Formulation and <Emphasis Type="Italic">In vitro</Emphasis> Characterization of Eudragit® L100 and Eudragit® L100-PLGA Nanoparticles Containing Diclofenac Sodium

The aim of this study was to formulate and characterize Eudragit® L100 and Eudragit® L100-poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing diclofenac sodium. Diclofenac generates severe adverse effects with risks of toxicity. Thus, nanoparticles were prepared to reduce these drawbacks in the present study. These nanoparticles were evaluated for surface morphology, particle size and size distribution, percentage drug entrapment, and in vitro drug release in pH 6.8. The prepared nanoparticles were almost spherical in shape, as determined by atomic force microscopy. The nanoparticles with varied size (241–274 nm) and 25.8–62% of entrapment efficiency were obtained. The nanoparticles formulations produced the release profiles with an initial burst effect in which diclofenac sodium release ranged between 38% and 47% within 4 h. The extent of drug release from Eudragit® L100 nanoparticles was up to 92% at 12 h. However, Eudragit®/PLGA nanoparticles showed an initial burst release followed by a slower sustained release. The cumulative release at 72 h was 56%, 69%, and 81% for Eudragit®/PLGA (20:80), Eudragit®/PLGA (30:70) and Eudragit®/PLGA (50:50) nanoparticles, respectively. The release profiles and encapsulation efficiencies depended on the amount of Eudragit in the blend. These data demonstrated the efficacy of these nanoparticles in sustaining the diclofenac sodium release profile.     

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

Melt-Extruded Eudragit® FS-Based Granules for Colonic Drug Delivery

The purpose of this study is to characterize the properties of Eudragit® FS-based granules prepared using melt extrusion process for colonic drug delivery. 5-Aminosalicylic acid (5-ASA), theophylline, and diclofenac sodium were used as the model compounds. Drug and polymer blends were melt-extruded into thin rods using a single screw extruder. Drugs were found to be dispersed as crystalline particles in the granules. A hammer mill was used to reduce the extrudate into 16–40 mesh granules, which were mixed with lactose and filled into hard gelatin capsules. Three-stage dissolution testing performed using USP paddle method was used to simulate drug release in gastrointestinal tract. In this study, melt extrusion has been demonstrated to be a suitable process to prepare granules for colonic delivery of 5-amino salicylic acid. At 30% drug loading, less than 25% 5-ASA was released from melt-extruded granules of 20–30 mesh in the first two stages (0.1 N hydrochloric acid solution and phosphate buffer pH 6.8) of the dissolution testing. All 5-ASA was released within 4 h when dissolution medium was switched to phosphate buffer pH 7.4. Drug loading, granule size, and microenvironment pH induced by the solubilized drug were identified as the key factors controlling drug release. Granules prepared with melt extrusion demonstrated lower porosity, smaller pore size, and higher physical strength than those prepared with conventional compression process. Eudragit® FS was found to be stable even when processed at 200°C.     

IMRT/VMAT dose distributions generated for HD® and Millennium® collimators TrueBeam® and Clinac® accelerators

AimThe aim of this study is to answer the question whether the calculated dose distributions for HD and Millennium collimators (Varian Medical Systems) are equivalent for large treatment volumes.BackgroundModern biomedical linacs are equipped with multileaf collimators where leaves can be of different widths. Thinner leaves allow better fit to desired (tumor) shape. At the same time, however, the maximum size of the field that can be obtained with the collimator is also reduced. Varian Medical Systems HD and Millennium collimators can be a good sample. They have 40 cm or 22 cm × 40 cm maximal field size at the isocenter, respectively.Materials and methodsThis paper presents the comparison of selected statistical and dosimetric parameters achieved for treatment plans where the beams for a HD collimator had to be merged because of the size of the tumor volume.Results and discussionAchieved results show that, independently from irradiated volume, there is no statistically significant difference for calculated dose distributions, integral doses, MU values and coefficients evaluating dose distributions for HD and Millennium collimators.ConclusionsResults show that both types of collimators can be used interchangeably for preparing the treatment plans for large tumor volume without quality reduction of the prepared treatment plan.     

Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima®

As biosimilars enter the market, comparisons of product quality are needed. Manufacturing differences may lead to differences in critical quality attributes, which affect efficacy. Therefore, critical quality attributes (structure and biological activity) of Remicade® and of 2 biosimilar products (Flixabi®/Renflexis® and Remsima®/Inflectra®) were determined. We assessed binding to tumor necrosis factor in a fluorescence competitive binding assay; potency in a luciferase reporter gene assay; percentages of galactosylated glycan, afucose plus high mannosylated glycans, and charged glycan; FcγRIIIa (CD16) binding (assessed by 3 methods); and antibody-dependent cell-mediated cytotoxicity (ADCC) in the NK92-CD16a cell line and in peripheral blood mononuclear cells (PBMC). The results of Fab-related activity were similar for all products. Compared with Remicade®, Flixabi® had a lower percentage of charged glycan, and Remsima® had a higher percentage of galactosylated glycan and a lower percentage of afucose plus high mannosylated glycans. Whereas Remsima® and Remicade® are expressed in a Sp2/0 cell line, Flixabi® is expressed in a CHO cell line. Despite this difference, galactosylated glycans from the 3 products were not correlated with the expression system. The results of all 3 methods used in this study indicated that FcγRIIIa binding was lower with Remsima® than with Remicade®. The percentage of ADCC in NK92-CD16a cells was lower with Remsima® and higher with Flixabi® compared with Remicade®, but was similar for all 3 products in PBMC. Surface expression of CD16 was 5.7-fold greater on NK92-CD16a cells than on PBMC. Combined percentages of afucosylated and high mannosylated glycans were positively correlated with FcγRIIIa binding and ADCC in NK92-CD16 cells, while no correlation was observed in PBMC.     

超临界流体强化溶液分散法制备Eudragit S100纳米颗粒

目的:制备Eudragit S100纳米颗粒。方法:采用超临界流体强化溶液分散(SEDS)法制备,考察了Eudragit S100浓度、超临界CO2流速、溶液流速、压力、温度对Eudragit S100纳米粒形貌和粒径的影响,并用场发射扫描式电子显微镜、激光粒度分析仪、差示扫描量热仪、傅里叶变换红外光谱仪对样品进行表征。结果:SEDS法可以制备球形的、粒径分布窄的Eudragit S100纳米粒,所得纳米粒的平均粒径在90~220 nm之间。降低Eudragit S100浓度和温度、升高压力有利于制备形貌好、粒径小的纳米粒;提高超临界CO2流速和降低溶液流速也有利于制备粒径小的纳米粒,但当超临界CO2流速升高至4 kg/h或溶液流速降低至0.5 ml/min时,纳米粒的产率较低。SEDS处理后Eudratit S100仍以无定形态存在,且SEDS过程没有对Eudratit S100的化学键造成破坏。结论:采用SEDS法可用于Eudragit S100纳米粒的制备,工艺简单可行。     

Osteoconductivity and osteoinductivity of NanoFUSE® DBM

Bone graft substitutes have become an essential component in a number of orthopedic applications. Autologous bone has long been the gold standard for bone void fillers. However, the limited supply and morbidity associated with using autologous graft material has led to the development of many different bone graft substitutes. Allogeneic demineralized bone matrix (DBM) has been used extensively to supplement autograft bone because of its inherent osteoconductive and osteoinductive properties. Synthetic and natural bone graft substitutes that do not contain growth factors are considered to be osteoconductive only. Bioactive glass has been shown to facilitate graft containment at the operative site as well as activate cellular osteogenesis. In the present study, we present the results of a comprehensive in vitro and in vivo characterization of a combination of allogeneic human bone and bioactive glass bone void filler, NanoFUSE^® DBM. NanoFUSE^® DBM is shown to be biocompatible in a number of different assays and has been cleared by the FDA for use in bone filling indications. Data are presented showing the ability of the material to support cell attachment and proliferation on the material thereby demonstrating the osteoconductive nature of the material. NanoFUSE^® DBM was also shown to be osteoinductive in the mouse thigh muscle model. These data demonstrate that the DBM and bioactive glass combination, NanoFUSE^® DBM, could be an effective bone graft substitute.     

Eudragit E100(®) potentiates the bactericidal action of ofloxacin against fluoroquinolone-resistant Pseudomonas aeruginosa

Sorbitol-fermenting Escherichia coli O157:NM (SF O157) is an emerging pathogen suggested to be more virulent than nonsorbitol-fermenting Escherichia coli O157:H7 (NSF O157). Important virulence factors are the Shiga toxins (stx), encoded by stx1 and/or stx2 located within prophages integrated in the bacterial genome. The stx genes are expressed from p(R) (') as a late protein, and anti-terminator activity from the Q protein is necessary for read through of the late terminator t(R) (') and activation of p(R) (') . We investigated the regulation of stx2(EDL933) expression at the genomic level in 17 Norwegian SF O157. Sequencing of three selected SF O157 strains revealed that the anti-terminator q gene and genes upstream of stx2(EDL933) were identical or similar to the ones observed in the E.?coli O111:H- strain AP010960, but different from the ones observed in the NSF O157 strain EDL933 (AE005174). This suggested divergent stx2(EDL933) -encoding bacteriophages between NSF O157 and the SF O157 strains (FR874039-41). Furthermore, different DNA structures were detected in the SF O157 strains, suggesting diversity among bacteriophages also within the SF O157 group. Further investigations are needed to elucidate whether the q(O111:H) (-) gene observed in all our SF O157 contributes to the increased virulence seen in SF O157 compared to NSF O157. An assay for detecting q(O111:H) (-) was developed.     

Glycosaminoglycans from Ateroid® and bovine duodenal mucosa and pancreas

Glycosaminoglycans (GG) were isolated from commercial Ateroid and compared with those from bovine duodenal mucosa and pancreas. The major GG in Ateroid is heparin. Heparan sulfate (HS) and dermatan sulfate were also found. HS, chondroitin sulfates, and heparin were isolated from duodenal mucosa after papain digestion, but a residue, non-digestible, was mostly heparin. Pancreas contains very little GG, and the GG composition is similar to that of mucosa. The heparin isolated from Ateroid and mucosa have similar lipoprotein lipase-releasing activity, but the former has considerably less anticoagulant activity. Interestingly, papain digestion of mucosa and pancreas did not release all heparin from the tissue, suggesting that the protein to which heparin is linked is not readily accessible to the enzyme.     

Formulation of Sustained-Release Dosage Form of Verapamil Hydrochloride by Solid Dispersion Technique Using Eudragit RLPO or Kollidon®SR

The release of verapamil hydrochloride from tablets with Eudragit RLPO or Kollidon®SR with different drug-to-polymer ratios were investigated with a view to develop twice-daily sustained-release dosage form by solid dispersion (SD) technique. The SDs containing Eudragit RLPO or Kollidon®SR at drug-polymer ratios of 1:1, 1:2, and 1:3 with verapamil hydrochloride were developed using solvent evaporation technique. The physical mixtures of drug and both polymers were prepared by using simple mixing technique at the same ratio as solid dispersion. The physicochemical properties of solid dispersion were evaluated by using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). The study of DSC, XRD, and FTIR could not show significant interaction between verapamil HCl and Kollidon®SR or Eudragit RLPO. The solid dispersions or physical mixtures were compressed to tablets. The tablets were prepared with solid dispersions containing Eudragit RLPO or Kollidon®SR, with all the official requirements of tablet dosage forms fulfilled. Tablets prepared were evaluated for the release of verapamil hydrochloride over a period of 12 h in pH 6.8 phosphate buffer using US Pharmacopoeia type II dissolution apparatus. The in vitro drug release study revealed that the tablet containing Eudragit has extended the release rate for 12 h whereas the tablet containing Kollidon®SR at the same concentration has extended the release rate up to 8 h. The in vitro release profile and the mathematical models indicate that release of verapamil hydrochloride can be effectively controlled from a tablet containing solid dispersions of Eudragit RLPO. The reduction of size fraction of the SD system from 200–250 to 75–125 μm had a great effect on the drug release.     

Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® cotton

Planta - Insertion of the gene encoding phosphinothricin acetyltransferase (PAT) has resulted in cotton plants resistant to the herbicide glufosinate. However, the lower expression and commensurate...     

Taxonomical classification and origin of Kamut® wheat

Bioagriculture and healthy lifestyle are trends of the twenty-first century. Bioagriculture involves the breeding of crops without using modern synthetic substances. Kamut brand wheat is one of the popular biocereals grown mainly in the USA and Europe. This cereal has the status of ancient wheat, not only because it has been grown since the era of the ancient Egyptian civilization, but also for its properties favorable for modern breeding programs and modern food marketing. In spite of Kamut’s^® interesting history and stable place in the market, it is not a common subject of genetic studies. It is also interesting that it has not been successfully taxonomically classified yet. There are a few studies which classify this tetraploid wheat as Triticum polonicum L., T. turanicum Jakubz., T. turgidum L. and T. durum Desf. These studies are based on cytological and comparative methods. We chose molecular (transposable element resistance gene analog polymorphism, diversity arrays technology, sequencing of genes SBEIIa, and ψLpx-A1_like) and statistical methods to classify Kamut^® wheat. According to our experiments we suggest that Kamut brand wheat originated as a natural hybrid between Triticum dicoccon conv. dicoccon and T. polonicum and is not original ancient Egyptian wheat. We suggest that Etruscan wheat has the same parents as Kamut^®.     

Interpolyelectrolyte Complexes of Eudragit? EPO with Hypromellose Acetate Succinate and Eudragit? EPO with Hypromellose Phthalate as Potential Carriers for Oral Controlled Drug Delivery

The objective of this study was to compare a novel controlled release tablet formulation based on interpolyelectrolyte complex (PEC). Interpolymer interactions between the countercharged polymers like Eudragit® EPO (polycation) and hypromellose acetate succinate (polyanion) and Eudragit® EPO and hypromellose phthalate (polyanion) were investigated with a view to their use in per oral controlled release drug delivery systems. The formation of inter-macromolecular ionic bonds between cationic polymer and anionic polymer was investigated using Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry. The FT-IR spectra of the tested polymeric matrices are characterized by visible changes in the observed IR region indicating the interaction between chains of two oppositely charged copolymers. The performance of the in situ formed PEC as a matrix for controlled release of drugs was evaluated, using acetaminophen as a model drug. The dissolution data of these matrices were fitted to different dissolution models. It was found that drug release followed zero-order kinetics and was controlled by the superposition of the diffusion and erosion. These profiles could be controlled by conveniently modifying the proportion of the polymer ratio, polymer type, and polymer concentration the in the tablets.KEY WORDS: Eudragit E, hypromellose acetate succinate, hypromellose phthalate polyelectrolyte complexation     

Anti-staphylococcal effect of enterocin in Sunar® and yogurt

Enterocin was used to control the growth of Staphylococcus aureus strains SA1 and Oxford 209P in Sunar (milk nourishment for suckling babies) and during the yogurt-making process. Reduction by three orders of magnitude was noted in the growth of SA1 strain in Sunar milk nourishment between the enterocin-containing (ES) and the control samples (CS) at 1-d cultivation. An inhibitory effect of enterocin was observed when surviving of SA1 cells were checked 6 h after the start of cultivation (2 h after enterocin application; enterocin was applied after 4 h). Decrease in the count of Oxford 209P strain in yogurt was detected in ES after 1 d of storage in comparison with CS (10(3) and 10(0) CFU mL-1 g-1). Thus a decrease by three orders of magnitude was found between ES and CS at the time mentioned. On the other hand, no bacteriocin activity was detected in ES after 1 d. Activity was detected only immediately after enterocin addition to ES (400 AU/mL) as well as after 1 and 3 1/2 h (200 AU/mL). Although the slight regrowth of the indicator was obtained up to 1 week of yogurt storage, the difference between ES and CS persisted. The lowest pH of the final yogurt product was noted in the reference yogurt sample but differences among the pH values of yogurt samples were not significant.     

Synthesis and characterization of pHLIP® coated gold nanoparticles

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP^®) were introduced. The presence of a tumor-targeting pHLIP^® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ～43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP^® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.     

Molecular dynamics studies of the Nafion®, Dow® and Aciplex® fuel-cell polymer membrane systems

The Nafion, Dow and Aciplex systems – where the prime differences lies in the side-chain length – have been studied by molecular dynamics (MD) simulation under standard pressure and temperature conditions for two different levels of hydration: 5 and 15 water molecules per (H)SO₃ end-group. Structural features such as water clustering, water-channel dimensions and topology, and the dynamics of the hydronium ions and water molecules have all been analysed in relation to the dynamical properties of the polymer backbone and side-chains. It is generally found that mobility is promoted by a high water content, with the side-chains participating actively in the H₃O⁺/H₂O transport mechanism. Nafion, whose side-chain length is intermediate of the three polymers studied, is found to have the most mobile polymer side-chains at the higher level of hydration, suggesting that there could be an optimal side-chain length in these systems. There are also some indications that the water-channel network connectivity is optimal for high water-content Nafion system, and that this could explain why Nafion appears to exhibit the most favourable overall hydronium/water mobility. Figure The simulation box for Aciplex with 5 water molecules per sulphonate end-group (yellow spheres). The polymer backbone is black; while side-chains are brown. The water-channel iso-surfaces are represented as blue clouds     

Advanced assessment of the physicochemical characteristics of Remicade® and Inflectra® by sensitive LC/MS techniques

In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.     

Impact of protein fouling on nanoparticle capture within the Viresolve® Pro and Viresolve® NFP virus removal membranes

Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters.     

Bioactivity and viscoelastic characterization of chitosan/bioglass® composite membranes

Membranes of chitosan (CTS) and composite membranes of CTS with bioglass are prepared by solvent casting. The composite membranes are shown to induce the precipitation of apatite upon immersion in SBF. The biomineralization process is followed by measuring the variation of the viscoelastic properties of the membranes immersed in SBF, both online and offline. Non-conventional DMA is used to measure the change in the storage modulus, E', and the loss factor, tan δ, as a function of the immersion in SBF. A simple model is used to estimate the E' of the apatite layer formed in vitro that is about 130?MPa. This work shows that innovate mechanical tests can be useful to characterize the mechanical performance of composites under physiological conditions.