International Journal of Pharmaceutics (v.478, #1)
EDITORIAL BOARD (iii).
Electrospun polyurethane/Eudragit® L100-55 composite mats for the pH dependent release of paclitaxel on duodenal stent cover application by Ludwig Erik Aguilar; Afeesh Rajan Unnithan; Altangerel Amarjargal; Arjun Prasad Tiwari; Seong Tshool Hong; Chan Hee Park; Cheol Sang Kim (1-8).
Display OmittedA nanofiber composite mat of PU and Eudragit® L100-55 was created using electrospinning process. The pH dependent release of paclitaxel was successfully done with the use of PU/EL100-55 nanocomposite mats as the controlling platform. The morphology of the nanofiber composites was surveyed using FESEM and ratios of the polymers affects the diameter of the nanofiber. Characterization of the nanofiber composite mat was done using FTIR, DSC-TGA method. The release rate of paclitaxel was determined and analyzed by in vitro drug release method. In order to mimic the condition of a human duodenum, the fibers were submersed on PBS of different pH levels (4.0, 6.0,) respectively, and then analyzed using high performance liquid chromatography (HPLC). Composite mats submersed in PBS with pH 4.0 showed lesser release profile compared to mats submersed in PBS with pH of 6.0. The composite mat has adequate mechanical properties and in vitro cell biocompatibility indicating that the material can be used for drug eluting stent cover application.
Keywords: Electrospinning; pH sensitive polymers; Drug eluting stent; Eudragit; Nanofiber;
Development of multicore hybrid particles for drug delivery through the precipitation of CO2 saturated emulsions by V.S.S. Gonçalves; S. Rodríguez-Rojo; A.A. Matias; A.V.M. Nunes; I.D. Nogueira; D. Nunes; E. Fortunato; A.P.Alves de Matos; M.J. Cocero; C.M.M. Duarte (9-18).
Display OmittedHybrid lipid–polymer particles are gaining increasing interest to be applied as drug delivery systems due to their greater stability in biological fluids and enhanced cellular uptake of drugs. However, a major drawback is the fact that these particles are usually produced through techniques that use organic solvents. This work studies the possibility of producing for the first time hybrid particles composed by lipid multicores enveloped in a polymeric layer through Particles from Gas Saturated Solutions (PGSS®), without using organic solvents. An oil-in-water emulsion, composed by Gelucire 43/01™ (GEL) as the discontinuous phase and by an aqueous polyethylene glycol 4000 (PEG) solution as the continuous phase, was successfully precipitated by PGSS®. Operating conditions that ensured the stability of the CO2 saturated emulsion were previously evaluated. The resulting PEG–GEL particles present a spherical-like morphology constituted by several lipid cores encapsulated into a polymeric shell. The applicability of these structured particles to be used as drug delivery system (DDS) was studied by using ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), as model drug. The particles provided an initial burst release of the drug due to the progressive dissolution of the external layer of PEG, followed by a controlled release of the NSAID from the GEL cores.
Keywords: Supercritical fluids; Particles from Gas Saturated Solutions; Emulsion; Multicore hybrid particles; Drug delivery systems;
A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy by Rong-Lin Xie; Yoon-jeong Jang; Lei Xing; Bing-Feng Zhang; Feng-Zhen Wang; Peng-Fei Cui; Myung-Haing Cho; Hu-Lin Jiang (19-30).
Display OmittedThe clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 μg/mL at different time (24 and 72 h), which much higher than PEI 25 K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras LA1 lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy.
Keywords: Citric acid; Hyperbranched polyspermine; Biocompatibility; Lung cancer; Aerosol delivery;
How to easily provide zero order release of freely soluble drugs from coated pellets by B. Dekyndt; J. Verin; C. Neut; F. Siepmann; J. Siepmann (31-38).
Display OmittedCoated pellets offer a great potential as controlled drug delivery systems. However, constant drug release rates are difficult to achieve with this type of dosage forms if the drug is freely water-soluble. This is because diffusional mass transport generally plays a major role and with time the drug concentration within the system decreases, resulting in decreased concentration gradients, which are the driving forces for drug release. Thus, generally “curve-shaped” release profiles with monotonically decreasing slopes are observed. This type of release kinetics might be inappropriate for an efficient and safe drug treatment. Despite the great practical importance of this potentially crucial formulation challenge, surprisingly little is yet known on how to effectively address it. In this study, a novel approach is presented based on sequential layers of drug and polymer (initially free of drug) to provide a non-homogeneous initial drug distribution, combined with lag-time effects, and partial initial drug diffusion towards the pellet’s core. Sugar and microcrystalline cellulose beads were used as starter cores, metoprolol succinate as freely soluble drug, ethylcellulose, and poly(vinyl acetate) as release rate controlling polymers. The type, number, thickness, and sequence of the drug and polymer layers were varied. Interestingly, a rather simple four layer system (two drug and two polymer layers) allowed providing about constant drug release during 8 h. Compared to previously proposed coated pellets aiming at constant release of freely water-soluble drugs based on non-homogeneous initial drug distribution, the total coating level in this study was very much reduced: to only about 20%. Hence, the suggested formulation approach is relatively simple and can help overcoming a potentially major hurdle in practice. Its applicability has also been demonstrated for another type of drug: propranolol hydrochloride.
Keywords: Film coating; Zero order release; Ethylcellulose; Poly(vinyl acetate); Pellet;
Topical formulation and dermal delivery of active phenolic compounds in the Thai medicinal plant – Clerodendrum petasites S. Moore by Premrutai Thitilertdecha; Michael G. Rowan; Richard H. Guy (39-45).
Display OmittedTo develop topical formulations of Clerodendrum petasites S. Moore (CP), and to optimize the skin permeability and topical bioavailability of the active phenolic compounds therein.The skin uptake and delivery of active compounds from two formulations, an oil-in-water cream and a lotion (both containing 10% w/w CP extracts), were examined (a) in vitro using pig skin, and (b) in vivo in human volunteers. Stratum corneum (SC) was collected by tape stripping and the compounds were detected and quantified by high performance liquid chromatography coupled with either mass spectrometry (HPLC–MS) or ultraviolet and photodiode array (HPLC–UV–PDA) detection.From the in vitro results, vanillic acid, verbascoside, nepetin and hispidulin, were chosen as potential phenolic actives for topical delivery optimization from the formulations. In vivo, vanillic acid, nepetin and hispidulin were appreciably taken up into the SC within 6 h, while verbascoside did not penetrate beyond the most superficial layers. No significant differences in delivery were observed between the two formulations, both of which were well tolerated.The detected topical absorption of hispidulin, nepetin, and vanillic acid, from the cream and lotion vehicles investigated, suggest that these compounds are potentially active compounds in Thai traditional medicine for the treatment of a wide range of skin diseases.
Keywords: Clerodendrum petasites S. Moore; Hispidulin; Skin permeation; Topical bioavailability; Mass spectrometry;
Counter-intuitive effect of non-crystallizing sugars on the crystallization of gemcitabine HCl in frozen solutions by Bhushan Munjal; Arvind K. Bansal (46-52).
Display OmittedIn this study, the effect of four non-crystallizing sugars, namely fructose, trehalose, sucrose and raffinose, was assessed on the crystallization of gemcitabine hydrochloride (GHCl) in frozen solutions. Aqueous solutions containing GHCl (50 mg/mL) and a sugar at varying concentrations (10–60 mg/mL) were frozen in situ in DSC and analyzed in the subsequent heating run. Crystallization propensity of GHCl was quantified in terms of reduced crystallization temperature (RCT) as a function of sugar type and concentration. Multivariate analysis option in JMP® software was employed for calculating correlation between the variables. All sugars inhibited GHCl crystallization in a concentration dependent manner. At equal concentration, fructose (with the lowest T g′) exerted the strongest inhibitory effect, whereas raffinose (with the highest T g′) exerted the weakest inhibitory effect. Additionally, RCT showed a poor correlation with T g′ (r = 0.2327). Thus, the inhibitory effect of sugars could not be described by their anti-plasticization effect. This counter-intuitive behavior was explained by the inhibitory effect of sugars on ice crystallization, which increased the unfrozen water content (UWC) in the freeze concentrate, thereby lowering the supersaturation of GHCl. This was established by observing a good correlation (r = 0.9666) between RCT and ln(1/UWC). Additionally, reduced diffusion kinetics of GHCl in presence of sugar molecules was also postulated. This study highlights the importance of unfrozen water towards governing the crystallization behavior of solutes in multi-component frozen systems.
Keywords: Unfrozen water; Calorimetry (DSC); Correlation; Supersaturation; Lyophilization; Bulking agent;
Extrinsic lactose fines improve dry powder inhaler formulation performance of a cohesive batch of budesonide via agglomerate formation and consequential co-deposition by Hanne Kinnunen; Gerald Hebbink; Harry Peters; Deborah Huck; Lisa Makein; Robert Price (53-59).
Display OmittedThe aim of the study was to investigate how the fine particle content of lactose carriers prepared with different types of lactose fines regulates dry powder inhaler (DPI) formulation performance of a cohesive batch of micronised budesonide. Budesonide formulations (0.8 wt%) were prepared with three different lactose carriers (Lactohale (LH) LH100, 20 wt% LH210 in LH100 and 20 wt% LH300 in LH100). Fine particle fraction of emitted dose (FPFED) and mean mass aerodynamic diameter (MMAD) of budesonide was assessed with a Next Generation Impactor (NGI) using a Cyclohaler at 90 l/min. Morphological and chemical characteristics of particles deposited on Stage 2 were determined using a Malvern Morphologi G3-ID. The results indicate that increasing concentration of lactose fines (<4.5 μm) not only increased the FPFED but also the MMAD of budesonide, suggesting drug deposition in agglomerates. Presence of agglomerates on Stage 2 was confirmed by morphological analysis of particles. Raman analysis of material collected on Stage 2 indicated that the more fine lactose particles were available the more agglomerates of budesonide and lactose were delivered to Stage 2. These results suggest drug-fines agglomerate formation is an important mechanism for how lactose fines improve and regulate DPI formulation performance.
Keywords: Lactose; Dry powder inhaler; Raman spectroscopy;
Optimization of process variables of zanamivir-loaded solid lipid nanoparticles and the prediction of their cellular transport in Caco-2 cell model by Li-Li Shi; Yue Cao; Xiao-Yin Zhu; Jing-Hao Cui; Qing-Ri Cao (60-69).
Display OmittedThe aim of this study was to optimize the process variables of zanamivir-loaded solid lipid nanoparticles (SLNs) and to predict their cellular transport across Caco-2 cell monolayers. Zanamivir-loaded SLNs were prepared by a double emulsion solvent evaporation method. The effects of process variables on the mean particle size and zeta potential of SLNs were investigated and the physicochemical properties of SLNs were characterized. In addition, the cytotoxicity and transport ability of SLNs were also studied in Caco-2 cell model. The mean particle size of drug-loaded SLNs was significantly affected by the process variables. The SLNs were prepared using glyceryl monosterate (1% polyvinyl alcohol (PVA) or 1.5% poloxamer 188 as surfactant) had a regular shape with a relatively lower mean particle size (324.2 and 224.9 nm, respectively), higher encapsulation efficacy (55.7 and 42.4%, respectively) and sustained drug release over 12 h. The crystalline form of drug had been partly changed and no physicochemical interaction had occurred between drug and carriers in SLNs. The cell viability was approximately 100% even at a high concentration of blank SLNs. There was no remarkable difference in drug permeation between drug-loaded SLNs and drug solution at 2 h, while drug-loaded SLNs showed a significant decrease in drug permeation compared with the drug solution at 4 h. The process variables of zanamivir-loaded SLNs were successfully optimized in this study. However, these SLNs significantly reduced the transport ability of zanamivir across Caco-2 cell monolayers. Further studies are needed to fully understand the real absorption mechanism of developed SLNs.
Keywords: Zanamivir; Solid lipid nanoparticles; Process variables; Cellular transport; Caco-2 cell;
A polycarboxylic/amino functionalized hyaluronic acid derivative for the production of pH sensible hydrogels in the prevention of bacterial adhesion on biomedical surfaces by Fabio Salvatore Palumbo; Antonella Bavuso Volpe; Maria Grazia Cusimano; Giovanna Pitarresi; Gaetano Giammona; Domenico Schillaci (70-77).
Display OmittedA graft copolymer derivative of hyaluronic acid bearing pendant amino and short polymethacrylate portions (HA-EDA-BMP-MANa) has been employed for the production of a pH sensible vancomycin releasing hydrogel and studied in vitro to test its potential anti adhesive property against Staphylococcus aureus colonization. The copolymer obtained through atom transfer radical polymerization bears chargeable (carboxyl and amino groups) portions and it could be formulated as a hydrogel at a concentration of 10% w/v. The HA-EDA-BMP-MANa hydrogels, produced at three different pH values (5, 6 and 7, respectively), were formulated with or without the addition of vancomycin (2% w/v). The vancomycin release profiles were detected and related to the starting hydrogel pH values, demonstrating that the systems were able to sustain the release of drug for more than 48 h. S. aureus adhesion tests were performed on glass culture plates and hydroxyapatite doped titanium surfaces, comparing the performances of HA-EDA-BMP-MANa hydrogel formulations (obtained with and without vancomycin) with similar formulations obtained using unmodified hyaluronic acid. The non fouling property of a selected HA-EDA-BMP-MANa hydrogel (without vancomycin) was also assayed with a BSA adsorption test. We found that the HA-EDA-BMP-MANa hydrogel even without vancomycin prevented bacterial adhesion on investigated surfaces.
Keywords: Hyaluronic acid; ATRP; Hydrogel; Non fouling; Vancomycin;
Microfluidic conceived pH sensitive core–shell particles for dual drug delivery by Ikram Ullah Khan; Lukas Stolch; Christophe A. Serra; Nicolas Anton; Roman Akasov; Thierry F. Vandamme (78-87).
Display OmittedIn current study, we report on the synthesis of core–shell microparticles for dual drug delivery by means of a two co-axial microfluidic device and online UV assisted free radical polymerization. Before developing pH-sensitive particles, ketoprofen loaded poly(methyl acrylate) core–ranitidine HCl loaded poly(acrylamide) shell particles were produced. Influence of inner and outer phases flow rates on particle size, shape, core diameter, shell thickness, and drug release properties was studied. All the particles were monodispersed with coefficient of variation below 5%. Furthermore, their diameter ranged from 100 to 151 μm by increasing continuous (Q c) to middle (Q m) phase flow rate ratio (Q c/Q m). Core diameter varied from 58 to 115 μm by decreasing middle (Q m) to inner (Q i) phase flow rate ratio (Q m/Q i) at constant continuous phase flow rate as confirmed by SEM images. It was observed that an optimum concentration of acrylamide (30 wt%) and an appropriate combination of surfactants were necessary to get core–shell particles otherwise Janus structure was obtained. FTIR confirmed the complete polymerization of core and shell phases. MTT assay showed variation in viability of cells under non-contact and contact conditions with less cytotoxicity for the former. Under non-contact conditions LD50 was 3.1 mg/mL. Release studies in USP phosphate buffer solution showed simultaneously release of ketoprofen and ranitidine HCl for non pH-sensitive particles. However, release rates of ranitidine HCl and ketoprofen were higher at low and high pH respectively. To develop pH-sensitive particles for colon targeting, the previous shell phase was admixed with few weight percentage of pH sensitive carboxyethyl acrylate monomer. Core and shell contained the same hydrophobic and hydrophilic model drugs as in previous case. The pH-sensitive shell prevented the release of the two entrapped molecules at low pH while increasing significantly their release rate at higher pH with a maximum discharge at colonic pH of 7.4.
Keywords: Microfluidics; Cytotoxicity; Dual drug delivery; Poly(acrylamide-co-carboxy ethyl acrylate); pH-sensitive.;
A substrate pharmacophore for the human sodium taurocholate co-transporting polypeptide by Zhongqi Dong; Sean Ekins; James E. Polli (88-95).
Display OmittedHuman sodium taurocholate co-transporting polypeptide (NTCP) is the main bile acid uptake transporter in the liver with the capability to translocate xenobiotics. While its inhibitor requirements have been recently characterized, its substrate requirements have not. The objectives of this study were (a) to elucidate NTCP substrate requirements using native bile acids and bile acid analogs, (b) to develop the first pharmacophore for NTCP substrates and compare it with the inhibitor pharmacophores, and (c) to identify additional NTCP novel substrates. Thus, 18 native bile acids and two bile acid conjugates were initially assessed for NTCP inhibition and/or uptake, which suggested a role of hydroxyl pattern and steric interaction in NTCP binding and translocation. A common feature pharmacophore for NTCP substrate uptake was developed, using 14 native bile acids and bile acid conjugates, yielding a model which featured three hydrophobes, one hydrogen bond donor, one negative ionizable feature and three excluded volumes. This model was used to search a database of FDA approved drugs and retrieved the majority of the known NTCP substrates. Among the retrieved drugs, irbesartan and losartan were identified as novel NTCP substrates, suggesting a potential role of NTCP in drug disposition.
Keywords: Bile acids; Sodium taurocholate co-transporting polypeptide (NTCP); Pharmacophore; Transporters; Irbesartan; Losartan;
Reduction of glycine particle size by impinging jet crystallization by Tímea Tari; Zoltán Fekete; Piroska Szabó-Révész; Zoltán Aigner (96-102).
Display OmittedThe parameters of crystallization processes determine the habit and particle size distribution of the products. A narrow particle size distribution and a small average particle size are crucial for the bioavailability of poorly water-soluble pharmacons. Thus, particle size reduction is often required during crystallization processes. Impinging jet crystallization is a method that results in a product with a reduced particle size due to the homogeneous and high degree of supersaturation at the impingement point.In this work, the applicability of the impinging jet technique as a new approach in crystallization was investigated for the antisolvent crystallization of glycine. A factorial design was applied to choose the relevant crystallization factors. The results were analysed by means of a statistical program. The particle size distribution of the crystallized products was investigated with a laser diffraction particle size analyser. The roundness and morphology were determined with the use of a light microscopic image analysis system and a scanning electron microscope. Polymorphism was characterized by differential scanning calorimetry and powder X-ray diffraction. Headspace gas chromatography was utilized to determine the residual solvent content.Impinging jet crystallization proved to reduce the particle size of glycine. The particle size distribution was appropriate, and the average particle size was an order of magnitude smaller (d(0.5) = 8–35 μm) than that achieved with conventional crystallization (d(0.5) = 82–680 μm). The polymorphic forms of the products were influenced by the solvent ratio. The quantity of residual solvent in the crystallized products was in compliance with the requirements of the International Conference on Harmonization.
Keywords: Impinging jet crystallization; Particle size; Glycine; Crystal habit; Polymorphism; Residual solvent content;
Improving the API dissolution rate during pharmaceutical hot-melt extrusion I: Effect of the API particle size, and the co-rotating, twin-screw extruder screw configuration on the API dissolution rate by Meng Li; Costas G. Gogos; Nicolas Ioannidis (103-112).
Display OmittedThe dissolution rate of the active pharmaceutical ingredients in pharmaceutical hot-melt extrusion is the most critical elementary step during the extrusion of amorphous solid solutions – total dissolution has to be achieved within the short residence time in the extruder. Dissolution and dissolution rates are affected by process, material and equipment variables. In this work, we examine the effect of one of the material variables and one of the equipment variables, namely, the API particle size and extruder screw configuration on the API dissolution rate, in a co-rotating, twin-screw extruder. By rapidly removing the extruder screws from the barrel after achieving a steady state, we collected samples along the length of the extruder screws that were characterized by polarized optical microscopy (POM) and differential scanning calorimetry (DSC) to determine the amount of undissolved API. Analyses of samples indicate that reduction of particle size of the API and appropriate selection of screw design can markedly improve the dissolution rate of the API during extrusion. In addition, angle of repose measurements and light microscopy images show that the reduction of particle size of the API can improve the flowability of the physical mixture feed and the adhesiveness between its components, respectively, through dry coating of the polymer particles by the API particles.
Keywords: Hot-melt extrusion; Particle size; Screw configuration; Dissolution rate; Flowability; Amorphous solid dispersions;
Novel non-viral gene delivery systems composed of carbosilane dendron functionalized nanoparticles prepared from nano-emulsions as non-viral carriers for antisense oligonucleotides by Cristina Fornaguera; Santiago Grijalvo; Marta Galán; Elena Fuentes-Paniagua; Francisco Javier de la Mata; Rafael Gómez; Ramon Eritja; Gabriela Calderó; Conxita Solans (113-123).
Schematic representation of the complete process of nanoparticle production from nano-emulsion and its surface functionalization to be used as non-viral gene delivery vectors.Display OmittedThe development of novel and efficient delivery systems is often the limiting step in fields such as antisense therapies. In this context, poly(d,l-lactide-co-glycolide) acid (PLGA) nanoparticles have been obtained by a versatile and simple technology based on nano-emulsion templating and low-energy emulsification methods, performed in mild conditions, providing good size control. O/W polymeric nano-emulsions were prepared by the phase inversion composition method at 25 °C using the aqueous solution/polysorbate80/[4 wt% PLGA in ethyl acetate] system. Nano-emulsions formed at oil-to-surfactant (O/S) ratios between 10/90–90/10 and aqueous contents above 70 wt%. Nano-emulsion with 90 wt% of aqueous solution and O/S ratio of 70/30 was chosen for further studies, since they showed the appropriate characteristics to be used as nanoparticle template: hydrodynamic radii lower than 50 nm and enough kinetic stability. Nanoparticles, prepared from nano-emulsions by solvent evaporation, showed spherical shape, sizes about 40 nm, negative surface charges and high stability. The as-prepared nanoparticles were functionalized with carbosilane cationic dendrons through a carbodiimide-mediated reaction achieving positively charged surfaces. Antisense oligonucleotides were electrostatically attached to nanoparticles surface to perform gene-silencing studies. These complexes were non-haemolytic and non-cytotoxic at the concentrations required. The ability of the complexes to impart cellular uptake was also promising. Therefore, these novel nanoparticulate complexes might be considered as potential non-viral carriers in antisense therapy.
Keywords: Nano-emulsion; PIC emulsification method; PLGA nanoparticles; Cationic dendron; Non-viral gene delivery systems; Antisense therapy;
Self-assembled drug delivery systems. Part 8: In vitro/in vivo studies of the nanoassemblies of cholesteryl-phosphonyl gemcitabine by Miao Li; Shuo Qi; Yiguang Jin; Junxing Dong (124-130).
Display OmittedA lipid derivative of gemcitabine (Gem), cholesteryl-phosphonyl gemcitabine (CPNG) was synthesized in this study. The amphiphilicity of CPNG was confirmed using a Langmuir monolayer method. Nanoassemblies were formed when the mixture of CPNG and a long-circulating material, CHS-PEG1500 (9:1, mol/mol) were injected into water. The nanoassemblies could be spherical vesicles according to the transmission electron microscopic images. Their mean size was 71.1 nm and the zeta potential was −17.6 mV. CPNG maintained stable in the weakly acidic and neutral environments although mouse plasma quickly degraded CPNG. The cytotoxicity of the nanoassemblies was 3–6 folds of Gem's cytotoxicity on five human cancer cell lines including 95C, 95D, A549, SW620, PANC-1 probably because of the phosphonyl substitution and amphiphilicity of CPNG. CPNG mainly distributed into the mononuclear macrophage system (including liver and spleen) after bolus intravenous administration of the nanoassemblies into mice though the expected significant long-circulating effect was not shown. The nanoassemblies with the high dose of CPNG showed the statistically higher in vivo anticancer effect than Gem. This study indicates that the N-substituted lipid derivative of Gem and the true long-circulating function are necessary for preparing a successful nanoassembly of Gem.
Keywords: Gemcitabine; Molecular self-assembly; Nanoassemblies; Phosphonate; Prodrug;
Preparation and in vitro–in vivo evaluation of teniposide nanosuspensions by Suna He; Hui Yang; Ruizhi Zhang; Yan Li; Lengxin Duan (131-137).
Display OmittedTeniposide (TEN) is a potent, broad spectrum antitumor agent, especially for cerebroma. But the application in clinic was limited because of its poor solubility. In this paper, teniposide nanosuspensions drug delivery system (TEN-NSDDS) for intravenous administration was developed for the first time. Specifically, TEN nanosuspensions were prepared by an anti-solvent sonication–precipitation method and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. TEN nanosuspensions prepared showed rod-like morphology and the size was 151 ± 11 nm with a narrow poly dispersion index 0.138 determined by dynamic light scattering. The obtained TEN nanosuspensions were physically stable at least 10 days at 4 °C. And the freeze-drying preparations were stable during 3 months. The cytotoxicity of TEN nanosuspensions were considerable to that of VUMON against U87MG and C6 cells in vitro. When tested in rats bearing C6 tumors, the TEN concentration in the tumors treated by the nanosuspensions was more than 20 times than that by the TEN solution at 2 h. The TEN nanosuspensions exhibited significant tumor growth inhibition. Overall, the results suggested that nanosuspensions was an alternative formulation for teniposide to be administered intravenously, and it would be a promising formulation in clinic.
Keywords: Teniposide; Nanosuspensions; Anti-solvent sonication–precipitation method; Stability; Antitumor activity;
Evaluation of transdermal salidroside delivery using niosomes via in vitro cellular uptake by Yongtai Zhang; Kai Zhang; Zhonghua Wu; Teng Guo; Beini Ye; Mingyun Lu; Jihui Zhao; Chunyun Zhu; Nianping Feng (138-146).
Display OmittedSpan 40-based niosomes were employed as nanocarriers to improve cutaneous absorption of salidroside. The niosomal formulation with a molar proportion of Span 40 to cholesterol of 4:3 showed the highest transdermal flux and skin deposition of salidroside. The transdermal flux of the 4:3 niosomal formulation was significantly greater than that of the aqueous solution. Salidroside-loaded niosomes showed good biocompatibility with skin tissue, human epidermal immortal keratinocytes (HaCaT), and human embryonic skin fibroblasts (CCC-ESF). The fluorescence intensity of HaCaT cells after uptake of coumarin 6-labeled niosomes was similar to that observed after uptake of the aqueous suspension. The fluorescence intensity of CCC-ESF cells was greater than that of the aqueous suspension after incubation for 10 min, but was not significantly different after 60 min. Further investigation revealed that internalization of niosomes by HaCaT cells may be achieved through pinocytotic vesicles and macropinocytosis, which consumes energy, rather than via lysosomes. In CCC-ESF cells, pinocytotic vesicles and lysosomes were both important mediators of endocytosis. The niosome formulations reported here could improve the dermal and transdermal salidroside delivery, and the in vitro cell uptake evaluation results serve as a basis for further research into the mechanisms through which niosomes enhance drug permeability.
Keywords: Nanocarrier; Topical administration; Cellular uptake; Endocytosis; Biocompatibility;
The enhancement of gene silencing efficiency with chitosan-coated liposome formulations of siRNAs targeting HIF-1α and VEGF by Emine Şalva; Suna Özbaş Turan; Fatih Eren; Jülide Akbuğa (147-154).
Display OmittedRNA interference (RNAi) holds considerable promise as a novel therapeutic strategy in the silencing of disease-causing genes. The development of effective delivery systems is important for the use of small interfering RNA (siRNA) as therapy. In the present study, we investigated the effect on breast cancer cell lines and the co-delivery of liposomes containing siHIF1-α and siVEGF. In order to achieve the co-delivery of siHIF1-α and siVEGF and to obtain lower cytotoxicity, higher transfection and silencing efficiency, in this study, we used chitosan-coated liposomal formulation as the siRNA delivery system. The obtained particle size and zeta potential values show that the chitosan coating process is an effective parameter for particle size and the zeta potential of liposomes. The liposome formulations loaded with siHIF1-α and siVEGF showed good stability and protected siRNA from serum degradation after 24-h of incubation. The expression level of VEGF mRNA was markedly suppressed in MCF-7 and MDA-MB435 cells transfected with chitosan-coated liposomes containing HIF1-α and VEGF siRNA, respectively (95% and 94%). In vitro co-delivery of siVEGF and siHIF1-α using chitosan-coated liposome significantly inhibited VEGF (89%) and the HIF1-α (62%) protein expression when compared to other liposome formulations in the MDA-MB435 cell. The co-delivery of siVEGF and siHIF1-α was greatly enhanced in the vitro gene silencing efficiency. In addition, chitosan-coated liposomes showed 96% cell viability. Considering the role of VEGF and HIF1-α in breast cancer, siRNA-based therapies with chitosan coated liposomes may have some promises in cancer therapy.
Keywords: siRNA; Chitosan; Liposome; VEGF; HIF-1α; Breast cancer;
Orodispersible films in individualized pharmacotherapy: The development of a formulation for pharmacy preparations by J. Carolina Visser; Herman J. Woerdenbag; Stefan Crediet; Edwin Gerrits; Marjan A. Lesschen; Wouter L.J. Hinrichs; Jörg Breitkreutz; Henderik W. Frijlink (155-163).
Display OmittedOrodispersible films (ODFs) are promising drug delivery systems for customized small scale pharmacy preparations. The aim of the present study was to develop a versatile casting solution suitable for the extemporaneous production of ODFs to which active pharmaceutical ingredients (APIs) can be added. Different combinations of film forming agents and other excipients and different casting heights were tested for their suitability for production of ODFs. The best suitable casting solution contained hypromellose, carbomer, glycerol, disodium EDTA and trometamol. This casting solution was used to prepare ODFs containing water-soluble APIs (enalapril maleate and prednisolone disodium phosphate) and a poorly water-soluble API (diazepam) for which ethanol 96% was used as co-solvent.The water-soluble APIs as well as ethanol influenced the viscosity of the casting solution, mechanical properties and disintegration time of the ODFs. All ODFs containing API met the requirements on uniformity of mass and uniformity of content set by the European Pharmacopoeia (2014) (Ph. Eur.) 8th edition. In conclusion, ODFs of good pharmaceutical quality can be prepared on small scale. Hereby opening the perspective of using ODFs for individualized pharmacotherapy.
Keywords: Orodispersible films; Casting solution; Extemporaneous pharmacy preparations; (Poorly) water-soluble API; Mechanical properties; Disintegration;
The influence of the adsorption of metoclopramide on the surface ionization of fumed silica by Tuba Buyuktimkin; Dale Eric Wurster (164-171).
Display OmittedThe effect of adsorbed metoclopramide on the surface ionization of fumed silica was studied using potentiometric titration. Adsorption isotherms of metoclopramide to unionized and negatively-charged silica surfaces were generated and compared to the titration data. The adsorption of metoclopramide caused the silica surface charge to become more negative with increasing pH that was independent of ionic strength which suggested that specific adsorbate-surface interactions were occurring. Adsorption studies showed that metoclopramide adsorbs to the unionized silica surface. Ionization caused drug adsorption to increase which was consistent with at least two distinct surface adsorption sites. The ratio of the additional amount of metoclopramide adsorbed to the surface ionized group density determined from the titration curves was approximately unity which showed conclusively that the negatively-charged silanols constitute one of the surface adsorption sites. Potentiometric titration has been shown to be a useful technique for determining the number and types of adsorption sites on the silica surface.
Keywords: Surface charge; Fumed silica; Metoclopramide; Silanol; Potentiometric titration;
Formation of hydrophilic nanofibers from nanoemulsions through electrospinning by V. Gordon; G. Marom; S. Magdassi (172-179).
Display OmittedThis study presents a method for one step incorporation of lipophilic compounds in hydrophilic nanofibers. By this method nanodroplets of oil and of volatile solvent are entrapped within polymer nanofibers during an electrospinning process. While performing the process with a volatile oil with dissolved lipophilic material, such as the drug celecoxib, nanofiber–nanoparticle composites are formed. The polymer used to form the fibers is a high molecular weight poly(vinyl alcohol) which enables rapid dissolution and release of the incorporated lipophilic material. The resulting celecoxib nanoparticles that are embedded within the nanofiber are amorphous and their average size is in between 21 and 93 nm, thus potentially lead to their increased dissolution rate. The preparation of such a solid matrix containing nanodroplets or nanoparticles may be applied as a fast dissolving delivery system for water insoluble materials.
Keywords: Electrospinning; Fast dissolving delivery system; Emulsion; Nanofibers; Lipophilic nanoparticles;
Cationic solid lipid nanoparticles enhance ocular hypotensive effect of melatonin in rabbit by Antonio Leonardi; Claudio Bucolo; Filippo Drago; Salvatore Salomone; Rosario Pignatello (180-186).
Display OmittedThe study was aimed at evaluating whether the ocular hypotensive effect of melatonin (MEL) was enhanced by its encapsulation in cationic solid lipid nanoparticles (cSLN), as well as at determining the tolerability of these formulations on the ocular surface. MEL was loaded in cSLN that had already been shown to be suitable for ophthalmic use. The formulations were prepared using Softisan® 100 as the main lipid matrix, with the presence of either stearic (SA) or palmitic acid (PA) as lipid modifiers. A fixed positive charge was provided by the addition of a cationic lipid (didecyldimethylammonium bromide). The ocular hypotensive effect was evaluated by measuring the intraocular pressure (IOP) during 24 h in albino rabbits. MEL elicited a significant (p < 0.01) IOP reduction in rabbit eye. All the formulations tested in vivo demonstrated a good tolerability. The nanocarrier containing SA was the most effective in terms of IOP reduction (maximum IOP reduction: −7 mmHg), and its effect lasted approximately 24 h.The experimental data indicate that the new formulations based on cSLN loaded with MEL represent a potent anti-glaucoma treatment with a safe profile, warranting further clinical evaluation of the proposed nanotechnological strategy.
Keywords: Melatonin; Nanoparticles; Eye drops; Glaucoma; Intraocular pressure;
Stability studies of As4S4 nanosuspension prepared by wet milling in Poloxamer 407 by Zdenka Bujňáková; Erika Dutková; Matej Baláž; Erika Turianicová; Peter Baláž (187-192).
Display OmittedIn this paper the stability of the arsenic sulfide (As4S4) nanosuspension prepared by wet milling in a circulation mill in the environment of copolymer Poloxamer 407 was studied. The obtained As4S4 particles in nanosuspension were of ∼100 nm in size. The influence of temperature and UV irradiation on the changes in physical and/or chemical properties was followed. Long-term stability was observed via particle size distribution and zeta potential measurements. Influence of UV irradiation was studied via UV–vis spectroscopy (UV–vis), photoluminicsence (PL) technique and Fourier transform infrared spectroscopy (FTIR) measurements. The best stability of the nanosuspension (24 weeks) was achieved when stored at 4 °C and in the dark.
Keywords: Nanosuspension; Stability; Nanomilling; Arsenic sulfide; Particle size;
Preparation and characterization of spironolactone-loaded nano-emulsions for extemporaneous applications by François Hallouard; Gilles Dollo; Nolwenn Brandhonneur; Fabien Grasset; Pascal Le Corre (193-201).
Display OmittedIn neonates as well as in adults having swallowing difficulty, oral medication is given through a nasogastric tube making liquid formulations preferable. In this study, we present the high potential of nanometric emulsions formulated by spontaneous surfactant diffusion, as extemporaneous formulations of hydrophobic drug. Spironolactone used as hydrophobic drug model, was incorporated in oil before formulation at a concentration of 13.5 mg/g oil. Then, all formulations were evaluated from pharmacotechnical and clinical standpoints, for their use in hospital or community pharmacy. The strength of this new liquid formulation lies on the simplicity, efficiency and reproducibility of their low energy process as on clinical aspects: high dose uniformity, facility to be administered through in nasogastric tube without any retention and a stability of 2 months at least compatible for an extemporaneous use. Moreover, this emulsion presented spironolactone content of 3.75 mg/ml among the most concentrated formulations published.
Keywords: Nano-emulsions; Nasogastric tube; Spironolactone; Spontaneous surfactant diffusion; Swallowing difficulty;
Formulation, characterization and anti-malarial activity of homolipid-based artemether microparticles by Chukwuma O. Agubata; Ifeanyi T. Nzekwe; Anthony A. Attama; Christel C. Mueller-Goymann; Godswill C. Onunkwo (202-222).
Display OmittedThe anti-malarial activity of artemether is dependent on its bioavailability. The purpose of the research is to improve the solubility, bioavailability and therapeutic efficacy of lipophilic artemether using homolipid-based microparticles. Irvingia fat was extracted from Irvingia gabonensis var. excelsa (Irvingia wombolu), and its lipid matrices (LM) with Phospholipon® 90G (P90G) were characterized by differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). Solid lipid microparticles were formulated, characterized, filled and compressed into capsules and tablets, respectively, and drug release studied. In vivo anti-plasmodial activity of artemether SLMs was evaluated in mice. The crystallinity of the phyto-lipid reduced in the presence of P90G, which was integrated into the irvingia fat crystal lattice. SLM dispersions with 3:1 irvingia fat/P90G composition showed higher diffusion and permeability through dialysis membrane while lower proportion of P90G (9:1 LM) favored increased dissolution rate of artemether from capsules (p < 0.05). Significant increase (p < 0.05) in % plasmodial growth inhibition and reduced parasitemia were observed in mice administered with the SLM dispersions compared with the controls. Therefore, SLMs prepared with composite mixtures of a homolipid and P90G could be used to improve the solubility, dissolution, permeability, bioavailability and anti-malarial efficacy of artemether.
Keywords: Homolipid; Microparticles; Anti-malarial activity;
Population pharmacokinetics of ketamine in children with heart disease by Mohammed H. Elkomy; David R. Drover; Gregory B. Hammer; Jeffery L. Galinkin; Chandra Ramamoorthy (223-231).
Display OmittedThis study aims at developing a population pharmacokinetic model for ketamine in children with cardiac diseases in order to rationalize an effective 2-h anesthetic medication, personalized based on cardiac function and age. Twenty-one children (6 months to 18 years old) were enrolled in this prospective, open label study. Ketamine 2 mg/kg IV was administered and blood samples were then collected over 8 h for ketamine assay. Pharmacokinetic data analysis using NONMEM, was undertaken. Ketamine pharmacokinetics was adequately described by a two-compartment linear disposition model. Typical population parameters were: total clearance: 60.6 × (weight/70)0.75 L/h, intercompartmental clearance: 73.2 × (weight/70)0.75 L/h, central distribution volume: 57.3 × (weight/70) L, and peripheral distribution volume: 152 × (weight/70) L. Ketamine clearance in children with pre-existing congenital heart disease was comparable to values reported in healthy subjects. Computer simulations indicated that an initial loading dose of ketamine 2 mg/kg IV over 1 min followed by a constant rate infusion of 6.3 mg/kg/h for 29 min, 4.5 mg/kg/h from 30 to 80 min, and 3.9 mg/kg/h from 80 to 120 min achieves and maintains anesthetic plasma level for 2 h in children 1 year or older (weight ≥10 kg).
Keywords: Children; Ketamine; Pharmacokinetics; NONMEM;
Effect of carbamazepine on viscoelastic properties and hot melt extrudability of Soluplus® by Simerdeep Singh Gupta; Tapan Parikh; Anuprabha K. Meena; Nidhi Mahajan; Imre Vitez; Abu T.M. Serajuddin (232-239).
Display OmittedThe purpose of this study was to apply viscoelastic properties of polymer and drug-polymer mixtures to determine processing conditions for the preparation of amorphous solid dispersion by melt extrusion. A poorly water-soluble drug, carbamazepine (CBZ), was mixed with Soluplus® as the carrier. Torque analysis using a melt extruder was performed at 10, 20 and 30% w/w drug concentrations and the effect of barrel temperature was studied. Viscosity of the mixtures either at fixed temperatures with different angular frequencies or as a function of temperature with the same frequency was studied using a rheometer. The viscosity of Soluplus® and the torque exerted on the twin screws decreased with the increase in CBZ concentration. The viscosity versus temperature plots for different CBZ concentrations were parallel to each other, without the drug melting transition, indicating complete drug-polymer miscibility. Thus, the drug-polymer mixtures could be extruded at temperature as low as 140 °C with 10% w/w drug load, 135 °C with 20% w/w drug and 125 °C with 30% w/w drug, which were, respectively, ∼50 °C, 55 °C and 65 °C below the melting point of 191 °C for CBZ. The differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD) analyses of the binary mixtures extruded at 125–150 °C showed absence of crystalline drug. A systematic study of miscibility and extrudability of drug-polymer mixtures by rheological and torque analysis as a function of temperature will help formulators select optimal melt extrusion processing conditions to develop solid dispersions.
Keywords: Hot melt extrusion; Rheology; Viscosity; Polymer; Drug-polymer miscibility; Torque analysis; Soluplus®;
Tumor homing cell penetrating peptide decorated nanoparticles used for enhancing tumor targeting delivery and therapy by Huile Gao; Qianyu Zhang; Yuting Yang; Xinguo Jiang; Qin He (240-250).
Display OmittedSpecific targeting ability and good tissue penetration are two critical requirements for tumor targeted delivery systems. Systematical selected peptides from a library may meet these two requirements. RLW was such a cell penetrating peptide that could specifically target to non-small cell lung cancer cells (A549). In this study, RLW was linked onto nanoparticles (RNPs) and then the RNPs were used for lung cancer targeting delivery. A traditional cell penetrating peptide, R8 (RRRRRRRR), was used as control. In vitro cellular uptake study demonstrated that modification with RLW specifically enhanced the uptake by A549 cells rather than human umbilical vein endothelial cells, while modification with R8 increased the uptake by both cells. Furthermore, the modification with RLW specifically elevated the penetration into A549 tumor spheroids rather than glioma cell (U87, used as in vivo control) spheroids. And the in vivo imaging further demonstrated RNPs could target to A549 xenografts rather than U87 xenografts. Importantly, the distribution of RNPs in normal organs was approximately the same as that of unmodified nanoparticles. However, R8 modified nanoparticles elevated the distribution in almost all the tissues. These results demonstrated that RLW was superior in A549 tumor targeted delivery. After loaded with docetaxel, an anti-microtube agent, different formulations could effectively induce the A549 cell apoptosis, and inhibit the growth of A549 spheroids in vitro. While in vivo, RNPs displayed the best antitumor effect. The tumor volume was significantly lower than other groups, which was only 33.3% as that of saline group. In conclusion, in vitro RLW could specifically target to A549 cells and enhance the cytotoxicity of docetaxel. In vivo, RLW could significantly enhance the A549 xenografts targeting delivery and led to improved antitumor effect.
Keywords: Tumor homing; Cell penetrating peptide; Non-small cell lung carcinoma; Targeted drug delivery;
Resveratrol-loaded polymeric nanoparticles suppress glucose metabolism and tumor growth in vitro and in vivo by Kyung-Ho Jung; Jin Hee Lee; Jin Won Park; Cung Hoa Thien Quach; Seung-Hwan Moon; Young Seok Cho; Kyung-Han Lee (251-257).
Display OmittedResveratrol (RSV) is a natural phenol with promising anti-tumor activities, but its use for in vivo cancer treatment is limited by low aqueous solubility and poor stability. In this study, we prepared RSV-loaded polyethylene glycol–polylactic acid (PEG–PLA; M.W. 5000-5000) polymer nanoparticles (NPs) for improved stability and controlled delivery, and investigated its metabolic and anti-tumor effect in vitro and in vivo. CT26 colon cancer cells displayed significantly reduced cell number to 5.6% and colony forming capacity to 6.3% of controls by 72 h treatment with 40 and 20 μM of RSV-NP, respectively. Flow cytometry and western blots demonstrated increased apoptotic cell death, and 18F FDG uptake and reactive oxygen species was significantly reduced by RSV-NP. All of these effects were comparable to or greater in potency compared to free RSV. When RSV-NP was intravenously administered to CT26 tumor bearing mice, there was a reduction of 18F FDG uptake on PET/CT by day 4. Longer treatment led to retardation of tumor growth accompanied by an improvement in survival compared to empty NP-injected controls. These results demonstrate that the in vitro and in vivo metabolic and anti-tumor effects of RSV is preserved by PEG–PLA NP loading, and provide an encouraging outlook on the potential of polymeric NPs as an effective method to deliver RSV for cancer therapy.
Keywords: Resveratrol; PEG–PLA; Glucose metabolism; Anticancer effect;
Intestinal uptake and toxicity evaluation of acetazolamide and its multicomponent complexes with hidroxypropyl-β-cyclodextrin in rats by María J. Mora; Juan P. Petiti; Marcela R. Longhi; Alicia I. Torres; Gladys E. Granero (258-267).
Display OmittedLarge oral doses of ACZ lower the intraocular pressure (IOP), but usually lead to a multitude of systemic side effects, including gastrointestinal upset. The present study was undertaken to evaluate the effect of ACZ on the histological structure of rat duodenal mucosa and to assess a possible protective role of the complex formation of ACZ with HP-β-CD, either separately or in combination with a third compound, on the gut epithelial layer by histological and ultrastructural examinations of sections of rat duodenum exposed to ACZ or its formulations. In addition, the transport process of ACZ and its binary or ternary complexes across the duodenal mucosa by means of the single-pass intestinal perfusion (SPIP) method in rats was evaluated. Evidence was found that ACZ alters intestinal permeability and induces damage to the rat small intestine. In contrast, ACZ-induced intestinal injury may be abrogated by ACZ complexation. In addition, the complexation of ACZ with HP-β-CD, alone or in combination with a third compound, facilitated significant levels of ACZ uptake across the rat duodenal segment. Ternary complexes of ACZ with HP-β-CD in combination with TEA (triethanolamine) or calcium ions were found to provide an excellent approach that enabled an increased apparent permeability of ACZ across the duodenal epithelium, with a concomitant ability to preserve the integrity of the gut epithelium from ACZ-induced injury. These results could be useful for the design and development of novel ACZ formulations that can reduce GI toxicity, while still maintaining their essential therapeutic efficacies.
Keywords: Acetozolamide; Cyclodextrins; Multicomponent complexes; Gut intestinal epithelium injury; Permeability;
Matrix based system of isotretinoin as nail lacquer to enhance transungal delivery across human nail plate by Monika Joshi; Vijay Sharma; Kamla Pathak (268-277).
Display OmittedThe project was aimed at development of isotretinoin nail lacquer and assessment of its penetration efficiency across human nail plate. Preliminary studies (hydration enhancement factor and SEM) aided the selection of thioglycolic acid as permeation and eugenol was selected as local anesthetic in the formulation. The nail lacquer was optimized by 32 factorial design and a total of nine formulations were prepared and screened. In vitro adhesion and ex vivo permeation (cumulative drug permeation per unit area (CDP/A) = 6.61 ± 0.57 mg/cm2) across bovine hoof guided the selection of F3 as optimized formulation that was improvised. Viscosity adjustments to improve handling characteristics were affected by incorporation of ethyl cellulose (6%; F3M1) that scaled the viscosity to 312.681 cp and insignificantly (p > 0.05) affected CDP/A (6.32 ± 0.45 mg/cm2). In comparison to marketed preparation (Retino-A cream) F3M1 afforded two fold increase in CDP/A. The permeation characteristics were defined by Higuchi model (r 2 = 0.964) and flux value of 176 μg/cm2/h. Confocal laser scanning microscopy, after 72 h of nail lacquer application, revealed extensive distribution of the fluorescent tracer across the human nail plate in comparison to control that was confined to the top layer. Conclusively, an efficacious and stable nail lacquer of isotretinoin was developed for potential clinical topical use to target the drug to nail bed in treatment of nail psoriasis.
Keywords: Isotretenoin; Nail lacquer; Nail plate permeation;
Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells by Luca Flebus; François Lombart; Chantal Sevrin; Jean-Olivier Defraigne; Pierre Peters; Ladan Parhamifar; Daniel G.M. Molin; Christian Grandfils (278-287).
Display OmittedPoly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA.In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein.The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood–brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p < 0.05) inhibition (40%) in presence of 2-dexoxy-d-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones.As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.
Keywords: Functional polymers; Polycations; Poly(2-dimethylamino-ethylmethacrylate); Biomedical applications; Cell trafficking;
Design of salmon calcitonin particles for nasal delivery using spray-drying and novel supercritical fluid-assisted spray-drying processes by Wonkyung Cho; Min-Soo Kim; Min-Sook Jung; Junsung Park; Kwang-Ho Cha; Jeong-Soo Kim; Hee Jun Park; Amjad Alhalaweh; Sitaram P. Velaga; Sung-Joo Hwang (288-296).
Display OmittedThe overall aim of this study was to prepare a nasal powder formulation of salmon calcitonin (sCT) using an absorption enhancer to improve its bioavailability. In this work, powder formulations for nasal delivery of sCT were studied using various absorption enhancers and stabilizers. Powders were prepared by two different methods: conventional spray-drying (SD) and novel supercritical fluid-assisted spray-drying (SASD) to investigate the role of CO2 in the particle formation process. The prepared sCT powder formulations were characterized by several analyses; powder X-ray diffractometry (PXRD), scanning electron microscopy (SEM), and the Fourier transform infrared (FT-IR) spectroscopy method. The particle size distribution was also evaluated. In vivo absorption tests were carried out in Sprague-Dawley rat using the prepared powder formulations, and the results were compared to those of raw sCT. Quantitative analysis by high-performance liquid chromatography (HPLC) indicated that sCT was chemically stable after both the SD and SASD processes. Results of PXRD, SEM, and FT-IR did not indicate a strong interaction or defragmentation of sCT. The in vivo absorption test showed that SD- and SASD-processed sCT powders increased the bioavailability of the drug when compared to the nasal administration of raw sCT. In addition, SASD-processed sCT exhibited higher nasal absorption when compared with SD-processed sCT in all formulations due to a reduction of particle size. The results from this study illustrate that the preparation of nasal powders using the SASD process could be a promising approach to improve nasal absorption of sCT.
Keywords: Salmon calcitonin; Supercritical fluid; Nanoparticle; Nasal; Formulation;
Matrix tablets for sustained release of repaglinide: Preparation, pharmacokinetics and hypoglycemic activity in beagle dogs by Wei He; Mengmeng Wu; Shiqing Huang; Lifang Yin (297-307).
Display OmittedRepaglinide (RG) is an efficient antihyperglycemic drug; however, due to its short half-life, patients are required to take the marketed products several times a day, which compromises the therapeutic effects. The present study was conducted to develop a hydrophilic sustained release matrix tablet for RG with the aims of prolonging its action time, reducing the required administration times and side effects and improving patient adherence. The matrix tablets were fabricated by a direct compression method, the optimized formulation for which was obtained by screening the factors that affected the drug release. Moreover, studies of the pharmacokinetics and hypoglycemic activity as measured by glucose assay kits were performed in dogs. Sustained drug releases profiles over 10 h and a reduced influence of medium pHs on release were achieved with the optimized formulation; moreover, the in vivo performance of extended release formulation was also examined, and better absorption, a one-fold decrease in C max, a two-fold increase of T max and a prolonged hypoglycemic effect compared to the marketed product were observed. In conclusion, sustained RG release and prolonged action were observed with present matrix tablets, which therefore provide a promising formulation for T2D patients who require long-term treatment.
Keywords: Repaglinide; Sustained release tablets; Pharmacokinetics; In vitro/in vivo correlation; Hypoglycemic effect;
Gemcitabine and γ-cyclodextrin/docetaxel inclusion complex-loaded liposome for highly effective combinational therapy of osteosarcoma by Liang Sun; Dong-Sheng Zhou; Peng Zhang; Qing-Hu Li; Ping Liu (308-317).
Display OmittedThe aim of the present investigation was to formulate a docetaxel (DTX) and gemcitabine (GEM) co-loaded PEGylated liposome (DTX/GEM-L) to increase the therapeutic efficacy in osteosarcoma (OS). 2-Hydroxypropyl-γ-cyclodextrin/DTX inclusion complex was made to increase DTX aqueous solubility. DTX/GEM-L was characterized for morphological shape and size parameters. Release study showed a sustained release pattern for both the drugs. The nanocarriers based combinational drug significantly increased the cytotoxic effect than the free drug combination at the same concentration. The cell cycle analysis showed a predominant G2/M phase arrest for combinational drug. Importantly, more than 20% of cells were in late apoptosis chamber for DTX/GEM-L treatment with significant proportion of cells in the early apoptosis and necrotic phases. The antitumor efficacy was tested in MG63 cancer cell bearing xenograft nude mice. Results showed that DTX/GEM-L significantly reduced the tumor burden comparing to that of free combination cocktail. The PEGylated liposome successfully delivered the anticancer drugs in the osteosarcoma tumor interstitial spaces via EPR effect. DTX/GEM-L showed excellent safety profile along with the remarkable tumor suppression ability. Overall, results suggest that nanocarriers-based delivery system remarkably enhanced the apoptosis and cytotoxicity and increased the potency of combinational drug regimen.
Keywords: Osteosarcoma; Liposome; Docetaxel; Gemcitabine; Cyclodextrin;
Evaluation of the coat quality of sustained release pellets by individual pellet dissolution methodology by Min Xu; Celine Valeria Liew; Paul Wan Sia Heng (318-327).
Display OmittedThis study explored the application of 400-DS dissolution apparatus 7 for individual pellet dissolution methodology by a design of experiment approach and compared its capability with that of the USP dissolution apparatus 1 and 2 for differentiating the coat quality of sustained release pellets. Drug loaded pellets were prepared by extrusion–spheronization from powder blends comprising 50%, w/w metformin, 25%, w/w microcrystalline cellulose and 25%, w/w lactose, and then coated with ethyl cellulose to produce sustained release pellets with 8% and 10%, w/w coat weight gains. Various pellet properties were investigated, including cumulative drug release behaviours of ensemble and individual pellets. When USP dissolution apparatus 1 and 2 were used for drug release study of the sustained release pellets prepared, floating and clumping of pellets were observed and confounded the release profiles of the ensemble pellets. Hence, the release profiles obtained did not characterize the actual drug release from individual pellet and the applicability of USP dissolution apparatus 1 and 2 to evaluate the coat quality of sustained release pellets was limited. The cumulative release profile of individual pellet using the 400-DS dissolution apparatus 7 was found to be more precise at distinguishing differences in the applied coat quality. The dip speed and dip interval of the reciprocating holder were critical operational parameters of 400-DS dissolution apparatus 7 that affected the drug release rate of a sustained release pellet during the individual dissolution study. The individual dissolution methodology using the 400-DS dissolution apparatus 7 is a promising technique to evaluate the individual pellet coat quality without the influence of confounding factors such as pellet floating and clumping observed during drug release test with dissolution apparatus 1 and 2, as well as to facilitate the elucidation of the actual drug release mechanism conferred by the applied sustained release coat onto the pellets.
Keywords: Sustained release pellets; Coat quality; Drug release profile; Individual pellet dissolution;
Relationships between surface free energy, surface texture parameters and controlled drug release in hydrophilic matrices by J. Saurí; J.M. Suñé-Negre; J. Díaz-Marcos; J. Vilana; D. Millán; J.R. Ticó; M. Miñarro; P. Pérez-Lozano; E. García-Montoya (328-340).
Display OmittedThe study of controlled release and drug release devices has been dominated by considerations of the bulk or average properties of material or devices. Yet the outermost surface atoms play a central role in their performance. The objective of this article has been to characterize the surface of hydrophilic matrix tablets using the contact angle (CA) method to ascertain the surface free energy, and atomic force microscopy (AFM) and confocal microscopy (CM) for the physical characterization of the surface of the hydrophilic matrix. The surface free energy results obtained show that hydroxypropylmethylcellulose K15M hinders the spreading of water on the surface of the tablet, such that the concentration of HPMC K15M increases the reaction rate of the hydrophobic interactions between the chains of HPMC K15M which increases with respect to the rate of penetration of water into the tablet. In this study, we developed a new method to characterize the swelling of the tablets and established a relationship between the new method based on microswelling and the swelling ratio parameter. The surface texture parameters have been determined and the morphology of the tablets of the different formulations and the evolution of the surface morphology after interacting with the water, swelling and forming a gel layer were characterized. This work represents significant progress in the characterization of matrix tablets.
Keywords: Surface free energy; Surface texture parameters; Microswelling; Surface analysis; Drug release; Hydrophilic matrices;
Improved oral absorption of dutasteride via Soluplus®-based supersaturable self-emulsifying drug delivery system (S-SEDDS) by Dong Hoon Lee; Dong Woo Yeom; Ye Seul Song; Ha Ra Cho; Yong Seok Choi; Myung Joo Kang; Young Wook Choi (341-347).
Display OmittedA novel supersaturable self-emulsifying drug delivery system (S-SEDDS) was formulated to improve the oral absorption of dutasteride (DTS), a 5α-reductase inhibitor that is poorly water-soluble. A supersaturable system was prepared by employing Soluplus ® (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer) as a precipitation inhibitor with a conventional SEDDS vehicle consisted of Capryol™ 90, Cremophor ® EL and Transcutol ® HP (DTS:SEDDS vehicle:Soluplus ® = 1.0:67.6:10.0 w/v/w). In an in vitro dissolution test in a non-sink condition, the drug dissolution rate from SEDDS was rapidly increased to 72% for an initial period of 5 min, but underwent rapid drug precipitation within 2 h, decreasing the amount of drug dissolved to one-seventh of its original amount. On the other hand, S-SEDDS resulted in a slower crystallization of DTS by virtue of a precipitation inhibitor, maintaining a 3 times greater dissolution rate after 2 h compared to SEDDS. In an in vivo pharmacokinetic study in rats, the S-SEDDS formulation exhibited 3.9-fold greater area-under-curve value than that of the drug suspension and 1.3-fold greater than that of SEDDS. The maximum plasma concentration of S-SEDDS was 5.6- and 2.0-fold higher compared to drug suspension and SEDDS, respectively. The results of this study suggest that the novel supersaturable system may be a promising tool for improving the physicochemical property and oral absorption of the 5α-reductase inhibitor.
Keywords: Dutasteride; Supersaturable self-emulsifying drug delivery system; Soluplus ® ; Oral absorption; Bioavailability;
Modelling drug degradation in a spray dried polymer dispersion using a modified Arrhenius equation by Adele Patterson; Ana P. Ferreira; Elizabeth Banks; Kirsty Skeene; Graham Clarke; Sarah Nicholson; Clare Rawlinson-Malone (348-360).
Display OmittedThe Pharmaceutical industry is increasingly utilizing amorphous technologies to overcome solubility challenges. A common approach is the use of drug in polymer dispersions to prevent recrystallization of the amorphous drug. Understanding the factors affecting chemical and physical degradation of the drug within these complex systems, e.g., temperature and relative humidity, is an important step in the selection of a lead formulation, and development of appropriate packaging/storage control strategies. The Arrhenius equation has been used as the basis of a number of models to predict the chemical stability of formulated product. In this work, we investigate the increase in chemical degradation seen for one particular spray dried dispersion formulation using hydroxypropyl methylcellulose acetate succinate (HPMC-AS). Samples, prepared using polymers with different substitution levels, were placed on storage for 6 months under a range of different temperature and relative humidity conditions and the degradant level monitored using high-performance liquid chromatography (HPLC). While the data clearly illustrates the impact of temperature and relative humidity on the degradant levels detected, it also highlighted that these terms do not account for all the variability in the data. An extension of the Arrhenius equation to include a term for the polymer chemistry, specifically the degree of succinoyl substitution on the polymer backbone, was shown to improve the fit of the model to the data.
Keywords: Arrhenius; Chemical stability; Hydroxypropyl methylcellulose acetate succinate; Pharmaceutics; Solid dispersion; Succinoyl substitution;
Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue by John R. Eisenbrey; Lorenzo Albala; Michael R. Kramer; Nick Daroshefski; David Brown; Ji-Bin Liu; Maria Stanczak; Patrick O’Kane; Flemming Forsberg; Margaret A. Wheatley (361-367).
Display OmittedRadiation therapy is frequently used in the treatment of malignancies, but tumors are often more resistant than the surrounding normal tissue to radiation effects, because the tumor microenvironment is hypoxic. This manuscript details the fabrication and characterization of an ultrasound-sensitive, injectable oxygen microbubble platform (SE61O2) for overcoming tumor hypoxia. SE61O2 was fabricated by first sonicating a mixture of Span 60 and water-soluble vitamin E purged with perfluorocarbon gas. SE61O2 microbubbles were separated from the foam by flotation, then freeze dried under vacuum to remove all perfluorocarbon, and reconstituted with oxygen. Visually, SE61O2 microbubbles were smooth, spherical, with an average diameter of 3.1 μm and were reconstituted to a concentration of 6.5 E7 microbubbles/ml. Oxygen-filled SE61O2 provides 16.9 ± 1.0 dB of enhancement at a dose of 880 μl/l (5.7 E7 microbubbles/l) with a half-life under insonation of approximately 15 min. In in vitro release experiments, 2 ml of SE61O2 (1.3 E8 microbubbles) triggered with ultrasound was found to elevate oxygen partial pressures of 100 ml of degassed saline 13.8 mmHg more than untriggered bubbles and 20.6 mmHg more than ultrasound triggered nitrogen-filled bubbles. In preliminary in vivo delivery experiments, triggered SE61O2 resulted in a 30.4 mmHg and 27.4 mmHg increase in oxygen partial pressures in two breast tumor mouse xenografts.
Keywords: Tumor hypoxia; Ultrasound contrast agent; Oxygen delivery; Surfactant; Microbubble;
Quantitative analysis of drug losses administered via nasogastric tube – In vitro study by Adriana Ruzsíková; Lenka Součková; Pavel Suk; Radka Opatřilová; Martina Kejdušová; Vladimír Šrámek (368-371).
Display OmittedDrug administration through nasogastric tube (NGT) is a standard practice but the real amount of the delivered drug is unknown. Therefore, we designed a study to determine the losses of various dosage forms administered by different methods through NGT.In vitro model was used. Five different administration methods (A–E) and six dosage forms (simple compressed tablets – T/S; film coated tablets – T/FC; enteric coated tablets – T/EC; capsules with powder filling – C/P; capsules containing extended release pellets – C/ER; capsules containing gastro-resistant pellets – C/GR) were investigated. Measurement was repeated six times for each drug-method combination. The overall losses were determined by gravimetry. In method A partial losses associated with each step of drug administration were also determined.Significant drug losses were measured (4–38%). Only methods A (crushing–beaker–syringe–water–NGT) and B (crushing–water–syringe–NGT) were suitable for administration of all tested dosage forms. Method B proved the most effective for all kinds of tablets and C/GR (p < 0.05) and tended to be more effective also for C/ER (p = 0.052) compared to method A. C/P showed minimal losses for both tested methods (B and E). Flushing of the drug through NGT causes major losses during drug administration compared to crushing and transfer (p < 0.05). All methods for intact pellets (C–E) were found inappropriate for clinical practice due to NGT clogging.Choosing a suitable administration method can significantly affect the amount of drugs delivered through NGT.
Keywords: Nasogastric tube; Intensive care; Dosage forms; Enteral feeding; Drug administration;
Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives by Magdalena Wytrwal; Chloe Leduc; Michal Sarna; Cristine Goncalves; Mariusz Kepczynski; Patrick Midoux; Maria Nowakowska; Chantal Pichon (372-382).
Display OmittedNon-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes.
Keywords: Gene delivery; Transfection efficiency; Non-viral gene vectors; Poly(allylamine); Nanocondensation;
Preactivated hyaluronic acid: A potential mucoadhesive polymer for vaginal delivery by Jessika Nowak; Flavia Laffleur; Andreas Bernkop-Schnürch (383-389).
Display OmittedThe objective of this study was to develop mucoadhesive polymeric excipients for vaginal drug delivery systems. Hyaluronic acid was thiolated and subsequently preactivated with 6-mercaptonicotinamide (HA-CYS–MNA) to enhance stability and mucoadhesive properties on vaginal mucosa. After determination of the thiol group content, disintegration studies and in vitro mucoadhesion studies (rotating cylinder and tensile) were performed. Furthermore, swelling behavior and cytotoxicity studies were performed in comparison with corresponding polymers. Both, disintegration and in vitro mucoadhesive studies revealed that modifying HA-CYS with MNA resulted in higher stability (3.6-fold prolonged disintegration time compared to unmodified hyaluronic acid) and prolonged mucoadhesion time. MTT assay and LDH revealed no toxicity for the polymeric excipients and safe for their use. Disintegration and swelling results conducted more pronounced stability of the preactivated thiomers compared to corresponding unmodified ones. According to these results preactivated hyaluronic acid might be a useful tool for vaginal delivery systems.
Keywords: Mucoadhesion; Hyaluronic acid; Preactivated thiomers; Thiomers; Vaginal delivery;
Overcoming drug crystallization in electrospun fibers – Elucidating key parameters and developing strategies for drug delivery by Salem Seif; Lutz Franzen; Maike Windbergs (390-397).
Display OmittedFor the development of novel therapeutics, uncontrolled crystallization of drugs within delivery systems represents a major challenge. Especially for thin and flexible polymeric systems such as oral films or dermal wound dressings, the formation and growth of drug crystals can significantly affect drug distribution and release kinetics as well as physical storage stability. In this context, electrospinning was introduced as a fabrication technique with the potential to encapsulate drugs within ultrafine fibers by rapid solvent evaporation overcoming drug crystallization during fabrication and storage. However, these effects could so far only be shown for specific drug-polymer combinations and an in-depth understanding of the underlying processes of drug-loaded fiber formation and influencing key parameters is still missing.In this study, we systematically investigated crystal formation of caffeine as a model drug in electrospun fibers comparing different polymers. The solvent polarity was found to have a major impact on the drug crystal formation, whereas only a minor effect was attributed to the electrospinning process parameters.Based on an in-depth understanding of the underlying processes determining drug crystallization processes in electrospun fibers, key parameters could be identified which allow for the rational development of drug-loaded electrospun fibers overcoming drug crystallization.
Keywords: Electrospinning; Controlled drug release; Drug crystallization; Nanofibers; Poly(vinyl alcohol); Polycaprolactone;
Development of an injectable PHBV microparticles-GG hydrogel hybrid system for regenerative medicine by Daniela P. Pacheco; Maria H. Amaral; Rui L. Reis; Alexandra P. Marques; Vítor M. Correlo (398-408).
Display OmittedUncontrollable displacements that greatly affect the concentration of active agents at the target tissues are among a major limitation of the use of microparticulate drug delivery systems (DDS). Under this context a biphasic injectable DDS combining poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microparticles (MPs) and a gellan gum (GG) injectable hydrogel is herein proposed for the localized delivery and long-term retention of MPs carrying hydrophilic and hydrophobic model active agents. A double emulsion-solvent evaporation method was adopted to develop the PHBV MPs, carrying bovine serum albumin (BSA) or dexamethasone (Dex) as hydrophilic and hydrophobic active agents’ models, respectively. Moreover, this method was modified, together with the properties of the hydrogel to tailor the delivery profile of the active agents. Variations of the composition of the organic phase during the process allowed tuning surface topography, particle size distribution and core porosity of the PHBV MPs and, thus, the in vitro release profile of Dex but not of BSA. Besides, after embedding hydrogels of higher GG concentration led to a slower and more sustained release of both active agents, independently of the processing conditions of the microparticulate system.
Keywords: Polyhydroxybutyrate-co-hydroxyvalerate; Gellan gum; Microparticulate systems; Injectable hydrogel; Regenerative medicine;
Modelling the response surface to predict the hydrodynamic diameters of theranostic magnetic siRNA nanovectors by Stephanie David; Hervé Marchais; Didier Bedin; Igor Chourpa (409-415).
Display OmittedShort interfering RNAs (siRNAs) appear to be a promising tool to treat various human diseases, such as cancer via the RNA interference (RNAi) mechanism. Since the systemic administration of siRNAs is limited by their capacity to attain the site of action, novel delivery systems are needed. Previously, we reported the formulation of magnetic siRNA nanovectors (MSN) using electrostatic assembly of the following components: (1) functionalized superparamagnetic iron oxide nanoparticles (SPIONs) able to act as agents for magnetic resonance imaging (MRI) and/or thermal therapy, (2) siRNAs as active molecules and (3) chitosan to protect siRNAs and to enhance their transfection efficacy.In this work, experimental design was used to further improve the formulation protocol and to optimize the component quantities. The aim was to obtain response surface plots that will help to optimize and predict the component quantities of the MSNs regarding their hydrodynamic diameter (D H). The influent parameters of the formulation process were determined using a Plackett–Burman design. The results show that the order of incorporation of the components is the most influent parameter on the D H of MSNs. A Box–Behnken design was used to optimize the component quantities. The model equations provided the parameters to obtain MSNs with D H smaller than 100 nm to allow their systemic administration.
Keywords: Physicochemical characterization; Superparamagnetic iron oxide nanoparticles (SPIONs); Design of experiments (DOE); Short interfering RNA (siRNA);
Novel applications of ubiquinone biopolymer nanocarriers for preventive and regenerative therapeutics: The Saccharomyces cerevisiae paradigm by Christoph Hoennscheidt; Argyrios Margaritis; Rainer Krull (416-425).
Display OmittedSince the last decade, nanodispersed drug delivery systems gain increasingly more importance for therapeutic research fields. The forced transport to the centers of inflammation is supposed to take advantage as a novel strategic approach. Thus, the focus of this study was to investigate the applicability of ubiquinone nanoformulations against oxidative stress. The physiological reduction of reactive oxygen species (ROS) seems to be a promising treatment to point out the potential effects of these sophisticated nano-constructs. Therefore, the yeast strain Saccharomyces cerevisiae N34 was used for in vitro studies as a representative for eukaryotic organisms. Growth parameters during sequential fed batch-cultivation were monitored online using focused beam reflectance measurement (FBRM) method. The ability to control diverse cellular processes makes this yeast strain to a valuable tool for the initial investigation by understanding the fundamental mechanisms of nanoparticulate formulations onto eukaryotic cells. Furthermore, the characteristic stress response of yeast cell culture was examined, so that drug effects could be determined quantitatively. As a chemical stressor, diamide was tested in the range of 1–1000 mg diamide per g cell dry weight (CDW). The ubiquinone nanoformulation demonstrated a total stress reduction of approximately 14% in the yeast culture, confirming the potential applicability of ubiquinone.
Keywords: Drug delivery system; Focused beam reflectance method; Oxidative stress; Saccharomyces cerevisiae; Sequential fed batch; Ubiquinone;