Imaging the intracellular degradation of biodegradable polymer nanoparticles.

In recent years, the development of smart drug delivery systems based on biodegradable polymeric nanoparticles has become of great interest. Drug-loaded nanoparticles can be introduced into the cell interior via endocytotic processes followed by the slow release of the drug due to degradation of the nanoparticle. In this work, poly(L-lactic acid) (PLLA) was chosen as the biodegradable polymer. Although common degradation of PLLA has been studied in various biological environments, intracellular degradation processes have been examined only to a very limited extent. PLLA nanoparticles with an average diameter of approximately 120 nm were decorated with magnetite nanocrystals and introduced into mesenchymal stem cells (MSCs). The release of the magnetite particles from the surface of the PLLA nanoparticles during the intracellular residence was monitored by transmission electron microscopy (TEM) over a period of 14 days. It was demonstrated by the release of the magnetite nanocrystals from the PLLA surface that the PLLA nanoparticles do in fact undergo degradation within the cell. Furthermore, even after 14 days of residence, the PLLA nanoparticles were found in the MSCs. Additionally, the ultrastructural TEM examinations yield insight into the long term intercellular fate of these nanoparticles. From the statistical analysis of ultrastructural details (e.g., number of detached magnetite crystals, and the number of nanoparticles in one endosome), we demonstrate the importance of TEM studies for such applications in addition to fluorescence studies (flow cytometry and confocal laser scanning microscopy).

Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model.

The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood-milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood-milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.

Complex encounters: nanoparticles in whole blood and their uptake into different types of white blood cells.

AIM: A whole blood assay for evaluating the uptake of nanoparticles into white blood cells in order to close the gap between basic studies in cell culture and pharmacokinetic studies in animals was developed. MATERIALS & METHODS: After drawing peripheral blood into standard blood collection vials with different anticoagulants, amino- and carboxy-functionalized polymeric styrene nanoparticles were added and uptake was evaluated by flow cytometry. RESULTS: By counterstaining surface markers of leukocytes (e.g., monocytes, neutrophil granulocytes, B or T lymphocytes), investigations of different cell types can be conducted in a single run by flow cytometry. The authors demonstrated that anticoagulation should be done with heparin, and not EDTA, in order to prevent hampering of uptake mechanisms. CONCLUSION: By using heparinized whole blood, the authors demonstrated differences and usefulness of this assay for screening cellular uptake as it should occur in the bloodstream. Nevertheless, animal studies are warranted for final assessment of the nanoparticles.

Suppressing unspecific cell uptake for targeted delivery using hydroxyethyl starch nanocapsules.

Synthesizing nanocarriers with stealth properties and delivering a "payload" to the particular organ remains a big challenge but is the prime prerequisite for any in vivo application. As a nontoxic alternative to the modification by poly(ethylene glycol) PEG, we describe the synthesis of cross-linked hydroxyethyl starch (HES, M(w) 200,000 g/mol) nanocapsules with a size range of 170-300 nm, which do not show nonspecific uptake into cells. The specific uptake was shown by coupling a folic acid conjugate as a model targeting agent onto the surface of the nanocapsules, because folic acid has a high affinity to a variety of human carcinoma cell lines which overexpress the folate receptor on the cell surface. The covalent binding of the folic acid conjugate onto HES capsules was confirmed by FTIR and NMR spectroscopy. The coupling efficiency was determined using fluorescence spectroscopy. The specific cellular uptake of the HES nanocapsules after folic acid coupling into the folate-receptor presenting cells was studied by confocal laser scanning microscopy (CLSM) and flow cytometry.

Synthesis of polyester nanoparticles in miniemulsion obtained by radical ring-opening of BMDO and their potential as biodegradable drug carriers.

5,6-Benzo-2-methylene-1,3-dioxepane (BMDO) is used to obtain degradable polymeric nanoparticles via a statistical free-radical copolymerization with MMA and styrene in direct miniemulsion. The nanoparticles are analyzed by means of IR, NMR, DLS, SEM, and TEM. They show excellent cellular uptake and drug delivery properties. The cellular uptake into HeLa cells of particles resulting from copolymerization of BMDO with styrene is drastically enhanced compared to pure polystyrene. As a model drug system, paclitaxel is incorporated in PBMDO particles and its release and the effect on HeLa cells is studied and compared to commercial drug formulations. It is found that a drug delivery system based on PBMDO shows an excellent pharmacological effect.

BSA adsorption on differently charged polystyrene nanoparticles using isothermal titration calorimetry and the influence on cellular uptake.

BSA adsorption onto negatively and positively charged polystyrene nanoparticles was investigated. The nanoparticles were characterized in terms of particle size, zeta potential, surface group density, and morphology. The adsorption behavior of BSA on the particle surface, as a function of pH and overall charge of the particle, was studied using ITC. Different thermodynamic data such as enthalpy changes upon binding and stoichiometry of the systems were determined and discussed. The degree of surface coverage with BSA was calculated using the thermodynamic data. The cellular uptake of particles before and after BSA adsorption was studied using HeLa cells in the presence and absence of supplemented FCS in the cell culture medium.

Species and age determination of central nervous system tissue by fatty acid patterns.

The banning of specified risk materials (SRMs) from food chain is one of the most important measures to protect the consumer against any exposition with the agent of transmissible spongiform encephalopathies (TSEs). In order to control the SRM-ban, suitable methods for the detection of SRMs have to be developed. In this study homogenized samples of central nervous system (CNS) tissue from cattle (n=38), sheep (n=38) and pig (n=40) were analyzed by gas chromatography-mass spectrometry (GC-MS). The analysis was focused on the identification of fatty acid ratios for species differentiation and age estimation. We found a novel fatty acid ratio (C24:1(n-9)/(n-7))/(2OH-C24:1(n-9)/(n-7) for species differentiation. For the first time, we used multivariate analysis for species differentiation, based on fatty acid ratios. It showed an excellent predictive ability (Q_(cum)(2))Q_(cum)(2)=0.842. For age estimation in cattle CNS we discovered a novel fatty acid ratio 2OH-C25:0/2OH-C24:1(n-7). The ratio is strongly correlated with age (coefficient of correlation (r)=0.935, Spearman-Rho). The corresponding regression analysis which allows the prediction of age by this ratio was acceptable (coefficient of determination (R(2))=0.831).

Diversity within the current algal species Prototheca zopfii: a proposal for two Prototheca zopfii genotypes and description of a novel species, Prototheca blaschkeae sp. nov.

In order to clarify the intraspecies taxonomic position of the non-photosynthetic algal species Prototheca zopfii, as well as the aetiology of bovine mammary protothecosis, a selection of P. zopfii strains isolated from clinical cases of protothecal mastitis and from various environmental habitats was characterized using a polyphasic molecular approach. Based on sequence analysis of the 18S rRNA gene, which showed distinct differences between the three currently known biotypes or 'variants', specific oligonucleotides were designed and used in biotype-specific PCRs. Furthermore, the pattern of cellular fatty acids was evaluated. Typing by means of these techniques revealed that the previously defined biotypes of P. zopfii were clearly different. Based on sequence analysis, the pattern of fatty acids and physiological characteristics, it is proposed that biotype 3 should be reclassified as representing a novel species, Prototheca blaschkeae sp. nov. (type strain, RZIII-3(T) = SAG 2064(T)). Furthermore, it is proposed that P. zopfii merits reclassification as a species comprising at least two genotypes that in future could probably be considered to represent two subspecies.

Alterations of mast cell mediator production and release by gamma-linolenic and docosahexaenoic acid.

The purpose of our investigations was to evaluate the supposed beneficial effects of gamma-linolenic (GLA) and docosahexaenoic acid (DHA) in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were cultured in a basic medium (DEH) and in DEH supplemented with 14.3 microM GLA (DEH-GLA) or 14.3 microM DHA (DEH-DHA) for 8 days. Chymase and tryptase activity, as well as histamine and prostaglandin (PG)E(2) release were measured. To stimulate histamine and PGE(2) release, cells were incubated with the wasp venom peptide mastoparan (50 microM) for 30 min. GLA increased tryptase activity and decreased histamine release after C2 stimulation. DHA diminished PGE(2) production in activated C2. These results support the prescription of GLA- and DHA-enriched diets to reduce inflammatory signs in canine atopic dermatitis.