International Journal of Pharmaceutics (v.474, #1-2)

Lipid nanoparticles protect from edelfosine toxicity in vivo by Beatriz Lasa-Saracíbar; María Ángela Aznar; Hugo Lana; Ismael Aizpún; Ana Gloria Gil; Maria J. Blanco-Prieto (1-5).
Display OmittedEdelfosine, an alkyl-lysophospholipid antitumor drug with severe side-effects, has previously been encapsulated into lipid nanoparticles (LN) with the purpose of improving their toxicity profile. LN are made of lipids recognized as safe by the Food and Drug Administration (FDA) and, therefore, these systems are generally considered as nontoxic vehicles. However, toxicity studies regarding the use of LN as vehicles for drug administration are limited. In the present study, we investigated the in vivo toxicity of free edelfosine, and the protection conferred by LN. The free drug, non-loaded LN and edelfosine-loaded LN were orally administered to mice. Our results show that the oral administration of the free drug at 4 times higher than the therapeutic dose caused the death of the animals within 72 h. Moreover, histopathology revealed gastrointestinal toxicity and an immunosuppressive effect. In contrast, LN showed a protective effect against edelfosine toxicity even at the higher dose and were completely safe. LN are, therefore, a safe vehicle for the administration of edelfosine by the oral route. The nanosystems developed could be further used for the administration of other drugs.
Keywords: Alkyl-lysophospholipids; Edelfosine; In vivo; Lipid nanoparticles; Toxicity;

In vivo pharmacokinetics and biodistribution of resveratrol-loaded solid lipid nanoparticles for brain delivery by S. Jose; S.S. Anju; T.A. Cinu; N.A. Aleykutty; S. Thomas; E.B. Souto (6-13).
Display OmittedResveratrol is a potent anticancer. However, because of its low half-life (<0.25 h) the molecule is difficult to achieve the therapeutic concentration at the site of action. The aim of this work was to check the brain targeting ability of glyceryl behenate-based solid lipid nanoparticles (SLN) for resveratrol. SLN were prepared by solvent evaporation technique employing high speed homogenization followed by ultrasonication. SLN were designed at varying drug–lipid ratios (1:5, 1:9, 1:10, 1:11, 1:12 and 1:15) using Tween 80 or a combination of Tween 80 and polyvinyl alcohol (PVA) as surfactants. The mean particle size and zeta potential of the optimized formulation (drug–lipid ratio of 1:10) were 248.30 ± 3.80 nm and −25.49 ± 0.49 mV, respectively. The particle size and the encapsulation efficiency (EE) increased when varying the drug–lipid ratio from 1:5 to 1:15. Scanning electron microscopic (SEM) analysis showed that SLN were spherical in shape and had a smooth surface. The X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analyses revealed that the matrix of drug-loaded SLN was in disordered crystalline phase. The in vitro release study in phosphate buffer pH 7.4 followed a sustained release pattern. The drug release data was found to fit best into Higuchi kinetic model suggesting the diffusion controlled mechanism of drug release. The cytotoxicity assay (MAT) showed that SLN were equally effective (P  < 0.5) as free resveratrol, as an anti-tumor agent. The in vivo biodistribution study using Wistar rats demonstrated that SLN could significantly (P  < 0.001) increase the brain concentration of resveratrol (17.28 ± 0.6344 μg/g) as compared to free resveratrol (3.45 ± 0.3961 μg/g). The results showed that our resveratrol-loaded SLN serve as promising therapeutic systems to treat neoplastic diseases located in the brain tissue.
Keywords: Resveratrol; Solid lipid nanoparticles (SLN); Brain targeting; Brain cancer; Cytotoxicity;

Display OmittedThe main goal of this study is to investigate the expression of sodium dependent vitamin C transport system (SVCT2). Moreover, this investigation has been carried out to define uptake mechanism and intracellular regulation of ascorbic acid (AA) in human breast cancer cells (MDA-MB231, T47D and ZR-75-1). Uptake of [14C] AA was studied in MDA-MB231, T47D and ZR-75-1 cells. Functional parameters of [14C] AA uptake were delineated in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was carried out with reverse transcription-polymerase chain reaction (RT-PCR). Uptake of [14C] AA was studied and found to be sodium, chloride, temperature, pH and energy dependent in all breast cancer cell lines. [14C] AA uptake was found to be saturable, with K m values of 53.85 ± 6.24, 49.69 ± 2.83 and 45.44 ± 3.16 μM and V max values of 18.45 ± 0.50, 32.50 ± 0.43 and 33.25 ± 0.53 pmol/min/mg protein, across MDA-MB231, T47D and ZR-75-1, respectively. The process is inhibited by structural analogs (l-AA and d-iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca++/calmodulin and protein kinase pathways appeared to play a crucial role in modulating AA uptake. A 626 bp band corresponding to a vitamin C transporter (SVCT2) based on the primer design was detected by RT-PCR analysis in all breast cancer cell lines. This research article describes AA uptake mechanism, kinetics, and regulation by sodium dependent vitamin C transporter (SVCT2) in MDA-MB231, T47D and ZR-75-1 cells. Also, MDA-MB231, T47D and ZR-75-1 cell lines can be utilized as a valuable in vitro model to investigate absorption and permeability of AA-conjugated chemotherapeutics.
Keywords: Ascorbic acid kinetics; Targeted drug delivery; In vitro cell culture models; MDA-MB231; T47D and ZR-75-1 cells; Nutrient transporter;

Kinetic and thermodynamic evaluation of phosphate ions binding onto sevelamer hydrochloride by Reem Elsiddig; Helen Hughes; Eleanor Owens; Niall J. O’ Reilly; David O’Grady; Peter McLoughlin (25-32).
Display OmittedSevelamer hydrochloride is the first non-aluminium, non-calcium-based phosphate binder developed for the management of hyperphosphatemia in end stage renal diseases. It is a synthetic ion-exchange polymer which binds and removes phosphate ions due to the high content of cationic charge associated with protonated amine groups on the polymer matrix. This is the first in-depth study investigating phosphate removal in vitro from aqueous solutions using commercially available sevelamer hydrochloride at physiological conditions of phosphate level, pH and temperature. The kinetic and thermodynamic parameters of phosphate binding onto the sevelamer hydrochloride particles were evaluated in order to define the binding process. A series of kinetic studies were carried out in order to delineate the effect of initial phosphate concentration, absorbent dose and temperature on the rate of binding. The results were analysed using three kinetic models with the best-fit of the experimental data obtained using a pseudo-second order model. Thermodynamic parameters provide in-depth information on inherent energetic changes that are associated with binding. Free energy ΔG°, enthalpy ΔH°, and entropy ΔS° changes were calculated in this study in order to assess the relationship of these parameters to polymer morphology. The binding reaction was found to be a spontaneous endothermic process with increasing entropy at the solid–liquid interface.
Keywords: Sevelamer hydrochloride; Ion binding; Kinetic; Thermodynamics; Binding capacity;

Formulation of solid lipid nanoparticles (SLN): The value of different alkyl polyglucoside surfactants by Cornelia M. Keck; Andjelka Kovačević; Rainer H. Müller; Snežana Savić; Gordana Vuleta; Jela Milić (33-41).
Display OmittedAlkyl polyglycosides (APGs) represent a group of nonionic tensides with excellent skin compatibility. Thus they seem to be excellent stabilizers for lipid nanoparticles for dermal application. To investigate this, different APGs were selected to evaluate their influence on the formation and characteristics of solid lipid nanoparticles (SLN). Contact angle analysis of the aqueous solutions/dispersions of the APGs on cetyl palmitate films revealed good wettability for all APG surfactants. Cetyl palmitate based SLN were prepared by hot high pressure homogenization and subjected to particle size, charge and inner structure analysis. 1% of each APG was sufficient to obtain SLN with a mean size between 150 nm and 175 nm and a narrow size distribution. The zeta potential in water was ∼ −50 mV; the values in the original medium were distinctly lower, but still sufficient high to provide good physical stability. Physical stability at different temperatures (5 °C, 25 °C and 40 °C) was confirmed by a constant particle size over an observation period of 90 days in all dispersions. In comparison to SLN stabilized with classical surfactants, e.g., Polysorbate, APG stabilized SLN possess a smaller size, improved physical stability and contain less surfactant. Therefore, the use of APGs for the stabilization of lipid nanoparticles is superior in comparison to classical stabilizers. Further, the results indicate that the length of the alkyl chain of the APG influences the diminution efficacy, the final particle size and the crystallinity of the particles. APGs with short alkyl chain led to a faster reduction in size during high pressure homogenization, to a smaller particle size of the SLN and to a lower recrystallization index, i.e., to a lower crystallinity of the SLN. The crystallinity of the SLN increased with an increase in the alkyl chain length of APGs. Therefore, by using the tested APGs differing in the alkyl chain length, not only small sized and physically stable but also SLN with different sizes and crystallinity can be obtained. An optimized selection of these stabilizers might therefore enable the production of lipid nanoparticles with “tailor-made” properties.
Keywords: Solid lipid nanoparticles; SLN; Alkyl polyglucosides; Size; Crystallinity; Physical stability;

In vitro PAMAM, phosphorus and viologen-phosphorus dendrimers prevent rotenone-induced cell damage by Katarzyna Milowska; Aleksandra Szwed; Maria Zablocka; Anne-Marie Caminade; Jean-Pierre Majoral; Serge Mignani; Teresa Gabryelak; Maria Bryszewska (42-49).
Display OmittedWe have investigated whether polyamidoamine (PAMAM), phosphorus (pd) and viologen-phosphorus (vpd) dendrimers can prevent damage to embryonic mouse hippocampal cells (mHippoE-18) caused by rotenone, which is used as a pesticide, insecticide, and as a nonselective piscicide, that works by interfering with the electron transport chain in mitochondria. Several basic aspects, such as cell viability, production of reactive oxygen species and changes in mitochondrial transmembrane potential, were analyzed. mHippoE-18 cells were treated with these structurally different dendrimers at 0.1 μM. A 1 h incubation with dendrimers was followed by the addition of rotenone at 1 μM, and a further 24 h incubation. PAMAM, phosphorus and viologen-phosphorus dendrimers all increased cell viability (reduced cell death-data need to be compared with untreated controls). A lower level of reactive oxygen species and a favorable effect on mitochondrial system were found with PAMAM and viologen-phosphorus dendrimers. These results indicate reduced toxicity in the presence of dendrimers.
Keywords: Dendrimers; Rotenone; Parkinson’s disease; Cytotoxicity;

Display OmittedTo enhance the aqueous solubility and thus oral bioavailability of a poorly water-soluble drug, probucol (PB), probucol–phospholipid complex (PB–PC) was formulated by solvent-evaporation or co-grinding methods. The complexes were characterized by differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), solubility, oil–water partition coefficient and in vitro dissolution. The DSC, IR and PXRD data confirmed the formation of phospholipid complex. Furthermore, the results indicated hydrogen bond formation between PB and PC molecules play an important role in the formation of PB–PC without the formation of a new compound. The water solubility of PB in the complexes was improved from 0.005 to 17.76 or 1.65 μg/mL (by solvent-evaporation or co-grinding methods respectively). As a result of it, the improved dissolution was shown in the prepared complexes. The PB–PC complexes by both solvent-evaporation and co-grinding methods exhibited higher peak plasma concentration (16,625.7 or 5343.3 vs. 2628.4 ng mL−1), increased AUC0–48 h (145,863.2 or 77,477.0 vs. 34,435.9 ng mL−1  h) when compared with the commercial product, suggesting improved bioavailability of the drug. The study therefore suggests that the phospholipid complexes have possibilities in enhancing the therapeutic efficacy of PB which may be due to its improved aqueous solubility, dissolution behavior and thus bioavailability.
Keywords: Probucol; Phospholipid complex; Solvent-evaporation method; Co-grinding method; Oral bioavailability;

Bis-quaternary gemini surfactants as components of nonviral gene delivery systems: A comprehensive study from physicochemical properties to membrane interactions by Ana M. Cardoso; Catarina M. Morais; Sandra G. Silva; Eduardo F. Marques; Maria C. Pedroso de Lima; Maria Amália S. Jurado (57-69).
Display OmittedGemini surfactants have been successfully used as components of gene delivery systems. In the present work, a family of gemini surfactants, represented by the general structure [C m H2m+1(CH3)2N+(CH2) s N+(CH3)2C m H 2m+1]2Br, or simply msm, was used to prepare cationic gene carriers, aiming at their application in transfection studies. An extensive characterization of the gemini surfactant-based complexes, produced with and without the helper lipids cholesterol and DOPE, was carried out in order to correlate their physico-chemical properties with transfection efficiency. The most efficient complexes were those containing helper lipids, which, combining amphiphiles with propensity to form structures with different intrinsic curvatures, displayed a morphologically labile architecture, putatively implicated in the efficient DNA release upon complex interaction with membranes. While complexes lacking helper lipids were translocated directly across the lipid bilayer, complexes containing helper lipids were taken up by cells also by macropinocytosis. This study contributes to shed light on the relationship between important physico-chemical properties of surfactant-based DNA vectors and their efficiency to promote gene transfer, which may represent a step forward to the rational design of gene delivery systems.
Keywords: Bis-quaternary gemini surfactant; Transfection efficiency; Complex preparation; Physico-chemical characterization; Cell membrane interactions;

Polyester-idarubicin nanoparticles and a polymer-photosensitizer complex as potential drug formulations for cell-mediated drug delivery by André-R. Blaudszun; Qiong Lian; Melanie Schnabel; Brigitta Loretz; Ute Steinfeld; Hyeck-H. Lee; Gerhard Wenz; Claus-M. Lehr; Marc Schneider; Anja Philippi (70-79).
Display OmittedCell-mediated transport of therapeutics has emerged as promising alternative to classical drug delivery approaches. To preserve viability and functions of carrier cells, encapsulation of active drugs in protective nanoparticles or the use of inducible therapeutics has been proposed. Here, we compared the effects of novel polymeric formulations of an active and a stimulus-sensitive anti-cancer drug on human T lymphocytes to identify suitable drug preparations for cell-mediated drug delivery. For the first approach, the chemotherapeutic agent idarubicin (IDA) was encapsulated in poly(lactic-co-glycolic-acid) (PLGA) and newly developed maleate-polyester (MPE) nanoparticles. PLGA- and MPE-encapsulated IDA was efficiently internalized by ex vivo activated human T lymphocytes; however, both encapsulations could not prevent premature T cell death resulting from IDA-uptake. In contrast, loading with a poly(styrene sulfonate) (PSS)-complex of the light-sensitive pharmaceutical 5,10,15,20-tetrakis(meso-hydroxyphenyl)porphyrin (mTHPP) did not affect T cell viability if upon loading the cells were kept in the dark. The photosensitizer was transferred from loaded T lymphocytes to co-cultivated carcinoma cells, and induced cancer cell death if co-cultures were exposed to light. Inducible drugs, such as photosensitizers, thus, may help to overcome the limitations of encapsulated active drugs and open up new perspectives for the use of cells as drug transporters in cancer therapy.
Keywords: Targeted drug delivery; T lymphocyte; Polymeric nanoparticle; Gold shell nanoparticle; Photosensitizer;

Effect of γ-cyclodextrin on solubilization and complexation of irbesartan: Influence of pH and excipients by Chutimon Muankaew; Phatsawee Jansook; Einar Stefánsson; Thorsteinn Loftsson (80-90).
Display OmittedIn effort to prepare an eye drop formulation of irbesartan, the effect of γ-cyclodextrin complexation on irbesartan solubilization in aqueous solutions was investigated. The optimum cyclodextrin concentration for formation of irbesartan/cyclodextrin inclusion complex was found to be 10% (w/v) and the solubility of ionized irbesartan/γ-cyclodextrin complex (at pH 7.2) was shown to be three fold greater than that of the unionized complex (at pH 4.3). The irbesartan flux through semipermeable membranes increased with increasing γ-cyclodextrin concentration at both pH values. However, the ionized complex displayed decrease in the drug permeation coefficient with increasing cyclodextrin concentration. The effect of four pharmaceutical excipients on the cyclodextrin solubilization was investigated. EDTA, hydroxypropyl methylcellulose, and tyloxapol increased complexation efficiency of γ-cyclodextrin while benzalkonium chloride had negligible effect. The largest solubilization was observed in the eye drop vehicle that contained all four excipients in addition to γ-cyclodextrin. Dynamic light scattering measurements disclosed that excipients had impact on size of complex aggregates and consequently on the drug flux through the semipermeable membranes. Complex of irbesartan/γ-cyclodextrin was characterized by FT-IR, 1H NMR, XRPD, and TEM techniques.
Keywords: Irbesartan; γ-Cyclodextrin; Solubilization; Complexation; pH; Excipient;

Folate receptor targeted self-assembled chitosan-based nanoparticles for SPECT/CT imaging: Demonstrating a preclinical proof of concept by András Polyák; István Hajdu; Magdolna Bodnár; Gabriella Dabasi; Róbert P. Jóba; János Borbély; Lajos Balogh (91-94).
Display OmittedA new biocompatible, biodegradable, self-assembled chitosan-based nanoparticulate product was successfully synthesized and radiolabeled with technetium-99m, and studied as a potential new SPECT or SPECT/CT imaging agent for diagnosis of folate receptor overexpressing tumors. In the present study we examined the conditions of a preclinical application of this labeled nanosystem in early diagnosis of spontaneously diseased veterinary patient using a human SPECT/CT device. The results confirmed that the nanoparticles accumulated in tumor cells overexpressing folate receptors, contrast agent revealed higher uptake in the tumor for a long time. Preclinical trials verified that the new nanoparticles are able to detect folate-receptor-overexpressing tumors in spontaneously diseased animal models with enhanced contrast.
Keywords: Chitosan; Nanoparticles; Nanomedicine; SPECT-CT; Folate receptor; 99mTc;

Display OmittedDelivering chemotherapeutics by nanoparticles into tumor was influenced by at least two factors: specific targeting and highly efficient penetrating of the nanoparticles. In this study, two targeting ligands, angiopep-2 and activatable cell penetrating peptide (ACP), were functionalized onto nanoparticles for tumor targeting delivery. In this system, angiopep-2 is a ligand of low-density lipoprotein receptor-related protein-1 (LRP1) which was highly expressed on tumor cells, and the ACP was constructed by the conjugation of RRRRRRRR (R8) with EEEEEEEE through a matrix metalloproteinase-2 (MMP-2) sensitive linker, enabling the ACP with tumor microenvironment-responsive cell penetrating property. 4 h incubation of ACP with MMP-2 leads to over 80% cleavage of ACP, demonstrating ACP indeed possessed MMP-2 responsive property. The constructed dual targeting nanoparticles (AnACNPs) were approximately 110 nm with a polydispersity index of 0.231. In vitro, ACP modification and angiopep-2 modification could both enhance the U-87 MG cell uptake because of the high expression of MMP-2 and LRP-1 on C6 cells. AnACNPs showed higher uptake level than the single ligand modified nanoparticles. The uptake of all particles was time- and concentration-dependent and endosomes were involved. In vivo, AnACNPs showed best tumor targeting efficiency. The distribution of AnACNPs in tumor was higher than all the other particles. After microvessel staining with anti-CD31 antibody, the fluorescent distribution demonstrated AnACNPs could distribute in the whole tumor with the highest intensity. In conclusion, a novel drug delivery system was developed for enhanced tumor dual targeting and elevated cell internalization.
Keywords: Glioma; Systemic targeting delivery; Activatable cell penetrating peptide; Angiopep-2;

Sustained and controlled release of lipophilic drugs from a self-assembling amphiphilic peptide hydrogel by Maria-Lucia Briuglia; Andrew J. Urquhart; Dimitrios A. Lamprou (103-111).
Display OmittedMaterials which undergo self-assembly to form supramolecular structures can provide alternative strategies to drug loading problems in controlled release application. RADA 16 is a simple and versatile self-assembling peptide with a designed structure formed of two distinct surfaces, one hydrophilic and one hydrophobic that are positioned in such a well-ordered fashion allowing precise assembly into a predetermined organization. A “smart” architecture in nanostructures can represent a good opportunity to use RADA16 as a carrier system for hydrophobic drugs solving problems of drugs delivery. In this work, we have investigated the diffusion properties of Pindolol, Quinine and Timolol maleate from RADA16 in PBS and in BSS-PLUS at 37 °C. A sustained, controlled, reproducible and efficient drug release has been detected for all the systems, which allows to understand the dependence of release kinetics on the physicochemical characteristics of RADA16 structural and chemical properties of the selected drugs and the nature of solvents used. For the analysis various physicochemical characterization techniques were used in order to investigate the state of the peptide before and after the drugs were added. Not only does RADA16 optimise drug performance, but it can also provide a solution for drug delivery issues associated with lipophilic drugs.
Keywords: Controlled release; Amphiphilic hydrogels; Spectroscopy; AFM; Lipophilic drugs;

Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer by Mei-Hua Qu; Rui-Fang Zeng; Shi Fang; Qiang-Sheng Dai; He-Ping Li; Jian-Ting Long (112-122).
Display OmittedCombination of more than one therapeutic strategy is the standard treatment in clinics. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a nanoparticulate system will suppress the tumor growth. In the present study, docetaxel (DTX) and BCL-2 siRNA was incorporated in a PEGylated liposome to systemically deliver in a lung cancer model (A549). The resulting nanoparticle (lipo-DTX/siRNA) was stable and exhibited a sustained release profile. The co-delivery of therapeutic moieties inhibited the cell proliferation (A549 and H226) in a time-dependent manner. Moreover, the co-delivery system of DTX and siRNA exhibited a remarkable apoptosis of cancer cells with elevated levels of caspase 3/7 activity (apoptosis markers). Cell cycle analysis further showed remarkable increase in sub-G0/G1 phase, indicating increasing hypodiploids or apoptotic cells. Pharmacokinetic study showed a long circulating profile for DTX from lipo-DTX/siRNA system facilitating the passive tumor targeting. In vivo antitumor study on A549 cell bearing xenograft tumor model exhibited a remarkable tumor regression profile for lipo-DTX/siRNA with 100% survival rate. The favorable tumor inhibition response was attributed to the synergistic effect of DTX potency and MDR reversing ability of BCL-2 siRNA in the tumor mass. Overall, experimental results suggest that co-delivery of DTX and siRNA could be promising approach in the treatment of lung cancers.
Keywords: Docetaxel; siRNA; Lung cancer; Antitumor; Liposomes;

Enhancement in bioavailability of ketorolac tromethamine via intranasal in situ hydrogel based on poloxamer 407 and carrageenan by Chenxi Li; Chunyan Li; Zheshuo Liu; Qiuhong Li; Xueying Yan; Yu Liu; Weiyue Lu (123-133).
Display OmittedThe objective of this study was to construct a new in situ gel system based on the combination of poloxamer 407 and carrageenan (carrageenan–poloxamer 407 hydrogel, CPH) for intranasal delivery of ketorolac tromethamine. CPH showed potassium ion concentration - dependent erosion characteristics which ensured slow erosion in aqueous environment containing potassium ion at the physiological level. Loading with ketorolac tromethamine influenced erosion, drug release and thermosensitive properties of CPH. CPH containing 15% ketorolac tromethamine showed suitable gelation temperature (near 35 °C) and in vitro sustained release profiles. Pharmacokinetic study of intranasal CPH containing 15% ketorolac tromethamine in rats demonstrated enhanced absolute bioavailability (68.8 ± 23.3%) and prolonged mean residence time (8.8 ± 3.5 h) in comparison with the intranasal solution group (24.8 ± 13.8%, 3.9 ± 0.6 h). Nasal ciliotoxicity evaluation on an in situ toad palate model preliminarily showed the safety of CPH for intranasal use. All results suggested the potential of CPH as a new sustained - release platform for the intranasal delivery of ketorolac tromethamine.
Keywords: Ketorolac tromethamine; In situ hydrogel; Carrageenan; Poloxamer 407; Bioavailability; Intranasal delivery;

Display OmittedThe aim of this work was to investigate the potential of an amphiphilic system comprising chitosan-grafted polylactide and carboxyl-functionalized polylactide acid as a carrier for the controlled release and co-release of two DNA alkylating drugs: doxorubicin and temozolomide. Polylactide and carboxyl-functionalized polylactide acid were obtained through direct melt polycondensation reaction, using methanesulfonic acid as a non-toxic initiator, and subsequently these were grafted to the chitosan backbone through a coupling reaction, utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a condensing agent. ATR-FTIR analysis and conductometric titration confirmed that a reaction between CS and PLA, PLACA2% and PLACA5% occurred. Chitosan-grafted-polylactide and polylactide-citric acid nanoparticles were prepared via the polyelectrolyte complex technique, applying dextran sulphate as a polyanion, and loaded with doxorubicin and temozolomide. The diameter of particles, ζ-potential and their relationship to temperature and pH were analysed in all formulations. Encapsulation, co-encapsulation efficiency and release studies were conducted in different physiological simulated environments and human serum. Results showed the continuous release of drugs without an initial burst in different physiological media.
Keywords: Nanoparticles; Drug delivery System; Doxorubicin; Temozolomide; Chitosan; Polylactide;

Amorphous isradipine nanosuspension by the sonoprecipitation method by Thao Truong-Dinh Tran; Phuong Ha-Lien Tran; Minh Ngoc Uyen Nguyen; Khanh Thi My Tran; Minh Nguyet Pham; Phuc Cao Tran; Toi Van Vo (146-150).
Display OmittedThe aims of this study are to increase and explain the mechanism of dissolution enhancement of isradipine using the sonoprecipitation method for stable nanosuspensions. There have been still few of published researches on formulation of isradipine using nanoparticle engineering. Nanosuspension systems were prepared upon various factors including amplitude and the time length of ultrasonication. The dissolution test was performed according to the USP paddle method in intestinal fluid (pH 6.8). The crystalline structure of drug, the molecular interaction, morphology and size of nanosuspension were also investigated to determine the mechanism of dissolution enhancement. The sonoprecipitation method with use of HPMC 6 showed its potential in enhancement of the drug release rate. Stable nanosuspension was significantly depended on amplitude and time of ultrasonication since these factors affected on the size of nanoparticles. The synergistic effects of reduction of drug crystallinity and particle size could increase the dissolution rate of isradipine by providing a stable nanosuspension. This work may contribute to a new strategy for improvement dissolution rate of isradipine.
Keywords: Nanosuspension; Sonoprecipitation method; Crystallinity; Dissolution enhancement;

Development of nanocrystal formulation of meloxicam with improved dissolution and pharmacokinetic behaviors by Masanori Ochi; Takaki Kawachi; Eri Toita; Issei Hashimoto; Kayo Yuminoki; Satomi Onoue; Naofumi Hashimoto (151-156).
Display OmittedThe present study aimed to develop nanocrystal formulations of meloxicam (MEL) in order to enhance its biopharmaceutical properties and provide a rapid onset of action. Nanocrystal formulations were prepared by wet-milling and lyophilization with hydrophilic polymers used as aggregation inhibitors. Aggregation inhibitors were selected based on high-throughput screening of crystal growth inhibition in supersaturated MEL solution. Supersaturation of MEL was observed in PVP K-30, HPC-SSL, and POVACOAT Type F solution. Although the particle size distributions of pulverized MEL with PVP K-30 (MEL/PVP), HPC-SSL (MEL/HPC), and POVACOAT Type F (MEL/POVA) were in the nanometer range following lyophilization, increases in micron-sized aggregates were observed after storage at 60 °C for 21 days. The order of increased amount of aggregates was MEL/POVA ≫ MEL/HPC > MEL/PVP. These findings showed that hydrophilic polymers that inhibited crystal growth in supersaturated MEL solutions tended to prevent aggregation. The dissolution behavior of all nanocrystal formulations tested was markedly enhanced compared with that of unpulverized MEL. Oral administration of MEL/PVP showed a 2.0 h shortened T max and a 5.0-fold increase in bioavailability compared with unpulverized MEL. These findings showed that the MEL/PVP mixture was physicochemically stable and provided a rapid onset of action and enhanced bioavailability after oral administration.
Keywords: Nanocrystals; Meloxicam; Stabilization; Supersaturation; Dissolution; Pharmacokinetic;

Mechanistic modeling of modular co-rotating twin-screw extruders by Andreas Eitzlmayr; Gerold Koscher; Gavin Reynolds; Zhenyu Huang; Jonathan Booth; Philip Shering; Johannes Khinast (157-176).
Display OmittedIn this study, we present a one-dimensional (1D) model of the metering zone of a modular, co-rotating twin-screw extruder for pharmaceutical hot melt extrusion (HME). The model accounts for filling ratio, pressure, melt temperature in screw channels and gaps, driving power, torque and the residence time distribution (RTD). It requires two empirical parameters for each screw element to be determined experimentally or numerically using computational fluid dynamics (CFD). The required Nusselt correlation for the heat transfer to the barrel was determined from experimental data. We present results for a fluid with a constant viscosity in comparison to literature data obtained from CFD simulations. Moreover, we show how to incorporate the rheology of a typical, non-Newtonian polymer melt, and present results in comparison to measurements. For both cases, we achieved excellent agreement. Furthermore, we present results for the RTD, based on experimental data from the literature, and found good agreement with simulations, in which the entire HME process was approximated with the metering model, assuming a constant viscosity for the polymer melt.
Keywords: Hot melt extrusion; Co-rotating twin-screw extruder; One-dimensional model; Non-Newtonian; Residence time distribution;

An in vitro test of the efficacy of an anti-biofilm wound dressing by Jawal Said; Michael Walker; David Parsons; Paul Stapleton; Anthony E. Beezer; Simon Gaisford (177-181).
Display OmittedBroad-spectrum antimicrobial agents, such as silver, are increasingly being formulated into medicated wound dressings in order to control colonization of wounds by opportunistic pathogens. Medicated wound dressings have been shown in-vitro to be effective against planktonic cultures, but in-vivo bacteria are likely to be present in biofilms, which makes their control and eradication more challenging. Recently, a functional wound dressing (AQUACEL® Ag+ Extra™ (AAg + E)) has been developed that in addition to silver contains two agents (ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC)) designed to disrupt biofilms. Here, the efficacy of AAg + E is demonstrated using a biofilm model developed in an isothermal microcalorimeter. The biofilm was seen to remain viable in the presence of unmedicated dressing, silver-containing dressing or silver nitrate solution. In the presence of AAg + E, however, the biofilm was eradicated. Control experiments showed that neither EDTA nor BC alone had a bactericidal effect, which means it is the synergistic action of EDTA and BC disrupting the biofilm with silver being bactericidal that leads to the product’s efficacy.
Keywords: Staphylococcus aureus; Biofilm; Isothermal calorimetry; Wound dressings; silver;

Hydroxypropyl-β-cyclodextrin grafted polyethyleneimine used as transdermal penetration enhancer of diclofenac sodium by Yan Yan; Jianfeng Xing; Wei Xu; Guilan Zhao; Kai Dong; Lu Zhang; Ke Wang (182-192).
These pictures are the schematic representation of synthesized HP-β-CD-PEI and transdermal penetration mechanism. This new kind of cationic polymer was used as the safety transdermal permeation enhancer that demonstrated its great potential application in transdermal hydrophilic drug delivery system.Display OmittedThe objective of this investigation was to develop a novel cationic polymer, hydroxypropyl-β-cyclodextrin grafted polyethyleneimine (HP-β-CD-PEI1800), as a penetration enhancer, and evaluate its viability on improving transdermal delivery of diclofenac sodium. In this study, HP-β-CD-PEI1800 was characterized by 1H NMR and DSC methods, respectively. The hydrophilic drug diclofenac sodium was chosen as model drug, and the transdermal permeation enhancement of HP-β-CD-PEI1800 was estimated in vitro by using Franz diffusion cells fitted with mouse dorsal skins, the in vivo kinetics of diclofenac sodium was analyzed by high-performance liquid chromatography (HPLC). The cumulative drug content deposited in epidermis and dermis was measured at the pre-determined time point of 3, 6, and 9 h, and the permeation profile was significantly higher than that of the control groups. In addition, the cytotoxicity and skin irritation of enhancer was evaluated by MTT assay and histological examination, respectively, and the results indicated that the polymer we prepared were non-toxic and non-irritant after exposure to skins. All the results suggested that HP-β-CD-PEI1800 could be a safe and efficient penetration enhancer of diclofenac sodium.
Keywords: Transdermal drug delivery; Penetration enhancer; Diclofenac sodium; Cytotoxicity; Irritation of the skin;

Routine application of Raman spectroscopy in the quality control of hospital compounded ganciclovir by Alexandre Amin; Philippe Bourget; Fabrice Vidal; Flavie Ader (193-201).
Display OmittedThis study compares the performance of a reference method of HPLC to Raman spectroscopy (RS) for the analytical quality control (AQC) of therapeutic objects. We assessed a model consisting of a widely used antiviral drug, i.e., ganciclovir, which was compounded in a medical device and then transferred in a vacuum glass vial prior to analyses. As the aim of the alternative RS method is to replace the destructive, time-consuming HPLC method, requiring sample preparation, it needs to be demonstrated that RS performs at least as good as the HPLC method. Therefore, the two methods were validated by calculating the accuracy profile and provided excellent results for the analytical validation key criteria, i.e., trueness, precision and accuracy, ranging from 0.8 to 10 mg/mL. The Spearman and Kendall correlation tests (p-value < 1.10–15) and the statistical studies performed on the graphs confirm a strong correlation in the results between RS and the standard HPLC under the experimental conditions. These results confirmed the potential of this option for future applications, owing to its analytical and practical quality and its contributions to the safety of the medication circuit. This method also greatly contributes to the protection of caregivers and their working environment.
Keywords: Raman spectroscopy; Analytical quality control; HPLC; Safety; Hospital environment; Ganciclovir;

Carboxymethyl-β-cyclodextrin conjugated nanoparticles facilitate therapy for folate receptor-positive tumor with the mediation of folic acid by Chang Su; Hongdan Li; Yijie Shi; Guan Wang; Liwei Liu; Liang Zhao; Rongjian Su (202-211).
Display OmittedCurrently, clinical operation treatments, chemotherapy and radiotherapy just could eliminate local tumor cells. However, chemotherapy and radiotherapy also injury normal cells and lead to serious side effects and toxicities. So, it is necessary to find an effective target cancer carrier that delivers the anticancer agents into tumor cells and reduces normal cells’ injury. Folic acid (FA) is a classical targeting agent mediates internalization of chemical drugs into tumor cells which over-express folate receptor (FR) on their surface. We herein report that based on host–guest interaction, NPs decorated by novel folate enhance antitumor drug delivery. BSA-NPs were prepared by desolvation method and carboxymethyl-β-cyclodextrin (CM-β-CD) was conjugated to the surface of NPs by carbodiimide coupling to hold FA. From in vitro cytotoxicity assay, cell apoptosis study, intracellular ATP level assay and western blot, we can see that FA-CM-β-CD-BSA NPs as good monodispersity, negative charge, and homogenous particle size have a high encapsulation efficiency. The results showed that MTT and cell apoptosis demonstrated that FA-decorated NPs exhibit stronger inhibition rate and induce obvious apoptosis in FR positive Hela cells as compared to free drug and FA undecorated NPs. Moreover, 5-fluorouracil (5-Fu) loaded FA-CM-β-CD-BSA NPs down-regulate ATP levels and increase the expression of caspase-3. Taken together, FA-CM-β-CD-BSA NPs enhance FR receptor-mediated endocytosis and lead to more intracellular uptake of drug, inducing the higher apoptosis ratio of cells than free 5-Fu.
Keywords: Folic acid; Nanoparticles; Carboxymethyl-β-cyclodextrin; 5-Fu; Apoptosis;

Lysine-based surfactants as chemical permeation enhancers for dermal delivery of local anesthetics by Raquel S. Teixeira; Tânia F.G.G. Cova; Sérgio M.C. Silva; Rita Oliveira; Maria J. Araújo; Eduardo F. Marques; Alberto A.C.C. Pais; Francisco J.B. Veiga (212-222).
Display OmittedThe aim of this study is to investigate the efficacy of new, biocompatible, lysine-based surfactants as chemical permeation enhancers for two different local anesthetics, tetracaine and ropivacaine hydrochloride, topically administered. Results show that this class of surfactants strongly influences permeation, especially in the case of the hydrophilic and ionized drug, ropivacaine hydrochloride, that is not easily administered through the stratum corneum. It is also seen that the selected permeation enhancers do not have significant deleterious effects on the skin structure. A cytotoxicity profile for each compound was established from cytotoxicity studies. Molecular dynamics simulation results provided a rationale for the experimental observations, introducing a mechanistic view of the action of the surfactants molecules upon lipid membranes.
Keywords: Dermal drug delivery; Chemical permeation enhancers; Cytotoxicity; Tetracaine; Ropivacaine; Molecular dynamic simulation;

Negative-charge-functionalized mesoporous silica nanoparticles as drug vehicles targeting hepatocellular carcinoma by Meng Xie; Yuanguo Xu; Haijun Shen; Song Shen; Yanru Ge; Jimin Xie (223-231).
Display OmittedIn this paper, a series of doxorubicin-loaded and negative-charge-functionalized mesoporous silica nanoparticles (DOX–MSN/COOH) was successfully prepared and used for imaging and targeting therapy of hepatocellular carcinoma. The nanoparticles were uniform and negatively charged, with a diameter of about 55 nm, and a zeta potential of −20 mV. In vitro study showed that the nanoparticles could easily be endocytosed by liver cancer cells (HepG2) and were well-accumulated in the liver by passive targeting. In vivo study proved the ability of DOX–MSN/COOH to inhibit the tumor growth and prolong the survival time of mice bearing hepatocellular carcinoma in situ, giving better results than free DOX. More importantly, histological examination showed no histopathological abnormalities of normal liver cells and heart cells after the administration of DOX–MSN/COOH, while the treatment with free DOX caused damage to those cells. In conclusion, DOX–MSN/COOH exhibited enhanced antitumor efficacy as well as reduced side effects for liver cancer therapy.
Keywords: Mesoporous silica nanoparticles; Doxorubicin; Biodistribution; Liver tumor in situ; Antitumor; Survival rate;

Anti-tumor drug delivery system based on cyclodextrin-containing pH-responsive star polymer: In vitro and in vivo evaluation by Qingqing Xiong; Mingming Zhang; Zhibao Zhang; Wei Shen; Lingrong Liu; Qiqing Zhang (232-240).
Display OmittedA cyclodextrin-containing pH-responsive star polymer, with cyclodextrin polymer and pH-sensitive poly(2-(dimethylamino)ethyl methacrylate) as the core and poly(ethylene glycol) as the arm, was evaluated as drug carriers in vitro and in vivo. Doxorubicin (DOX) was successfully loaded into the star polymer to form nanoparticles (DOX-NPs) via host–guest interaction. The physicochemical properties such as drug loading content, size, morphology, stability and physical state of DOX-NPs were characterized in detail by 1H NMR, DLS, SEM and DSC. Uniform and stable DOX-NPs with high encapsulation efficiency of 77.1% were obtained, and they also exhibited sustainable and controllable release of DOX in vitro. The cellular uptake of DOX-NPs was in concentration-, time- and cell type-dependent manners, and the cytotoxicity of DOX-NPs was significantly high toward HeLa and HepG2 cancer cells. Furthermore, in vivo anti-tumor experiment on BALB/c mice bearing cervical tumor showed that DOX-NPs could effectively suppress the growth of tumor without significant side effect. These findings suggest that the cyclodextrin-containing pH-responsive star polymer has a promising potential in developing novel drug delivery system for cancer therapy.
Keywords: Cyclodextrin-containing polymer; Star polymer; 2-(Dimethylamino)ethyl methacrylate; pH-response; Cellular uptake; Anti-tumor drug delivery;

A novel antigen-carrier system: The Mycobacterium tuberculosis Acr protein carried by raw starch microparticles by S.A. Moreno-Mendieta; D. Guillén; C. Espitia; R. Hernández-Pando; S. Sanchez; R. Rodríguez-Sanoja (241-248).
Display OmittedMicroparticles have been used as promising carriers for in vivo vaccine delivery. However, the processes for immobilizing peptides or proteins on microparticles usually require the use of undesirable compounds and complex protocols. In this work, we propose a new immobilization and delivery system with raw starch microparticles and a starch binding domain (SBD) tag fusion protein. The heat shock protein alpha crystallin from Mycobacterium tuberculosis was used as model. The immunogenicity of the system was investigated in BALB/c mice inoculated with purified Acr-SBDtag protein (pAcr-SBDtag) and starch immobilized Acr-SBDtag protein (μAcr-SBDtag) by oral and intranasal routes. We demonstrated mucosal immunization with the μAcr-SBDtag protein induced systemic antibodies that were predominantly immunoglobulin G2a (IgG2a). An analysis of the cytokines from spleen cells and lung homogenates revealed that loaded microparticles induced the secretion of interferon-γ (INF-γ), suggesting an adjuvant effect from the immobilization. The immune responses induced by immobilized protein were primarily affected by the route of administration. These results demonstrate that the system exhibits the necessary characteristics to improve antigen release and presentation to antigen presenting cells (APCs) in the mucosae. Because no extra adjuvants were used, we posit that the system may be suitable for delivery and presentation to the field of subunit vaccine development.
Keywords: Starch microparticles; Starch Binding Domain tag; Mucosal immunization; Microparticle vaccines; Protein delivery;