using porcine articular cartilage by Jin Seon Kwon; So Mi Yoon; Sun Woo Shim; Ji Hoon Park; Kyung Jun Min; Hyun Ju Oh; Jae Ho Kim; Young Jick Kim; Jun Jin Yoon; Byung Hyune Choi; Moon Suk Kim (183-191).
A BSA-loaded porcine articular cartilage powder (PCP) suspension at room temperature easily gelled upon s.c. injection into rats and exhibited sustained release of BSA over extended experimental periods.This work was first development of a delivery system capable of maintaining a sustained release of protein drugs at specific sites by using potentially biocompatible porcine articular cartilage. The prepared porcine articular cartilage powder (PCP) was easily soluble in phosphate-buffered saline. The PCP suspension easily entrapped bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in pharmaceutical formulations at room temperature. The aggregation of PCP and BSA-FITC was confirmed by dynamic light scattering. When the BSA-FITC-loaded PCP suspension was subcutaneously injected into rats, it gelled and formed an interconnecting three-dimensional PCP structure that allowed BSA to penetrate through it. The amount of BSA-FITC released from the PCP hydrogel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated sustained release of BSA-FITC for 20 days in vivo. In addition, the PCP hydrogel induced a slight inflammatory response. In conclusion, we showed that the PCP hydrogel could serve as a minimally invasive therapeutics depot.
Keywords: Porcine cartilage powder; Injectable; Hydrogel; BSA; Protein;

Mixed protein–DNA gel particles for DNA delivery: Role of protein composition and preparation method on biocompatibility by M.C. Morán; D.R. Nogueira; M.P. Vinardell; M.G. Miguel; B. Lindman (192-203).
Mixtures of two cationic proteins were used to prepare protein–DNA gel particles, employing associative phase separation and interfacial diffusion (Morán et al., 2009a). By mixing the two proteins, we have obtained particles that displayed higher loading efficiency and loading capacity values than those obtained in single-protein systems. However, nothing is known about the adverse effects on haemocompatibility and cytotoxicity of these protein–DNA gel particles. Here, we examined the interaction of protein–DNA gel particles obtained by two different preparation methods, and their components, with red blood cells and established cells. From a haemolytic point of view, these protein–DNA gel particles were demonstrated to be promising long-term blood-contacting medical devices. Safety evaluation with the established cell lines revealed that, in comparison with proteins in solution, the cytotoxicity was reduced when administered in the protein–DNA systems. In comparison with large-sized particles, the cytotoxic responses of small-sized protein–DNA gel particles showed to be strongly dependent of both the protein composition and the cell line being the tumour cell line HeLa more sensitive to the deleterious effects of the mixed protein-based particles. The observed trends in haemolysis and cell viabilities were in agreement with the degree of complexation values obtained for the protein–DNA gel particles prepared by both preparation methods.
Keywords: DNA gels; Particles; Size; Haemolysis; In vitro cytotoxicity;

Non-covalent functionalization of single-walled carbon nanotubes with modified polyethyleneimines for efficient gene delivery by Behzad Behnam; Wayne T. Shier; Azadeh Hashem Nia; Khalil Abnous; Mohammad Ramezani (204-215).
Functionalized carbon nanotubes (CNTs) have been recently emerged as important class of vectors for delivery of DNA and other biomolecules into various cells. In this study, single-walled carbon nanotubes (SWNTs) were functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to polyethyleneimines (PEIs). PEIs of three molecular weights (25, 10 and 1.8 kDa) were used. CNTs were functionalized with the PEI series either through phospholipid moiety (via a polyethyleneglycol linker) or through directly-attached long (18 carbons) or intermediate (10 carbons) hydrophobic alkyl moieties. All PEI-functionalized CNTs exhibited good stability and dispersibility in biological media. Visualizing of functionalized CNTs and lack of aggregation were confirmed by atomic force microscopy. The PEI derivatives bound to CNTs retained the ability to fully condense plasmid DNA at low N/P ratios and substantial buffering capacity in the endosomal pH range. PEI-functionalized CNTs exhibited increased transfection efficiency compared to underivatized PEIs up to 19-fold increase being observed in the functionalized CNT with the smallest PEI tested, the smallest hydrophobic attachment moiety tested and no linker. Also PEI-functionalized CNTs were effective gene delivery vectors in vivo following tail vein injection in mice with the largest expression occurring with the vector PEI-functionalized through a polyethyleneglycol linker.
Keywords: Single-walled carbon nanotubes; Polyethyleneimine; Gene delivery; Non-viral vector; In vivo;

To exploit the therapeutic potential of growth factors in tissue regeneration, it is necessary to design a porous scaffold in order to concurrently accommodate cells and release angiogenic factors in a controlled manner. In an attempt to address these issues, we developed a nanocomposite scaffold based on silk/calcium phosphate/PLGA by freeze-drying and electrospinning in order to control the release of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The highly porous scaffold possessed appropriate chemical and physical structure as confirmed by FTIR, XRD, SEM, and Zeta potential analysis. Furthermore, the incorporation of PDGF and VEGF in the scaffold was confirmed using Raman spectroscopy while their bioactivity was maintained by 82% and 89% for up to 28 days, respectively. The release of PDGF was slower than VEGF as respected. Additionally, the scaffold could promote proliferation, alkaline phosphatase production and attachment of human osteoblast cells. Histological examination established new bone matrix formation with neovascularization in the angiogenic factors loaded scaffold after 10 weeks of implantation in rabbit model. Finally, it was considered that the fabricated nanocomposite could be useful for bone tissue engineering applications.
Keywords: Silk/calcium phosphate/PLGA; Controlled release; PDGF; VEGF; Bone tissue engineering;

Studies on the kinetics of killing and the proposed mechanism of action of microemulsions against fungi by Ibrahim S.I. Al-Adham; Hana Ashour; Elham Al-Kaissi; Enam Khalil; Martin Kierans; Phillip J. Collier (226-232).
Microemulsions are physically stable oil/water clear dispersions, spontaneously formed and thermodynamically stable. They are composed in most cases of water, oil, surfactant and cosurfactant. Microemulsions are stable, self-preserving antimicrobial agents in their own right. The observed levels of antimicrobial activity associated with microemulsions may be due to the direct effect of the microemulsions themselves on the bacterial cytoplasmic membrane. The aim of this work is to study the growth behaviour of different microbes in presence of certain prepared physically stable microemulsion formulae over extended periods of time. An experiment was designed to study the kinetics of killing of a microemulsion preparation (17.3% Tween-80, 8.5% n-pentanol, 5% isopropyl myristate and 69.2% sterile distilled water) against selected test microorganisms (Candida albicans, Aspergillus niger, Schizosaccharomyces pombe and Rhodotorula spp.). Secondly, an experiment was designed to study the effects of the microemulsion preparation on the cytoplasmic membrane structure and function of selected fungal species by observation of 260 nm component leakage. Finally, the effects of the microemulsion on the fungal membrane structure and function using S. pombe were studied using transmission electron microscopy. The results showed that the prepared microemulsions are stable, effective antimicrobial systems with effective killing rates against C. albicans, A. niger, S. pombe and Rhodotorula spp. The results indicate a proposed mechanism of action of significant anti-membrane activity, resulting in the gross disturbance and dysfunction of the cytoplasmic membrane structure which is followed by cell wall modifications, cytoplasmic coagulation, disruption of intracellular metabolism and cell death.
Keywords: Microemulsions; Antimicrobial;

Water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency of aminoglycoside antibiotics by Alexandru Mihai Grumezescu; Ecaterina Andronescu; Alina Maria Holban; Anton Ficai; Denisa Ficai; Georgeta Voicu; Valentina Grumezescu; Paul Cătălin Balaure; Carmen Mariana Chifiriuc (233-240).
The aim of this study was to obtain a nano-active system to improve antibiotic activity of certain drugs by controlling their release. Magnetic composite nanomaterials based on magnetite core and cross-linked chitosan shell were synthesized via the co-precipitation method and characterized by Fourier transform infrared spectroscopy (FT-IR), infrared microscopy (IRM), scanning electron microscopy (SEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The prepared magnetic composite nanomaterials exhibit a significant potentiating effect on the activity of two cationic (kanamycin and neomycin) drugs, reducing the amount of antibiotics necessary for the antimicrobial effect. The increase in the antimicrobial activity was explained by the fact that the obtained nanosystems provide higher surface area to volume ratio, resulting into higher surface charge density thus increasing affinity to microbial cell and also by controlling their release. In addition to the nano-effect, the positive zeta potential of the synthesized magnetite/cross-linked chitosan core/shell magnetic nanoparticles allows for a more favorable interaction with the usually negatively charged cell wall of bacteria. The novelty of the present contribution is just the revealing of this synergistic effect exhibited by the synthesized water dispersible magnetic nanocomposites on the activity of different antibiotics against Gram-positive and Gram-negative bacterial strains. The results obtained in this study recommend these magnetic water dispersible nanocomposite materials for applications in the prevention and treatment of infectious diseases.
Keywords: Magnetic chitosan beads; Entrapped drugs; Antimicrobial therapy; Cross-linked;

Systemic in vitro and in vivo evaluation for determining the feasibility of making an amorphous solid dispersion of a B-Raf (rapidly accelerated fibrosarcoma) inhibitor by Yong Cui; Po-Chang Chiang; Edna F. Choo; Jason Boggs; Joachim Rudolph; Jonas Grina; Steve Wenglowsky; Yingqing Ran (241-248).
It is well acknowledged that oral bioavailability of a drug candidate is often influenced by factors such as the permeability, physico-chemical properties, and metabolism of the drug. Among the physico-chemical properties, solubility and dissolution rate are considered the most critical factors affecting the oral bioavailability of a compound G–F is a potent and selective B-Raf inhibitor with poor solubility and adsorption is limited by solubility at high doses. In order to overcome this issue using a spray-dried amorphous dispersion (SDD) formulation was evaluated. A combination of theoretical solubility prediction and in vitro dissolution, were used to predict the in vivo exposure of G–F. The predicted value was found to have good agreement with the in vivo exposure from dosing the crystalline and amorphous form of G–F.In general, this combined approach demonstrated that the amorphous form of G–F offers an advantage over the crystalline form of G–F in terms of solubility; in vitro dissolution and in vivo absorption were predictable and consistent with the literature. This systemic approach provides a great value for compound development.
Keywords: Solubility; Prediction; Dissolution; B-Raf; SDD; Bioavailability;

Hypericin (HY) is a naturally-occurring, potent photosensitizer. However, its lipophilicity limits its therapeutic applications. Our attempt is, thus, to develop a biodegradable nanocarrier for hypericin capable of preserving its antibacterial photoactivity. Amphiphilic block copolymers were synthesized to prepare hypericin-laden nanoparticles (HY-NPs). The antimicrobial photoactivity of HY-NPs was assessed; in vitro against biofilm and planktonic cells of methicillin resistant Staphylococcus aureus (MRSA) clinical isolates and in vivo on infected wounds in rats. Nanoparticles of 45 nm in diameter ensured higher amounts of reactive oxygen species upon irradiation. HY-NPs demonstrated superior inhibition of biofilm over planktonic cells. In vivo wound healing studies in rats revealed faster healing, better epithelialization, keratinization and development of collagen fibers when HY-NPs were applied. Determination of growth factors and inflammatory mediators in the wound area confirmed superior healing potential of nanoencapsulated hypericin suggesting that hypericin can join the era of antibiotic-free antimicrobial therapy.
Keywords: Hypericin; Photodynamic therapy; MRSA biofilms; Nanoparticles; Amphiphilic copolymers;

Hydrotalcite composites for an effective fluoride buccal administration: A new technological approach by Luana Perioli; Morena Nocchetti; Paola Giannelli; Cinzia Pagano; Maria Bastianini (259-268).
The aim of this work was to develop new mucoadhesive buccal patches containing an inorganic fluorinated compound, MgAl-F, intended for decay prevention.Firstly MgAl-F was synthesized and characterized, then the patches were prepared starting from a physical blend of mucoadhesive polymers (NaCMC and polycarbophil) in which MgAl-F was dispersed in different amounts in order to obtain the films. The prepared mucoadhesive patches were characterized in terms of swelling capacity, mucoadhesion force and time, surface morphology and in vitro release studies. Moreover, the organoleptic properties and acceptability have been evaluated by in vivo application.The performed studies demonstrated that the proposed formulations are practical, manageable, flexible and adaptable to the biological substrate showing, at the same time, good organoleptic properties. Moreover, the presence of the MgAl-F is able to decrease the strong adhesion of the employed polymers, reducing pain and irritations resulting in a high patient acceptability. Data obtained from release studies revealed that the application of small patch portions is enough able to release, for a prolonged time, an amount of fluoride ions able to reach the efficacious dose. These observations suggest the applicability of such formulations for buccal administration of different active ingredients.
Keywords: Buccal patches; Mucoadhesion; Hydrotalcite; Fluoride; Decay;

Study on formability of solid nanosuspensions during nanodispersion and solidification: I. Novel role of stabilizer/drug property by Peng-Fei Yue; Yu Li; Jing Wan; Ming Yang; Wei-Feng Zhu; Chang-Hong Wang (269-277).
Few or no attempts have been made so far to understand the feasibility of solid nanosuspension formulation during nanodispersion and solidification in terms of drug properties and stabilizer characterizations. In order to establish a knowledge base about the effect of physicochemical property of drug compounds and stabilizers on solid nanosuspension production during nanodispersion and solidification, a comparative study was firstly performed on 10 different stabilizers at 3 concentrations for 8 structurally different drug compounds. Synthetic polymers (HPMC, PVP K30, CMS-Na and MC) displayed a poor stabilizing performance (10% success rate on average) during nanodispersion, but polymers showed better potential when higher concentrations was applied during freezing and lyophilization. Meanwhile, an effect for the surfactants group was even more pronounced during nanodispersion. However, the solid nanosuspension stabilized by surfactants showed the worst formability potential when be applied in setted concentrations during freezing and lyophilization. From the point of view of drug property, it was found that the surface hydrophobicity and cohesive energy of drug, were responsible for the formability of the solid nanosuspension during nanodispersion and solidification. Wetting index (k) and ΔE were concluded to have a direct correlation on the feasibility of formation of a stable solid nanosuspension, which can give a formulation design strategy from where candidate drugs and stabilizers with a set of properties.
Keywords: Solid nanosuspension; Formability; Nanodispersion; Solidification; Contact angle; Cohesive energy; Redispersibility;

Preparation, characterization, and evaluation in vivo of Ins-SiO2-HP55 (insulin-loaded silica coating HP55) for oral delivery of insulin by Xiuhua Zhao; Chang Shan; Yuangang Zu; Ying Zhang; Weiguo Wang; Kunlun Wang; Xiaoyu Sui; Ruiqiang Li (278-284).
Insulin is the most effective and durable drug in the treatment of advanced stage diabetes. However, oral delivering insulin was a tough task for rapid enzymatic degradation. In this work, we designed and developed a delivery system composed of enteric nanosphere for oral delivery of insulin. The silica was selected for loading insulin, which surface has a lot of pores with a powerful adsorption capacity, advantages for permeability and slow-release. The insulin-loaded silica (Ins-SiO2) was prepared by adsorption in HCl solution. The Ins-SiO2 obtained was coated with the hydroxypropyl methylcellulose phthalate (HP55) by desolvation method, which is a good enteric coating material. The Ins-SiO2-HP55, an enteric nanosphere of insulin obtained were characterized by transmission electron microscope (TEM), surface area, Fourier-transform infrared (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The results showed that insulin was loaded most in the pores of silica, while the HP55 coated on the extent of Ins-SiO2. In vitro drug release results revealed that the release of insulin from Ins-SiO2-HP55 was markedly reduced in simulated gastric fluid (SGF). By contrast, the release amount of insulin from Ins-SiO2-HP55 was increased significantly in simulated intestinal fluid (SIF). In vivo evaluation on diabetic animals showed the blood glucose level of diabetic rats could be effectively reduced after oral administration Ins-SiO2-HP55. There is marked hypoglycemic effect after 1 h of taking the Ins-SiO2-HP55. After 3 h, the GLU of rats of the Ins-SiO2-HP55 stably kept from 4.85 to 2.67 mmol/L that was significantly less than the normal level (6.7 mmol/L). However, that of rats taking raw insulin kept from 8.03 to 6.56 mmol/L that is higher than the normal level. These results suggested that Ins-SiO2-HP55 could have potential value in oral administration systems of diabetes chemotherapy.
Keywords: Insulin; Silica; Oral delivery; In vivo evaluation; Enteric capsule; Controlled release;

Following the quest for new composite materials for bone tissue engineering, a novel elastic hydroxyapatite–glucan composite loaded with two aminoglycoside antibiotics was prepared. The porosity of the composite and the drug release profiles in closed-loop and semi-open systems were tested. The antibacterial activity of the drug was estimated against two Gram-positive and two Gram-negative bacterial strains causing orthopedic infections. It was found that the loaded antibiotic acted in a biphasic mode. The majority of the drug was released within 48–119 h in a pore-dependent manner and inhibited the bacterial growth in the culture medium. However, a small residual amount of the drug was bound to the composite microstructure via ionic interactions and acted as a short-lived barrier against bacterial adhesion to the composite, although the surrounding medium was no longer protected against bacterial infection. Sub-inhibitory concentrations of the released drug were observed in the medium only during the last two days of the experiment (minimized risk of occurrence of drug-resistant strains). Thus the novel drug-loaded elastic hydroxyapatite–glucan composite, demonstrating a biphasic mode of antibacterial action, may be recommended for antibiotic prophylaxis in bone substitute implantation, with less emphasis on the treatment of bone infections.
Keywords: Elastic bone-replacing material; Ceramics; Gentamicin; Amikacin; Multiphasic release; Bacterial strains;

Curcuma increasing antitumor effect of Rhizoma paridis saponins through absorptive enhancement of paridis saponins by Shuli Man; Yuanyuan Li; Wei Fan; Wenyuan Gao; Zhen Liu; Nan Li; Yao Zhang; ChangXiao Liu (296-301).
Rhizoma paridis saponins (RPS) played a good antitumor role in many clinical applications. However, low oral bioavailability limited its application. In this research, water extract of Curcuma (CW) significantly increased antitumor effect of Rhizoma paridis saponins (RPS). GC–MS was used to identify its polar composition. HPLC was applied for determination of the content of curcuminoids in CW. As a result, 47 analytes with 0.65% of curcuminoids were identified in CW. According to the in vivo anti-tumor data, the best proportion of curcuminoids in CW with RPS was 16:500 (w/w). Using this ratio, curcuminoids significantly increased absorption of RPS in the everted rat duodenum sac system. In addition, curcuminoids decreased the promotion of RPS on rhodamine 123 efflux. The effect of curcuminoids was similar to that of the P-gp inhibitor, cyclosporin A in combination with RPS. In conclusion, drug combination of water extract of Curcuma with RPS was a good method to increase the antitumor effect of RPS. This combination would be a potent anticancer agent used in the prospective application.
Keywords: Curcuma; Curcuminoids; Rhizoma paridis saponins; Absorption; Antitumor effect;

Selection of high efficient transdermal lipid vesicle for curcumin skin delivery by Ying-Zheng Zhao; Cui-Tao Lu; Yi Zhang; Jian Xiao; Ya-Ping Zhao; Ji-Lai Tian; Yan-Yan Xu; Zhi-Guo Feng; Chong-Yong Xu (302-309).
Curcumin shows effective anti-inflammatory activities but is seldom used in clinic because of its poor solubility in water and vulnerablity to sunshine ultraviolet effect. Novel lipid vesicles have been developed as carriers for skin delivery. In this paper, lipid vesicles—propylene glycol liposomes (PGL), Ethosomes and traditional liposomes, were prepared as curcumin carriers respectively. Their morphology, particle size and encapsulation efficiency and drug release behavior in vitro were evaluated. Transdermal efficiency and deposition quantity in abdominal skin were also measured with Franz diffusion device. Carrageenan-induced paw edema was established to evaluate the anti-inflammatory effect. From the result, the particle size order of lipid vesicles was: PGL (182.4 ± 89.2 nm) < Ethosomes (289 ± 132.1 nm) < traditional liposomes (632.9 ± 184.1 nm). The order of particle dispersion coefficient was as the same as that of particle size. The sequence of encapsulation efficiency was: PGL > Ethosomes > traditional liposomes. PGL had the best encapsulation efficiency of 92.74 ± 3.44%. From anti-inflammatory experiment, PGL showed the highest and longest inhibition on the development of paw edema, followed by Ethosomes and Traditional liposomes. With the elevated entrapment efficiency, good transdermic ability and sustained-release behavior, PGL may represent an efficient transdermal lipid vesicle for skin delivery.
Keywords: Transdermal vector; Curcumin; Encapsulation efficiency; Anti-inflammatory effect;

The current study involved investigation of the effect of storage temperature and moisture on the performance of poly(lactide-co-glycolide) (PLGA) microsphere/poly(vinyl-alcohol) (PVA) hydrogel composites. Physical aging occurred in composites stored at 25 °C due to structural relaxation. The glass transition temperature (Tg) and enthalpy of relaxation of the composites increased leading to a slower cumulative % release. The Tg of composites incubated at 40 °C, 75% RH decreased significantly due to the plasticization effect of absorbed water, whereas no change was observed in the Tg of microspheres alone; indicating that the hydrogel component enhanced water absorption. PLGA degradation occurred leading to significantly faster dexamethasone release following incubation at 40 °C, 75% RH for 1 month. No significant change was observed in the in vitro release profiles of composites after 6 months storage at 25 °C, 60% RH, however, release was accelerated following 12 months storage. Accordingly, exposure of the composites to ambient temperature/moisture during storage, shipping or handling may cause physical aging, plasticization, and degradation and hence, their performance may be affected. The extent to which the performance of the composite is affected by storage temperature and moisture is a net effect of physical aging and moisture induced plasticization/hydrolytic degradation.
Keywords: Storage temperature; Moisture; Physical aging; Plasticization; Hydrolytic degradation;

Lipid nanoparticles for brain targeting III. Long-term stability and in vivo toxicity by Paolo Blasi; Aurélie Schoubben; Giovanna Traina; Giuseppe Manfroni; Lanfranco Barberini; Paolo Francesco Alberti; Carlo Cirotto; Maurizio Ricci (316-323).
The aim of the work was to assess the long-term stability and the safety of lipid nanoparticles intended for brain drug delivery.Lipid nanoparticles, prepared by high pressure homogenization, were stored at room temperature and 4 °C and monitored for their mean hydrodynamic diameter and Gaussian distribution width over time. Cetylpalmitate and polysorbate® 80 chemical integrity were investigated by nuclear magnetic resonance on diagnostic signals. Nanoparticle toxicity was assessed in chicken embryos by chorioallantoic membrane assay and in rodents by brain histological evaluation.Data showed nanoparticle stability at 4 °C over a period of time of 4 years with only a limited particle size increase while at room temperature destabilization was observed after 9 months. Nuclear magnetic resonance investigation confirmed the absence (<5%) of chemical degradation of the lipid matrix and the surfactant after 4 years of storage at 4 °C. Chorioallantoic membrane assay and rat brain histology showed the absence of acute toxicity corroborating previously published data.Cetylpalmitate nanoparticle long-term physical and chemical stability, together with the in vivo safety, corroborate the existing evidences of the high value of colloidal lipids as parenteral formulations and carriers for brain drug delivery.
Keywords: Nanoparticles (NPs); Solid lipid nanoparticles (SLN); Long-term stability; Chorioallantoic membrane (CAM) assay; Acute toxicity;

Consolidation trend design based on Young's modulus of clarithromycin single crystals by B. Janković; M. Škarabot; Z. Lavrič; I. Ilić; I. Muševič; S. Srčič; O. Planinšek (324-332).
The key aim of this study was to determine single mechanical properties of clarithromycin polymorphic forms in order to select some of them as more suitable for the tableting process. For this purpose, AFM single-point nanoindentation was used. The Young's moduli of clarithromycin polymorphs were substantially different, which was consistent with the structural variations in their packing motifs. The presence of the adjacent layers, which can easily slide over each other due to the low energy barrier (the lowest Young's modulus was 0.25 GPa) resulted in better bulk compressibility (the highest Heckel coefficient) of clarithromycin Form I. We also addressed the importance of tip geometry screening because the stress during the force mode often results in tip apex fracture. Even the initial manufacture of the diamond-coated tips can result in defects such as double-apex tips.
Keywords: Polymorphism; Clarithromycin; Atomic force microscopy; Nanoindentation; Young's modulus;

Ultrasonic approach for viscoelastic and microstructure characterization of granular pharmaceutical tablets by Armin Saeedi Vahdat; Chaitanya Krishna Prasad Vallabh; Bruno C. Hancock; Cetin Cetinkaya (333-343).
The mechanical properties of a solid dosage, defined by its granular micro-structure and geometry, play a key role in its dissolution profile and performance. An ultrasonic method for extracting the viscoelastic material properties and granular structure of drug tablet compacts is introduced and its utility is demonstrated for tablet compacts made of microcrystalline cellulose (MCC), lactose monohydrate, and sodium starch glycolate as well as magnesium stearate as lubricant. The approach is based on the effect of viscoelasticity and internal micro-structures on the frequency-dependent attenuation of an ultrasonic wave propagating in a granular medium. The models for viscoelastic (a two-parameter Zener model) and scattering attenuation (Rayleigh model) mechanisms are employed. The material parameters including viscoelastic and scattering parameters (average Young's modulus, stress and strain relaxation time constants, and the Rayleigh scattering material parameter) and grain size distribution with a known distribution profile are extracted by an optimization algorithm based on the least square method. The results also indicate good agreement between experimentally and computationally determined phase and group velocities in compacted samples. It is found that the effects of both attenuation mechanisms are present and the extracted grain size distribution parameters are in good agreement with the optically determined values.
Keywords: Granular pharmaceutical solid dosage; Mechanical properties; Grain size distribution; Viscoelasticity; Rayleigh scattering; Non-destructive characterization techniques;

Effect of water on exenatide acylation in poly(lactide-co-glycolide) microspheres by Rongcai Liang; Xiang Li; Yanan Shi; Aiping Wang; Kaoxiang Sun; Wanhui Liu; Youxin Li (344-353).
Peptide or protein degradation often occurs when water flows into the dosage form. The aim of this study was to investigate the effect of water on exenatide acylation in poly(lactide-co-glycolide) (PLGA) microspheres. Exenatide-loaded PLGA microspheres were incubated at different relative humidities (RH) as well as in solutions of different pH for 20 days. The stability of exenatide was monitored using HPLC and HPLC–MS analysis. The alteration of exenatide conformation caused by water was investigated by FT-IR spectroscopy. Exenatide and glycolide were incubated in DMSO–water solutions to verify the effect of exenatide conformation state on the peptide acylation. Exenatide was relatively stable in microspheres at lower RH, and the absorbed water could act as a plasticizer and thus promote the peptide acylation by PLGA. However, when the microspheres were incubated at 100% RH, the excessively absorbed water could cause conformation recovery of exenatide and play an inhibitory effect on acylation. The formation of acylated exenatide incubated in acetate buffer saline of pH 6.0 was more than that of pH 4.5 and 3.0. Stability studies of exenatide in glycolide solutions showed that exenatide in nonnative monomer state was easier to be acylated by eletrophiles than that in aggregation state.
Keywords: Exenatide; Acylation; Poly(lactide-co-glycolide) (PLGA); Microsphere; Solution model;

This study was to design an innovative nanostructured lipid carrier (NLC) for drug delivery of genistein applied after cataract surgery for the prevention of posterior capsular opacification. NLC loaded with genistein (GEN-NLC) was produced with Compritol 888 ATO, Gelucire 44/14 and Miglyol 812N, stabilized by Solutol® HS15 by melt emulsification method. A 24 central composite design of 4 independent variables was performed for optimization. Effects of drug concentration, Gelucire 44/14 concentration in total solid lipid, liquid lipid concentration, and surfactant concentration on the mean particle size, polydispersity index, zeta potential and encapsulation efficiency were investigated. Analysis of variance (ANOVA) statistical test was used to assess the optimization. The optimized GEN-NLC showed a homogeneous particle size of 90.16 nm (with PI = 0.33) of negatively charged surface (−25.08 mv) and high encapsulation efficiency (91.14%). Particle morphology assessed by TEM revealed a spherical shape. DSC analyses confirmed that GEN was mostly entrapped in amorphous state. In vitro release experiments indicated a prolonged and controlled genistein release for 72 h. In vitro growth inhibition assay showed an effective growth inhibition of GEN-NLCs on human lens epithelial cells (HLECs). Preliminary cellular uptake test proved a enhanced penetration of genistein into HLECs when delivered in NLC.
Keywords: Nanostructured lipid carrier; Genistein; Posterior capsular opacification; Central composite design; Inhibition; Cellular uptake;

Nano-transfersomes as a novel carrier for transdermal delivery by Hema Chaudhary; Kanchan Kohli; ViKash Kumar (367-380).
Photomicrographs of rat skin (A) vehicle, (B) optimized formulation (100×) and fluorescence photomicrograph of rat skin after application of fluorescence probe Rhodamine B. (C) vehicle, (D) optimized formulation (100×). *FL: flourescence labeled.The aim of this study was to design and optimize a nano-transfersomes of Diclofenac diethylamine (DDEA) and Curcumin (CRM). A 33 factorial design (Box–Behnken) was used to derive a polynomial equation (second order) to construct 2-D (contour) and 3-D (Response Surface) plots for prediction of responses. The ratio of lipid to surfactant (X 1), weight of lipid to surfactant (X 2) and sonication time (X 3) (independent variables) and dependent variables [entrapment efficiency of DDEA (Y 1), entrapment efficiency of CRM (Y 2), effect on particle size (Y 3), flux of DDEA (Y 4), and flux of CRM (Y 5)] were studied. The 2-D and 3-D plots were drawn and a statistical validity of the polynomials was established to find the compositions of optimized formulation. The design established the role of the derived polynomial equation, 2-D and 3-D plots in predicting the values of dependent variables for the preparation and optimization of nano-transfersomes for transdermal drug release.
Keywords: Drug design; Factorial design; Formulation; Nano-transfersomes; Drug transport; Transdermal delivery;

The effect of capsule-filling machine vibrations on average fill weight by Marcos Llusa; Eva Faulhammer; Stefano Biserni; Vittorio Calzolari; Simon Lawrence; Massimo Bresciani; Johannes Khinast (381-387).
The aim of this paper is to study the effect of the speed of capsule filling and the inherent machine vibrations on fill weight for a dosator-nozzle machine. The results show that increasing speed of capsule filling amplifies the vibration intensity (as measured by Laser Doppler vibrometer) of the machine frame, which leads to powder densification. The mass of the powder (fill weight) collected via the nozzle is significantly larger at a higher capsule filling speed. Therefore, there is a correlation between powder densification under more intense vibrations and larger fill weights. Quality-by Design of powder based products should evaluate the effect of environmental vibrations on material attributes, which in turn may affect product quality.
Keywords: Vibration; Powder density; Fill weight;

Zein-based oral drug delivery system targeting activated macrophages by Sungmun Lee; Noaf Salah Ali Alwahab; Zainab Muhammad Moazzam (388-393).
Reactive oxygen species (ROS) play an important role in the pathogenesis of rheumatoid arthritis (RA). ROS such as hydrogen peroxide and superoxide are overproduced by activated macrophages in RA. As scavengers of ROS, enzymatic proteins such as catalase and superoxide dismutase (SOD) have a great therapeutic potential; however, in vivo application is limited especially when they are orally administered. Although, the oral route is the most convenient for drug administration, therapeutic proteins are easily degraded in vivo by the harsh conditions of gastrointestinal (GI) tract. Here, we introduce a novel drug delivery system composed of zein, a plant storage protein derived from maize. We demonstrate that zein nanoparticles can protect therapeutic proteins, catalase and SOD, from the harsh conditions of GI tract. Folate-conjugated catalase or SOD in zein nanoparticles can target the activated macrophages and scavenge the ROS generated by macrophages in vitro. This novel drug delivery system will be applicable to other orally administered treatments based on the protective property in the harsh conditions of GI tract.
Keywords: Zein; Oral drug delivery; Reactive oxygen species; Inflammatory disease;

Development of noncytotoxic PLGA nanoparticles to improve the effect of a new inhibitor of p53–MDM2 interaction by Ana M. Paiva; Rita A. Pinto; Maribel Teixeira; Carlos M. Barbosa; Raquel T. Lima; M. Helena Vasconcelos; Emília Sousa; Madalena Pinto (394-402).
One possible approach to overcome solubility complications and enhance the biological activity of drugs is their incorporation into drug delivery systems. Within this scope, several nanosphere and nanocapsule formulations of a new inhibitor of p53–MDM2 interaction (xanthone 1) were developed and their physicochemical properties analyzed. Through the investigation of the effect of several empty nanoparticles on the growth of MCF-7 cells, it was possible to observe that four out of five formulations were cytotoxic and that some correlations between the toxic potential of these polymeric nanoparticles and their properties/composition could be extrapolated. One empty formulation of nanocapsules developed by emulsification/solvent evaporation and containing PLGA, PVA and Mygliol® 812 was found to be noncytotoxic to this cell line. The corresponding compound 1-loaded nanocapsules showed an incorporation efficiency of 77% and revealed to be more potent than the free drug against cell growth inhibition, which may be related to the enhancement in its intracellular delivery. In an integrative study, the intracellular uptake of nanocapsules was confirmed using fluorescent 6-coumarin and well as compound 1 release from nanocapsules. Overall, it was possible to enhance the effect of the hit inhibitor of p53–MDM2 interaction through the development of suitable noncytotoxic polymeric nanoparticles.
Keywords: Antitumor; Poly(d,l-lactide-co-glycolide); Polymeric nanoparticles; Xanthones;

The main objective of this study was to develop carboxylated ordered mesoporous carbon microparticles (c-MCMs) loaded with a poorly water-soluble drug, intended to be orally administered, able to enhance the drug loading capacity and improve the oral bioavailability. A model drug, carvedilol (CAR), was loaded onto c-MCMs via a procedure involving a combination of adsorption equilibrium and solvent evaporation. The physicochemical properties of the drug-loaded composites were systematically studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and HPLC. It was found that c-MCM has a high drug loading level up to 41.6%, and higher than that of the mesoporous silica template. Incorporation of CAR in both drug carriers enhanced the solubility and dissolution rate of the drug, compared to the pure crystalline drug. After loading CAR into c-MCMs, its oral bioavailability was compared with the marketed product in dogs. The results showed that the bioavailability of CAR was improved 179.3% compared with that of the commercial product when c-MCM was used as the drug carrier. We believe that the present study will help in the design of oral drug delivery systems for enhanced oral bioavailability of poorly water-soluble drugs.
Keywords: Drug delivery; Carboxylated ordered mesoporous carbon; Poorly water-soluble drugs; Carvedilol; Crystalline state; Bioavailability;

In the literature various solutions exist for the calculation of the diametral compression tensile strength of doubly-convex tablets and each approach is based on experimental data obtained from single materials (gypsum, microcrystalline cellulose) only. The solutions are represented by complex equations and further differ for elastic and elasto-plastic behaviour of the compacts. The aim of this work was to develop a general equation that is applicable independently of deformation behaviour and which is based on simple tablet dimensions such as diameter and total tablet thickness only. With the help of 3D-FEM analysis the tensile failure stress of doubly-convex tables with central cylinder to total tablet thickness ratios W/D between 0.06 and 0.50 and face-curvature ratios D/R between 0.25 and 1.85 were evaluated. Both elastic and elasto-plastic deformation behaviour were considered. The results of 80 individual simulations were combined and showed that the tensile failure stress σ t of doubly-convex tablets can be calculated from σ t  = (2P/πDW)(W/T) = 2P/πDT with P being the failure load, D the diameter, W the central cylinder thickness, and T the total thickness of the tablet. This equation converts into the standard Brazilian equation (σ t  = 2P/πDW) when W equals T, i.e. is equally valid for flat cylindrical tablets. In practice, the use of this new equation removes the need for complex measurements of tablet dimensions, because it only requires values for diameter and total tablet thickness. It also allows setting of standards for the mechanical strength of doubly-convex tablets. The new equation holds both for elastic and elasto-plastic deformation behaviour of the tablets under load. It is valid for all combinations of W/D-ratios between 0.06 and 0.50 with D/R-ratios between 0.00 and 1.85 except for W/D  = 0.50 in combination with D/R-ratios of 1.85 and 1.43 and for W/D-ratios of 0.40 and 0.30 in combination with D/R  = 1.85. FEM-analysis indicated a tendency to failure by capping or even more complex failure patterns in these exceptional cases. The FEM-results further indicated that in general W/D-ratios between 0.15 and 0.20 are favourable when the overall size and shape of the tablets is modified to give maximum tablet tensile strength. However, the maximum tensile stress of doubly-convex tablets will never exceed that of a flat-face cylindrical tablet of similar W/D-ratio. The lowest tensile stress depends on the W/D-ratio. For the thinnest central cylinder thickness, this minimum stress occurs at D/R  = 0.50; for W/D-ratios between 0.10 and 0.20 the D/R-ratio for the minimum tensile stress increases to 0.67, and for all other central cylinder thicknesses the minimum tensile stress is found at D/R  = 1.00.
Keywords: Doubly-convex cylindrical tablets; Diametral compression test; Finite element method; Tablet tensile strength;

A series of novel amphiphilic chitosan derivatives, cholic acid modified N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (HTCC-CA) with different quaternization degrees and cholic acid substitutions were synthesized in this study. HTCC-CA is biocompatible and forms particles in aqueous solution. The binding with superoxide dismutase (SOD) at pH 6.8 destroys the original aggregates of HTCC-CA and produces smaller SOD/HTCC-CA complex nanoparticles via electrostatic and hydrophobic interactions. The SOD loading efficiency and loading capacity of HTCC-CA can reach to more than 90% and 45%, respectively. Confocal laser scanning microscopy observation and flow cytometry analysis reveal that SOD/HTCC-CA complex nanoparticles greatly enhance the cellular internalization of the loaded SOD. The SOD activities and malonaldehyde concentrations in the serum and organs of the rats, administrated intravenously with free SOD, free HTCC-CA, and SOD/HTCC-CA nanoparticles, were assayed to evaluate the antioxidant efficiency in vivo. The results demonstrate that free HTCC-CA is effective to scavenge superoxide radicals in the blood circulation and SOD/HTCC-CA nanoparticles have better antioxidant efficiency than free SOD as well as free HTCC-CA.
Keywords: Chitosan; Drug delivery; Nanoparticles; Protein;

Biodegradable multiblock poly(N-2-hydroxypropyl)methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment by Nate Larson; Jiyuan Yang; Abhijit Ray; Darwin L. Cheney; Hamidreza Ghandehari; Jindřich Kopeček (435-443).
The synthesis, characterization, and in vitro evaluation of a combination delivery of multiblock poly(N-2-hydroxypropyl)methacrylamide (HPMA), gemcitabine (GEM) and paclitaxel (PTX) conjugates is described in this study. Multiblock copolymer conjugates of a large molecular weight (M w  > 200 kDa) were studied and compared to traditional, small molecular weight (M w  < 45 kDa) conjugates. Stability of the conjugates in different pH was assessed, and their cytotoxicity in combination toward A2780 human ovarian cancer cells was evaluated by combination index analysis. Treatment duration (4 and 72 h) and sequence of addition were explored. In addition, an HPMA copolymer conjugate with both GEM and PTX in the side chains was evaluated in a similar manner and compared to a physical mixture of individual conjugates. Conjugates with narrow molecular weight distribution (M w/M n  < 1.1) were obtained via RAFT polymerization, and drug loadings of between 5.5 and 9.2 wt% were achieved. Conjugates demonstrated moderate stability with less than 65% release over 24 h at pH 7.4, and near complete drug release in the presence of the lysosomal enzyme cathepsin B in 3 h. In combination, the cytotoxic effects of a mixture of the conjugates were primarily additive. Synergistic effects were observed when A2780 human ovarian cancer cells were treated simultaneously for 4 h with multiblock conjugates (CI < 0.7). When both GEM and PTX were conjugated to the same copolymer backbone, moderate antagonism (CI 1.3–1.6) was observed. These results demonstrate that multiblock HPMA copolymer–GEM and –PTX conjugates, when delivered as a mixture of individual agents, are promising for the treatment of ovarian cancer.
Keywords: HPMA copolymers; Biodegradable polymers; Combination therapy; Ovarian cancer; Gemcitabine; Paclitaxel;

Molecular expression and functional activity of efflux and influx transporters in hypoxia induced retinal pigment epithelial cells by Ramya Krishna Vadlapatla; Aswani Dutt Vadlapudi; V.K. Chaithanya Ponnaluri; Dhananjay Pal; Mridul Mukherji; Ashim K. Mitra (444-452).
A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs.
Keywords: Efflux and influx transporters; Hypoxia; Molecular expression and functional activity; Retinal pigment epithelial cells;

Facile synthesis of camptothecin intercalated layered double hydroxide nanohybrids via a coassembly route by Xiaowen Wu; Haiping Li; Shue Song; Renjie Zhang; Wanguo Hou (453-461).
A method has been developed for the synthesis of intercalated layered double hydroxide (LDH) nanohybrids of the charge-neutral and poorly water-soluble anticancer drug camptothecin (CPT) using a coassembly route. For this route, CPT molecules were initially incorporated into the micelles of a biocompatible surfactant, such as sodium cholate (SCh) or sodium deoxycholate (SDC). The resulting negatively charged CPT-loaded micelles and the positively charged LDH nanosheets were then coassembled together into the CPT intercalated LDH nanohybrids. The resulting nanohybrids were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) and elemental analyses. The results revealed that the loading of CPT in the nanohybrids could reach as high as 13%, indicating that this route could be used to achieve the effective intercalation of charge-neutral and poorly water-soluble drugs into the LDH gallery. The in vitro release of CPT from the nanohybrids was examined, and the results showed that the release was a diffusion-controlled process and that the diffusion process through the LDH particles was the rate-limiting step. The parabolic diffusion equation effectively described the kinetic process associated with the release of CPT from the nanohybrids.
Keywords: Layered double hydroxide; Camptothecin; Nanohybrid; Coassembly; Release;

Scanty solubility and permeability of diosmin (DSN) are perpetrators for its poor oral absorption and high inter-subject variation. This article investigated the potential of novel DSN nanosuspensions to improve drug delivery characteristics. Bottom-up nanoprecipitation technique has been employed for nanosuspension development. Variables optimized encompassed polymeric stabilizer type, DSN: stabilizer ratio, excess stabilizer removal, spray drying, and mannitol incorporation. In vitro characterization included particle size (PS), infrared spectroscopy (IR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM), and dissolution profile. Ex vivo permeation was assessed in rats using non-everted sac technique and HPLC. Optimal DSN nanosuspension (DSN:hydroxypropylmethyl cellulose HPMC 2:1) was prepared with acid base neutralization technique. The formula exhibited the lowest PS (336 nm) with 99.9% drug loading and enhanced reconstitution properties after mannitol incorporation. SEM and TEM revealed discrete, oval drug nanocrystals with higher surface coverage with HPMC compared to MC. DSN nanosuspension demonstrated a significant enhancement in DSN dissolution (100% dissolved) compared to crude drug (51%). Permeation studies revealed 89% DSN permeated from the nanosuspension after 120 min compared to non-detected amounts from drug suspension. Conclusively, novel DSN nanosuspension could successful improve its dissolution and permeation characteristics with promising consequences of better drug delivery.
Keywords: Nanosuspension; Diosmin; Permeation; Dissolution; Nanocrystals;

Development of intravenous lipid emulsion of vinorelbine based on drug–phospholipid complex technique by Yanzhen Li; Wenxian Jin; Hui Yan; Huan Liu; Chenggang Wang (472-477).
In order to reduce the severe venous toxicity, we developed an intravenous lipid emulsion of vinorelbine and investigated its preclinical stability, toxicity and antitumor efficacy. Vinorelbine–phospholipid complex was prepared to enhance the lipophilicity of vinorelbine thus facilitating the encapsulation into lipid emulsion. After complexation more than 70% of vinorelbine was encapsulated into the oil phase. Meanwhile, the lipid emulsion showed good stability without drug leakage. Local irritation after injection of the lipid emulsion was investigated in rabbits and compared with Navelbine® (the commercial product). The antitumor therapeutic efficacies were evaluated in tumor-bearing mouse models inoculated with A549 human lung cancer cells and BCAP-37 human breast cancer cells and compared as well. Results showed that the lipid emulsion significantly reduced the injection irritation compared with that of Navelbine®, while maintained the antitumor activity in A549 and BCAP-37 cells xenograft tumor mouse models. Taken together, lipid emulsion loaded with vinorelbine–phospholipid complex is a promising vinorelbine intravenous injection with reduced venous irritation.
Keywords: Vinorelbine; Drug–phospholipid complex; Injection irritation; Antitumor activity;

A versatile drug delivery system using streptavidin-tagged pegylated liposomes and biotinylated biomaterials by Ming-Han Chen; Yasushi Soda; Kiyoko Izawa; Seiichiro Kobayashi; Kenzaburo Tani; Kazuo Maruyama; Arinobu Tojo; Shigetaka Asano (478-485).
Here we have developed a versatile liposome-mediated drug delivery system (DDS) allowing a strong bridge between the streptavidin-tagged liposome (SAL) and biotin (Bi)-tagged biomaterials which has strong affinity to surface proteins expressed in restricted cell lineages. This DDS was effective and specific for many leukemia cells in vitro and in vivo. When examining 6 human leukemia cell lines using calcein-encapsulated SALs in combination with Bi-granulocyte colony-stimulating factor (G-CSF), Bi-anti-CD33 monoclonal antibody (MAb) or Bi-anti-CD7 MAb, the fluorescent positive rate of each cell line was in almost proportion to degree of G-CSF receptor, CD33 or CD7 expression, respectively. More importantly, the binding ability was shown to be well maintained in a mouse xenograft model. Furthermore the cytosine arabinoside (AraC)-encapsulated SALs could kill the corresponding cells much more effectively in combination with Bi-biomaterials than free AraC, as expected. These findings strongly indicate that our SAL/Bi-biomaterial system could allow various types of medical agents to be delivered reliably and stably to the cells targeted.
Keywords: Drug delivery system; Immunoliposome; Tumor-targeting; Internalization; Streptavidin; Biotin;

The main purposes of this study are to demonstrate the synergistic anticancer drug systems with the combined doxorubicin (D) and paclitaxel (P) via the aid of cell penetrating and cell targeting moieties for enhancing the cancer therapeutic effect. Firstly, the synergistic effect of combined free drugs (D/P) was investigated to obtain the suitable dose combination for subsequent studies. The combination of free drugs D/P at molar ratio of 1/0.2 shows synergistic therapeutic effect compared with the treatment of a free single drug D or P. Secondly, sustainable release systems of two single drug-loaded micelles, (i) co-delivered D-FOL micelle & P-FOL micelle system and (ii) co-delivered D-TAT/FOL micelle & P-TAT/FOL micelle system, at D/P molar ratio of 1/0.2 were investigated. The results show synergistic effect with the higher efficacy of the TAT/FOL system compared to FOL only system. Finally, a dual D/P-loaded system with sustainable release rate, synergistic drug interaction, selective targeting to cancer cells and high cell penetrating ability was designed. The D/P-TAT/FOL micelles exhibit an IC50 value of 0.172 μM D/0.043 μM P, which is much lower than the IC50 values of the single drug-loaded micelles without functionalization (3.873 μM for D-micelles and 0.790 μM for P-micelles). Overall, this newly developed dual encapsulation of D and P in the multifunctional carrier would be a promising technology for cancer treatment.
Keywords: Dual drug synergy; Doxorubicin; Paclitaxel; TAT; FOL;

Towards more effective advanced drug delivery systems 1 by Daan J.A. Crommelin; Alexander T. Florence (496-511).
This position paper discusses progress made and to be made with so-called advanced drug delivery systems, particularly but not exclusively those in the nanometre domain. The paper has resulted from discussions with a number of international experts in the field who shared their views on aspects of the subject, from the nomenclature used for such systems, the sometimes overwrought claims made in the era of nanotechnology, the complex nature of targeting delivery systems to specific destinations in vivo, the need for setting standards for the choice and characterisation of cell lines used in in vitro studies, to attention to the manufacturability, stability and analytical profiling of systems and more relevant studies on toxicology. The historical background to the development of many systems is emphasised. So too is the stochastic nature of many of the steps to successful access to and action in targets. A lacuna in the field is the lack of availability of data on a variety of carrier systems using the same models in vitro and in vivo using standard controls. The paper asserts that greater emphasis must also be paid to the effective levels of active attained in target organs, for without such crucial data it will be difficult for many experimental systems to enter the clinic. This means the use of diagnostic/imaging technologies to monitor targeted drug delivery and stratify patient groups, identifying patients with optimum chances for successful therapy. Last, but not least, the critical importance of the development of science bases for regulatory policies, scientific platforms overseeing the field and new paradigms of financing are discussed.
Keywords: Drug delivery systems; Nanotechnology; Optimisation; Progress;

This commentary enlarges on some of the topics addressed in the Position Paper “Towards more effective advanced drug delivery systems” by Crommelin and Florence (2013). Inter alia, the role of mathematical modeling and computer-assisted device design is briefly addressed in the Position Paper. This emerging and particularly promising field is considered in more depth in this commentary. In fact, in-silico simulations have become of fundamental importance in numerous scientific and related domains, allowing for a better understanding of various phenomena and for facilitated device design. The development of novel prototypes of space shuttles, nuclear power plants and automobiles are just a few examples. In-silico simulations are nowadays also well established in the field of pharmacokinetics/pharmacodynamics (PK/PD) and have become an integral part of the discovery and development process of novel drug products. Since Takeru Higuchi published his seminal equation in 1961 the use of mathematical models for the analysis and optimization of drug delivery systems in vitro has also become more and more popular. However, applying in-silico simulations for facilitated optimization of advanced drug delivery systems is not yet common practice. One of the reasons is the gap between in vitro and in vivo (PK/PD) simulations. In the future it can be expected that this gap will be closed and that computer assisted device design will play a central role in the research on, and development of advanced drug delivery systems.

Advanced drug delivery in motion by Enrico Mastrobattista (517-520).
After 50 years of research on advanced drug delivery systems the time has come to critically reflect upon the past achievements. Despite some successes, many hurdles still need to be overcome before we can quantitatively deliver therapeutically relevant amounts of drug molecules to any desired location within the human body. In this commentary, I give my opinion on how to improve the current generation of nanocarriers for drug delivery. In addition, I speculate on which direction the drug delivery field should be going in order to fulfill the “magic bullet” dream in the long run.

Inflated claims to both design and mechanistic novelty in drug delivery and imaging systems, including most nanotechnologies, are not supported by the generally poor translation of these systems to clinical efficacy. The “form begets function” design paradigm is seductive but perhaps over-simplistic in translation to pharmaceutical efficacy. Most innovations show few clinically important distinctions in their therapeutic benefits in relevant preclinical disease and delivery models, despite frequent claims to the contrary. Long-standing challenges in drug delivery issues must enlist more realistic, back-to-basics approaches to address fundamental materials properties in complex biological systems, preclinical test beds, and analytical methods to more reliably determine fundamental pharmaceutical figures of merit, including drug carrier purity and batch-batch variability, agent biodistribution, therapeutic index (safety), and efficacy.
Keywords: Drug delivery; Stimuli sensitive; Animal models; Analytical methods; Translational medicine; Smart materials;

There is “hype” surrounding passive and active drug targeting of diseased tissues in vivo. The most common example of passive targeting is the utilisation of the “enhanced permeation and retention” phenomenon to target solid tumours and inflamed tissues. Alternatively, targeting moieties or “ligands” could be conjugated to the delivery system offering “actively” targeted delivery systems. The targets are usually receptors that are up-regulated in the biological areas of interest. Targeted drug delivery has been proposed to treat many diseases, such as cardiovascular diseases and diabetes. However, the most important application of targeted drug delivery is to treat cancerous tumours. Standardisation of in vitro and in vivo assays currently in place to assess the targeting efficiency of such systems is of utmost importance since heterogeneity in targeting assays and target validation could easily bias conclusions made affecting the future perspective of the field of active drug targeting.

Recent advances in nanotechnology and material science have re-ignited interest in drug delivery research. Arguably, however, hardly any of the systems developed and strategies proposed are really relevant for shaping the future (clinical) face of the nanomedicine field. Consequently, as outlined in this commentary, instead of making ever more carrier materials, and making nanomedicine both science-fiction and fiction-science, we should try to come up with rational and realistic concepts to make nanomedicines work, in particular in patients.
Keywords: Drug delivery; Drug targeting; Nanomedicine; Cancer; EPR;

Will nanomedicine deliver on its promise of changing therapeutics or remain an interesting and important research tool in cell biology and physiology? by Richard Kirsh; Steve Hood; Chris Brook; Aidan Gilmartin; Philip Dell’orco; Tom Meek (530-531).

Preparation, characterization and pharmacological evaluation of tolterodine hydrogels for the treatment of overactive bladder by Fengying Sun; Cheng Sui; Yulin Zhou; Ximing Liu; Yanan Shi; Yi Wu; Youxin Li (532-538).
Effects of tolterodine hydrogels and tolterodine tablets on the cumulative urinary output in rats.In this study, transdermal gel formulations for tolterodine were developed to investigate the effects of gel matrix and chemical enhancers on drug skin permeation from tolterodine hydrogels. In vitro permeation studies of tolterodine through excised mouse skin were carried out using Franz-type diffusion cells. In the optimum gel formulation, Carbopol 940 was selected as the gel matrix. Compared to gels without enhancer, tolterodine hydrogels with N-methyl pyrrolidone (NMP) showed significant enhancing effect on transdermal permeation of tolterodine (p  < 0.05). The results of in vitro percutaneous delivery experiment showed that the relationship of the steady accumulative percutaneous amount (Q, μg cm−2) of tolterodine hydrogels and time was Q 4–12h  = 770.19t 1/2  − 966.99. Tolterodine permeated at the steady-state speed of 770.19 μg cm−2  h−1 and its release coincided with Higuchi Equation. The pharmacokinetic properties of the optimized tolterodine formulation were studied in rabbits. The absolute bioavailability of tolterodine was 11.47%. Since the absence of hepatic first-pass metabolism, only a single active compound-tolterodine was detected in the plasma. A skin irritation study was also carried out on rabbits, and the results showed tolterodine hydrogels had no skin irritation. In the pharmacodynamic study, the significant effects of tolterodine hydrogels on the inhibition of pilocarpine-induced rat urinary bladder contraction were last to 12 h, indicating that tolterodine hydrogels could produce prolonged pharmacological responses. In conclusion, tolterodine hydrogels were formulated successfully using Carbopol 940 and NMP and these results helped in finding the optimum formulation for percutaneous drug release. It is quite evident that tolterodine hydrogels may offer a possibility to avoid the first-pass effect, resulting in a single active compound of tolterodine in plasma, which may profit on the patient under the dose control and the reduction of potential adverse effect from two active compounds in the body.
Keywords: Tolterodine; Hydrogels; Transdermal; Pharmacokinetics; Pharmacodynamics;

Strategies for the design of orally bioavailable antileishmanial treatments by T.T.H. Pham; P.M. Loiseau; G. Barratt (539-552).
Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. The most serious, life-threatening form is visceral leishmaniasis (VL). No vaccine is yet available for human use and chemotherapy is the main mean of dealing with this disease. This review focuses on the development of drug delivery systems (DDS) for treatment of leishmaniasis. After an overview of the significance of leishmaniasis in 2013, current chemotherapy and its limitations are considered, leading to possible strategies to improve the treatment of VL: new drugs, combinations of existing drugs and DDS, particularly for oral administration. Nanostructured biomaterials such as lipid-based or polymeric nanoparticles have unique physicochemical properties, ultra-small and controllable size, large surface area to mass ratio and the possibility of surface modification which can be used to advantage for the oral administration of antileishmanial drugs. They can improve the rate of dissolution of poorly water-soluble drugs, increase intestinal residence time by bioadhesion and, especially when lipid additives are used, influence the route and efficiency of absorption. These recent advances in this very active field should lead to better management of this serious disease.
Keywords: Visceral leishmaniasis; Drug combinations; Oral administration; Nanoparticles;

Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site by Xiaowei Qian; Hongxia Dong; Hong Tian; Yue Tong; Linfeng Guo; Xiaojing Hu; Xiangdong Gao; Wenbing Yao (553-558).
PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine–sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine–SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.
Keywords: Exendin-4; PEGylation site; Tryptic digestion; Amino acid analysis (AAA); Cysteine;

A permeation method for detection of self-aggregation of doxorubicin in aqueous environment by Zoltán Fülöp; Ruxandra Gref; Thorsteinn Loftsson (559-561).
For pharmaceutical scientists, it is important to know if dissolved drug molecules are present only as monomers or in the form of aggregates in a test solution or formulation. Amphiphilic or hydrophobic drugs frequently self-associate to form dimers, trimers or higher order aggregates. Doxorubicin aggregation was examined by a previously developed permeation technique to detect oligosaccharide aggregation in aqueous solutions. At very low doxorubicin concentrations dimers and trimers have been observed, but in aqueous 0.5 mg/ml doxorubicin solutions aggregates containing about 40 molecules were observed. The permeation studies were supported by TEM studies. The results indicate that neutral doxorubicin molecules aggregate more readily than the protonated ones. Doxorubicin aggregation is a stepwise process resulting in formation of aggregates of variable sizes are enhanced aggregation with increasing doxorubicin concentration.
Keywords: Self-association; Aggregation; Doxorubicin; Permeation;

I. Technological approaches to improve the dissolution behavior of nateglinide, a lipophilic insoluble drug: Nanoparticles and co-mixing by Lauretta Maggi; Giovanna Bruni; Mariarosa Maietta; Andrea Canobbio; Andrea Cardini; Ubaldo Conte (562-567).
Dissolution profiles of nateglinide H samples of different particle size (NH = about 18 μm, 2 μm and 0.5 μm), alone, compared to the same samples co-mixed with amorphous silica (AS) in 1:4 (w:w) ratio, dose: 20 mg. By reducing the drug particle size no significant difference in dissolution rate can be evidenced as the particles tend to aggregate in water. The drug dissolution rate can be improved only when an hydrophilic excipient, amorphous silica, is used. The small hydrophilic silica particles produce a very fine dispersion of the binary system with the silica surrounding the drug particles, thus preventing their aggregation.Nateglinide is a non-sulphonylurea insulinotropic oral antidiabetic agent. The main problem in formulating an oral dosage form is its low solubility in aqueous media. This problem is particularly critical for an anti-diabetic drug because it should be administered just before the meals and be quickly bioavailable to cover the post-prandial glycemic peak. In this work, some technological approaches have been studied to improve the dissolution rate of nateglinide. Furthermore, two different polymorphs of nateglinide (H and B) have been tested to evaluate the influence of the crystal habitus on the dissolution behavior of the drug. The results have clearly demonstrated that wettability plays a key role in the dissolution behavior of nateglinide. As a matter of fact the physical dispersion of the drug with colloidal silica or hydrophilic swellable polymers strongly enhances the dissolution rate of nateglinide. The two polymorphs tested did not show significant differences in terms of dissolution behavior.
Keywords: Nateglinide; Insoluble drug; Dissolution rate; Co-mixing, silica; Hydrophilic swellable polymers;

II. Technological approaches to improve the dissolution behavior of nateglinide, a lipophilic insoluble drug: Co-milling by Lauretta Maggi; Giovanna Bruni; Mariarosa Maietta; Andrea Canobbio; Andrea Cardini; Ubaldo Conte (568-572).
Enhancement of the nateglinide dissolution rate obtained from drug:super-disintegrant co-milled (CM) systems in 1:4 w:w ratio, compared to physical mixtures and nateglinide alone (NH). Dose: 20 mg. Cross-linked polyvinylpyrrolidone (PVPC) and sodium starch glycolate (SSG) ensure a rapid and complete dissolution of the insoluble drug in very short times, in water.Nateglinide is an oral antidiabetic agent that should be administered 10–30 min before the meal, but it shows low and pH-dependent solubility that may reduce its oral bioavailability.To improve nateglinide dissolution rate, the active was co-milled with three different super-disintegrants or with some hydrophilic excipients, in 1:1, 1:2, and 1:4 drug to carrier ratio (w:w). The three super-disintegrants were crosslinked polyvinylpyrrolidone (PVPC), sodium starch glycolate (SSG) and crosslinked carboxymethyl cellulose (CMCC). The three hydrophilic excipient were amorphous silica (AS), mannitol (M) and Poloxamer (PO).A strong enhancement of drug dissolution rate was obtained from the nateglinide:super-disintegrant co-milled systems in 1:4 ratio, which can be explained by a combination of several factors: an increase in wettability, due to the hydrophilic nature of the carriers, a possible reduction of particle size and a more intimate dispersion of the drug onto the carrier, as a result of the mechanical treatment.
Keywords: Nateglinide; Insoluble drug; Dissolution rate; Hydrophilic swellable polymers; Superdisintegrant; Co-milling;

Tamoxifen citrate (Tmx) was formulated in nanostructured lipid carrier system (NLC) using long chain solid lipids (LCSL) and oils (LCO) with the aim to target lymphatic system to improve its bioavailability in plasma and lymphnode (initial sites for metastasis) and reduce its drug associated toxicity. Tamoxifen loaded NLC (Tmx-NLC) was formulated using solvent diffusion technique. Preformulation studies comprised evaluation of drug–excipients compatibility. Solubility of Tmx was screened in LCSL and LCO, surfactants and co-surfactants to identify NLC components. Surfactant co-surfactant combinations were studied for their ability to stabilize the system. Tmx-NLC was physicochemically characterized by TEM, DSC, XRD, and FTIR studies. Drug–excipients chemical compatibility study facilitated anticipation of excipients induced oxidative degradation of Tmx. Suitable storage condition below 30 °C could stabilize Tmx. Tmx-NLC with >90% entrapment efficiency and 215.60 ± 7.98 nm particle size were prepared and freeze dried. Freeze dried Tmx-NLC could withstand various gastrointestinal tract (GI) media (pH 1.2, pH 3.5, pH 4.5, pH 6.8, pH 7.4). Dissolution profile of Tmx-NLC in various media showed sustained release pattern irrespective of pH of medium. No significant change in characteristics of Tmx-NLC was observed after 3 months of accelerated stability studies.
Keywords: Tamoxifen; Preformulation; Solubility; Nanostructured lipid carrier; Stability;

Long chain lipid based tamoxifen NLC. Part II: Pharmacokinetic, biodistribution and in vitro anticancer efficacy studies by Harshad Shete; Sushmita Chatterjee; Abhijit De; Vandana Patravale (584-592).
Long chain lipid (LCL) based tamoxifen loaded nanostructured lipid carriers (Tmx-NLCs) meant to target intestinal lymphatic systems (ILSs) was developed and characterized previously. The aim of the present work was to evaluate in vitro efficacy of developed Tmx-NLC against breast cancer cell lines and to confirm the hypothesis of targeting ILS after single dose oral administration. In vitro anticancer activity of Tmx-NLC was assessed in human estrogen receptor expressing breast cancer cell lines viz. MCF-7 and ZR-75-1. The study revealed relatively improved activity for Tmx-NLC compared to free Tmx against MCF-7 cells. However, the activity was compromised against ZR-75-1 cells which could be attributed to its up regulation of MUC1 gene. Confocal and flow cytometric analysis revealed remarkable intracellular uptake of Tmx-NLC and its localization in nuclear and perinuclear region of cells. Tmx-NLC exhibited distinctly different pharmacokinetic profile compared to Tamoxifen suspension (Tmx-susp) and exhibited an increment in the bioavailability by 2.71-fold and prolonged the T 1/2 by 7.10-fold. Moreover, detectable drug concentration in mesenteric lymph nodes justifies our hypothesis of targeting ILS and explains the major uptake of Tmx to occur via lymphatic system.
Keywords: Tamoxifen; Nanostructured lipid carriers; Lymphatic system; Pharmacokinetics;