Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

Reactive Nanoparticles Derived from Polysaccharide Phenyl Carbonates.

Polysaccharide (PS) based nanoparticles (NP) are of great interest for biomedical applications. A key challenge in this regard is the functionalization of these nanomaterials. The aim of the present work was the development of reactive PS-NP that can be coupled with an amino group containing compounds under mild aqueous conditions. A series of cellulose phenyl carbonates (CPC) and xylan phenyl carbonates (XPC) with variable degrees of substitution (DS) was obtained by homogeneous synthesis. The preparation of PS-NP by self-assembling of these hydrophobic derivatives was studied comprehensively. While CPC mostly formed macroscopic aggregates, XPC formed well-defined spherical NP with diameters around 100 to 200 nm that showed a pronounced long-term stability in water against both particle aggregation as well as cleavage of phenyl carbonate moieties. Using an amino group functionalized dye it was demonstrated that the novel XPC-NP are reactive towards amines. A simple coupling procedure was established that enables direct functionalization of the reactive NP in an aqueous dispersion. Finally, it was demonstrated that dye functionalized XPC-NP are non-cytotoxic and can be employed in advanced biomedical applications.

Biocompatibility, uptake and subcellular localization of bacterial magnetosomes in mammalian cells.

Magnetosomes represent biogenic, magnetic nanoparticles biosynthesized by magnetotactic bacteria. Subtle biological control on each step of biomineralization generates core-shell nanoparticles of high crystallinity, strong magnetization and uniform shape and size. These features make magnetosomes a promising alternative to chemically synthesized nanoparticles for many applications in the biotechnological and biomedical field, such as their usage as biosensors in medical diagnostics, as drug-delivery agents, or as contrast agents for magnetic imaging techniques. Thereby, the particles are directly applied to mammalian cells or even injected into the body. In the present work, we provide a comprehensive characterization of isolated magnetosomes as potential cytotoxic effects and particle uptake have not been well studied so far. Different cell lines including cancer cells and primary cells are incubated with increasing particle amounts, and effects on cell viability are investigated. Obtained data suggest a concentration-dependent biocompatibility of isolated magnetosomes for all tested cell lines. Furthermore, magnetosome accumulation in endolysosomal structures around the nuclei is observed. Proliferation rates are affected in the presence of increasing particle amounts; however, viability is not affected and doubling times can be restored by reducing the magnetosome concentration. In addition, we evidence magnetosome-cell interactions that are strong enough to allow for magnetic cell sorting. Overall, our study not only assesses the biocompatibility of isolated magnetosomes, but also evaluates effects on cell proliferation and the fate of internalized magnetosomes, thereby providing prerequisites for their future in vivo application as biomedical agents.

Towards standardized purification of bacterial magnetic nanoparticles for future in vivo applications.

Bacterial magnetosomes (MS) are well-defined membrane-enveloped single-domain iron oxide (magnetite) nanoparticles, which are susceptible to genetic and chemical engineering. Additionally, the possibility to manipulate these particles by external magnetic fields facilitates their application in biomedicine and biotechnology, e.g. as magnetic resonance imaging probes or for drug delivery purposes. However, current purification protocols are poorly characterized, thereby hampering standardized and reproducible magnetosome production and thus, reliable testing for in vivo applications. In that context, the establishment of reproducible particle isolation procedures as well as the identification of high quality control parameters and the evaluation of potential cytotoxic effects of purified particles are of major importance. In this study, we characterize a multi-step purification protocol for MS with regard to purity, iron content, size and polydispersity of magnetite particles. In addition, we address potential cytotoxic effects of isolated MS when incubated with mammalian cells. Overall, we provide a detailed overview of the process-structure relationship during the isolation of MS and thus, identify prerequisites for high-yield MS production and their future application in the biomedical and biotechnological field. STATEMENT OF SIGNIFICANCE: Magnetic nanoparticles are of increasing interest for a variety of biomedical and biotechnological applications. Due to their unprecedented material characteristics, bacterial magnetosomes represent a promising alternative to chemically synthesized iron oxide nanoparticles. As applications require well-defined, highly purified and fully characterized nanoparticles, reliable protocols are necessary for efficient and reproducible magnetosome isolation. In our study, we evaluate an improved magnetosome extraction procedure and monitor quality parameters such as particle size distribution, membrane integrity and purity of the suspension by a combination of physicochemical and biochemical methods. Furthermore, the cytotoxicity of the isolated magnetosomes is assessed using different cell lines. In summary, our study helps to establish prerequisites for many real-world applications of magnetosomes in the field of biotechnology and biomedicine.

Seeding of epithelial cells into circulation during surgery for breast cancer: the fate of malignant and benign mobilized cells.

BACKGROUND: Surgery of malignant tumors has long been suspected to be the reason for enhancement of growth of metastases with fatal outcome. This often prevented surgeons from touching the tumor if not absolutely necessary. We have shown in lung cancer patients that surgery, itself, leads to mobilization of tumor cells into peripheral blood. Some of the mobilized cells finding an appropriate niche might grow to form early metastases. Monitoring of tumor cell release during and the fate of such cells after surgery for breast cancer may help to reveal how metastases develop after surgery. METHOD: We used the MAINTRAC analysis, a new tool for online observation of circulating epithelial cells, to monitor the number of epithelial cells before, 30 min, 60 min, three and seven days after surgery and during subsequent variable follow up in breast cancer patients. RESULTS: Circulating epithelial cells were already present before surgery in all patients. During the first 30-60 min after surgery values did not change immediately. They started increasing during the following 3 to 4 days up to thousand fold in 85% of treated patients in spite of complete resection of the tumor with tumor free margins in all patients. There was a subsequent re-decrease, with cell numbers remaining above pre-surgery values in 58% of cases until onset of chemotherapy. In a few cases, where no further therapy or only hormone treatment was given due to low risk stage, cell numbers were monitored for up to three years. They remained elevated with no or a slow decrease over time. This was in contrast to the observation in a patient where surgery was performed for benign condition. She was monitored before surgery with no cells detectable. Epithelial cells increased up to more than 50,000 after surgery but followed by a complete reduction to below the threshold of detection. CONCLUSION: Frequently before but regularly during surgery of breast cancer, epithelial cells are mobilized into circulation. Part of these cells, most probably normal or apoptotic cells, are cleared from the circulation as also shown to occur in benign conditions. After resection even if complete and of small tumors, cells can remain in the circulation over long times. Such cells may remain "dormant" but might settle and grow into metastases, if they find appropriate conditions, even after years.

Monitoring circulating epithelial tumour cells (CETC) to gauge therapy: in patients with disease progression after trastuzumab persisting CETC can be eliminated by combined lapatinib treatment.

BACKGROUND: In breast cancers, the gene for the growth factor receptor HER2 can be amplified leading to increased aggressiveness and metastasis formation. The monoclonal antibody trastuzumab prolongs relapse-free survival highly significantly but eventually many patients relapse. METHOD: In this study, CETC were monitored using the Maintrac method during adjuvant trastuzumab treatment and during subsequent treatment with capecitabine/lapatinib. RESULTS: In one patient, trastuzumab led to marginal reduction in CETC with disease progress. The combination of capecitabine/lapatinib was preliminarily capable to eliminate all CETC, however, CETC reappeared. The second patient received adjuvant taxane together with trastuzumab and 1 year of further trastuzumab during which CETC increased. After stopping trastuzumab skin metastases occurred. Capecitabine/lapatinib led to complete CETC elimination with stable disease. CONCLUSIONS: In patients with lack of CETC reduction in spite of trastuzumab treatment correlated with disease progression the combination of capecitabine/lapatinib highly efficiently led to rapid elimination of CETC warranting further monitoring during such studies.

Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse.

PURPOSE: To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse. PATIENTS AND METHODS: In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. RESULTS: Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of chemotherapy. The following three typical patterns of response were observed: (1) decrease in cell numbers (> 10-fold); (2) marginal changes in cell numbers (< 10-fold); and (3) an (sometimes saw-toothed) increase or an initial decrease with subsequent reincrease (> 10-fold) in numbers of CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33 patients from response group 3. The difference in relapse-free survival was highly significant for CETC (hazard ratio = 4.407; 95% CI, 1.739 to 9.418; P < .001) between patients with decreasing cell numbers and those with marginal changes and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model). CONCLUSION: These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.