Colloid biology - Develop and analyse microbial interactions

Prof. Dr. Ales Lapanje, from the Jozef Stefan Institute (Slovenia)

Colloid physics and colloid chemistry are well established fields within the colloid sciences. Both of these two disciplines are directed toward studying or engineering inanimate particles that are interacting in a physical way, such as electrostatics, rheology, stability, or their surfaces are either chemically modified or chemically reactive, respectively. In contrast, the new discipline the colloid biology is implementing the methods of colloid science on the living cells to either change their surface, cause aggregation, surface attachment as well entrapment or encapsulation. However, the biological properties of these particles are always adding emergent properties to the particles either through the constant adaptation of the cell surfaces, mass gaining, motility or other ways that cannot be predicted only by the physical properties of a particular cell. Oppositely, the method of modification of cells based on the colloid physics can likewise change the biological properties of the cell such as growth rate, expression profiles as well as interaction with other cells in the population or communities and can affect ecological relations, changing it from competitive to the collaborative or symbiotic coexistence. Therefore, it is a substantial need to establish the third pillar of colloid science, colloid biology. Therefore, the main objective of this chapter is to understand the basic concepts of colloid biology that are differentiating as well as linking with the colloid physics and colloid chemistry concepts and resulting in interdisciplinary science.

Literature:

1. Lapanje, A., Wimmersberger, C., Furrer, G., Brunner, I. and Frey, B., 2012. Pattern of elemental release during the granite dissolution can be changed by aerobic heterotrophic bacterial strains isolated from Damma Glacier (central Alps) deglaciated granite sand. Microbial ecology, 63, pp.865-882.

2. van Tatenhove-Pel, R.J., Rijavec, T., Lapanje, A., van Swam, I., Zwering, E., Hernandez-Valdes, J.A., Kuipers, O.P., Picioreanu, C., Teusink, B. and Bachmann, H., 2021. Microbial competition reduces metabolic interaction distances to the low µm-range. The ISME journal, 15(3), pp.688-701.

3. Rybkin, I., Gorin, D., Sukhorukov, G. and Lapanje, A., 2019. Thickness of polyelectrolyte layers of separately confined bacteria alters key physiological parameters on a single cell level. Frontiers in bioengineering and biotechnology, 7, p.378.

4. Gusev, A., Zakharova, O., Muratov, D.S., Vorobeva, N.S., Sarker, M., Rybkin, I., Bratashov, D., Kolesnikov, E., Lapanje, A., Kuznetsov, D.V. and Sinitskii, A., 2019. Medium-dependent antibacterial properties and bacterial filtration ability of reduced graphene oxide. Nanomaterials, 9(10), p.1454.

5. Gusev, A., Zakharova, O., Vasyukova, I., Muratov, D.S., Rybkin, I., Bratashov, D., Lapanje, A., Il'inikh, I., Kolesnikov, E. and Kuznetsov, D., 2019. Effect of GO on bacterial cells: Role of the medium type and electrostatic interactions. Materials Science and Engineering: C, 99, pp.275-281.

Dr. Bogdan Parakhonskiy, from Ghent University.

Hydrogels, which are versatile three-dimensional structures containing polymers and water, are very attractive for use in biomedical fields, but they suffer from rather weak mechanical properties. In this regard, design a hydrogel based biomaterials with cell-binding sites, tunable mechanical properties and complex architectures have emerged as a powerful tool to control cell adhesion and proliferation for tissue engineering.

In this chapter we highlighted the usage of the bioceramic colloidal micro/ nanoparticles as key component of such system. Particles containing various ratios of Ca2+/Mg2+ with sizes ranging from 1 to 8 μm were prepared and mixed with gellan gum (GG) solution to study the in-situ formation of hydrogel-particle composites. The particles provide multiple functionalities: 1) they efficiently crosslink GG to induce hydrogel formation through the release of the divalent cations (Ca2+/Mg2+) known to bind to GG polymer chains; 2) they enhance mechanical properties of the hydrogel from 2 up to 100 kPa; 3)particles provide a delivery function, where loading efficiency and loading capacity are depending on particle size, time of enzyme loading, and various container compositions and enzyme concentrations 4) the samples most efficiently promoting cell growth were found to contain two types of minerals: calcium carbonate and hydroxymagnesite, which enhanced cells proliferation and hydroxyapatite formation.

Our results reveal that the size of particles influences their morphology and this, in turn, affects the activity of the encapsulated enzymes. The presence of therapeutic effect on osteoblastic cells coupled with a relatively high loading capacity, biocompatibility, and ease of fabrication suggests that the developed carriers are promising candidates for efficient drug delivery, especially in the field of bone reconstruction.

In conclusion, the development and construction of biocarriers based on mineralized hydrogels is an exciting area of research with significant potential for advancements in drug and cellular delivery. The use of mineralized hydrogels can enhance the properties and biocompatibility of hydrogels, providing new opportunities for the development of effective controlled release systems and tissue engineering applications.

References:

1. Y. Huang, L. Cao, B. V. Parakhonskiy, and A. G. Skirtach, “Hard, Soft, and Hard-and-Soft Drug Delivery Carriers Based on CaCO3 and Alginate Biomaterials: Synthesis, Properties, Pharmaceutical Applications,” Pharmaceutics, vol. 14, no. 5, p. 909, Apr. 2022, doi: 10.3390/pharmaceutics14050909.

2. A. Abalymov et al., “The influence of Ca/Mg ratio on autogelation of hydrogel biomaterials with bioceramic compounds,” Mater. Sci. Eng. C, vol. 133, no. December 2021, p. 112632, Jan. 2022, doi: 10.1016/j.msec.2021.112632.

3. B. V Parakhonskiy et al A method of drug delivery to tumors based on rapidly biodegradable drug-loaded containers, Appl. Mater. Today. 25 (2021) 101199.

4. S. Sovova, A. Abalymov, M. Pekar, A.G. Skirtach, B. Parakhonskiy, Calcium carbonate particles: synthesis, temperature and time influence on the size, shape, phase, and their impact on cell hydroxyapatite formation, J. Mater. Chem. B. 9 (2021) 8308–8320.

5. A.A. Abalymov, L. Van der Meeren, D. Van de Walle, K. Dewettinck, A.G. Skirtach, B. V. Parakhonskiy, Meshes-to-Fibrils Transition of Gellan Gum Hydrogel Architecture by Thermal Annealing, Macromol. Mater. Eng. 305 (2020) 1–5.

The biocolloid structures are formed through the interactions between the colloid particles, which are instead of the inanimate particles here used as alive microbial cells. To engage the physical interaction between cells it is needed to initiate the interaction through the modification of their surfaces that result in increasing either attraction or repulsion. The modification of surfaces based on the use of polyelectrolytes has been previously developed. In our approaches, we enhanced the method to get a spontaneous aggregation of bacterial cells at the minimum and to have individual cells with various surface charges that represent individual “lego brick” for forming complex multicellular structures. Moreover, the use of this method can result in control of the growth and activity of the individual cell. At the right conditions, cells can be then either deposited on surfaces in multilayers or aggregated by forming simple aggregates when non-specific aggregation is started or forming very defined structures through specific aggregation. Within large structures formed by specific aggregation, the physicochemical parameters within the aggregates are changing due to the physical properties of the structure or due to the metabolic activities of different cells and as a result of both those factors the specific niches are defined that a particular cell type can occupy. In some cases, the distances between cells in the aggregates can change ecological interactions from competition to collaboration. Besides aggregates, there can be also formed multilayered complex structures on the surface of the inanimate as well as alive structures, which enable either engineering the biofilm succession or preventing attachment of naturally occurring microbes. Moreover, the surface modification of bacterial cells cannot only change electrostatic properties, but can also add magnetic properties or some others that are needed for particular biotechnological applications. In the end, the artificially formed structures, aggregates and biofilms, are not static but represent the initial stage of the further development of the structure through biological activity, which gives various emerging properties that can be then used in remediation, medicine, prevention of biologically induced corrosion and others.

Literature:

1. Rybkin, I., Pinyaev, S., Sindeeva, O., German, S., Koblar, M., Pyataev, N., Čeh, M., Gorin, D., Sukhorukov, G. and Lapanje, A., 2022. Modification of bacterial cells for in vivo remotely guided systems. Frontiers in Bioengineering and Biotechnology, 10.

2. Deev, D., Rybkin, I., Rijavec, T. and Lapanje, A., 2021. When beneficial biofilm on materials is needed: electrostatic attachment of living bacterial cells induces biofilm formation. Frontiers in Materials, 8, p.624631.

3. van Tatenhove-Pel, R.J., Rijavec, T., Lapanje, A., van Swam, I., Zwering, E., Hernandez-Valdes, J.A., Kuipers, O.P., Picioreanu, C., Teusink, B. and Bachmann, H., 2021. Microbial competition reduces metabolic interaction distances to the low µm-range. The ISME journal, 15(3), pp.688-701.

4. Rijavec, T., Zrimec, J., van Spanning, R. and Lapanje, A., 2019. Natural microbial communities can be manipulated by artificially constructed biofilms. Advanced Science, 6(22), p.1901408.

5. Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity

Dr. Cornelius Fischer, Helmholtz-Zentrum Dresden-Rossendorf, Institute of Resource Ecology, Reactive Transport Department, Leipzig (Germany).

Vertical Scanning Interferometry (VSI) is a surface sensitive technique to analyze very precisely the topography of materials. Roughness differences in the nanometer range can be quantified. Compared to atomic force microscopy, VSI is a very fast method that allows the analysis of large fields of view up to the mm range. It therefore works complementary to AFM approaches or micro computed tomography surface analysis.

Furthermore, sequences of surface maps can be analyzed to create difference maps (1). These allow the calculation of rate maps (2), e.g. for the dissolution of crystals (3) or the degradation of plastics (4). These rate maps show the rate distribution of reacting chemically homogeneous materials. This is important information for parameterizing reactive transport models, which previously worked with only one reaction rate in such cases. This analytical information therefore also helps to improve the reliability of the predictions of modern numerical approaches (5, 6).

References:

1. Fischer, C.; Arvidson, R. S.; Lüttge, A., How predictable are dissolution rates of crystalline material? Geochimica et Cosmochimica Acta 2012, 98, 177-185.

2. Fischer, C.; Luttge, A., Pulsating dissolution of crystalline matter. PNAS 2018, 115 (5), 897-902.

3. Fischer, C.; Luttge, A., Beyond the conventional understanding of water–rock reactivity. Earth and Planetary Science Letters 2017, 457, 100-105.

4. Lippold, H.; Kahle, L.; Sonnendecker, C.; Matysik, J.; Fischer, C., Temporal and spatial evolution of enzymatic degradation of amorphous PET plastics. npj Materials Degradation 2022, 6 (1), 93.

5. Karimzadeh, L.; Fischer, C., Implementing Heterogeneous Crystal Surface Reactivity in Reactive Transport Simulations: The Example of Calcite Dissolution. ACS Earth and Space Chemistry 2021, 5 (9), 2408-2418.

6. Schabernack, J.; Fischer, C., Improved kinetics for mineral dissolution reactions in pore-scale reactive transport modeling. Geochimica et Cosmochimica Acta 2022, 334, 99-118.

Dr. Stefan Schymura (HZDR)

The use of vertical scanning interferometry to characterize a reacting surface is illustrated using the case study of demineralization and remineralization of human teeth. Human teeth are mainly composed of calcium and phosphate that are present in the dental hard tissue in the form of hydroxyapatite of varying crystallinity and purity. Low pH environments (< pH 5.5) that can be caused by acidic food and drink or microbially from nutritional sugar can cause the erosion of the dental hard tissue by dissolving the hydroxyapatite structures. These processes are naturally counteracted by calcium and phosphate ions contained in the saliva and dairy products such as curd. At the higher pH of the saliva the eroded structures are remineralized, with saliva proteins playing a crucial role as calciumphosphate carrier proteins. This process can be enhanced by increasing the calcium and phosphate concentration in the oral cavity, for example by addition of Ca-caseinate, a calciumphosphate nanocluster containing protein derived from milk production. The dynamics of the de- and remineralization can easily be tracked with nanometer precision by the use of vertical scanning interferometry. To characterize the reacting surface the height data obtained by measurements taken after each step of a series of de- and remineralization steps is transformed into roughness data using converged Sq values. For every pixel of the height data the Sq value - the standard deviation of the height data - is calculated in a 7x7 pixel window to yield detailed roughness maps that can be evaluated according to their changes during de- and remineralization using the histograms of the Sq maps. Using this workflow several conclusions concerning dental health can be derived from a series of facile experiments, concerning the erosion of dental hard tissue, the treatment of such tooth wear with remineralization agents and the effectiveness of fluoridation.

Literature:

Schymura et al. 2023, Dental Materials, in rev.

Lechner et al. 2015, ACS Appl. Mater. Interfaces, 7, 18937−18943. DOI: 10.1021/acsami.5b04790

Fischer & Luttge 2007, American Journal of Science, 307, 955–973, DOI: 10.2475/07.2007.01

Pignatelli et al. 2016, Dental Materials, 32(10), 251-261, DOI: 10.1016/j.dental.2016.07.004

Lussi & Carvalho 2014, Monographs in Oral Sciences, 25, 1-15, DOI: 10.1159/000360380

Dr. Andre Skirtach, from Ghent University.

Developing surfaces is important for assuring effective interaction or repulsion of microbial organisms. Various methods for developing and analyzing the bacterial biofilm formation and microbial cell-surface interface in general take a special place in this process.

Traditionally used fluorescence microscopy had been used to investigate and visualize the formation of bacterial biofilms and to monitor cell-surface interactions. But the addition of labels can substantially limit investigation of molecules and molecular interactions.

In this Chapter, we highlight application of other and alternative techniques, such as Raman microscopy and atomic force microscopy (AFM) to investigate microbial cell-surface and surface-colloid interactions. In Raman microscopy, unlike to what is the case in fluorescence microscopy, vibrations of molecules reveal the composition of microbial cells. Raman microscopy is based on scattering and not on absorption of light, as such, the scattering finger-prints of molecules are rather weak. That prompts to use visible or near-infrared lasers with a high power which may even lead to temperature rise upon illumination. But due to the fact that biological samples are typically located in an aqueous solution, the heat accumulation does not pose essential problem. Thus, molecules and organisms can be visualized in a label-free way, but enhancement of rather weak scattering signals is desired. One way to obtain such enhancement is based on surface-enhanced Raman spectroscopy, in which gold or silver nanoparticles are placed in vicinity of molecules whose vibrational scattering signal needs to be amplified.

Another technique that is quite unique for investigation of cell-surface and surface-colloidal interactions is atomic force microscopy (AFM). In this technique, a small probe, also called a cantilever, is moved over the surface of samples revealing topography of samples at the nanoscale level. In addition, AFM can be used to characterize the surface charges and mechanical properties. Although AFM is a seemingly slow technique, latest developments, including already those available commercially, provide rather fast scanning capabilities. One of the limitations of AFM is a finite thickness of samples scanned by a cantilever.

In conclusion, novel techniques, in addition to traditional fluorescence microscopy based techniques provide important information, otherwise not possible to obtain using fluorescent labels. Raman microscopy, based on scattering of light from molecular vibrations. Application of these techniques should lead to uncovering novel mechanisms in microbial cell-surface and surface-colloid interactions.

References:

1. Alexey Yashchenok, Admir Masic, Dmitry Gorin, Bong Sup Shim, Nicholas A Kotov, Peter Fratzl, Helmuth Möhwald, Andre Skirtach. Nanoengineered Colloidal Probes for Raman‐based Detection of Biomolecules inside Living Cells. Small 2013, 3, 351-356. DOI: 10.1002/smll.201201494.

2. Pieter C Wuytens, Ananth Z Subramanian, Winnok H De Vos, Andre G Skirtach, Roel Baets. Gold nanodome-patterned microchips for intracellular surface-enhanced Raman spectroscopy. Analyst 2015, 140, 8080-8087. DOI: 10.1039/C5AN01782C.

3. Cristina García-Timermans, Peter Rubbens, Frederiek-Maarten Kerckhof, Benjamin Buysschaert, Dmitry Khalenkow, Willem Waegeman, Andre G Skirtach, Nico Boon. Label-free Raman characterization of bacteria calls for standardized procedures. Journal of microbiological methods 2018, 151, 69-75. Doi: 10.1016/j.mimet.2018.05.027.

4. Louis Van der Meeren, Joost Verduijn, Dmitri V Krysko, Andre G Skirtach. AFM analysis enables differentiation between apoptosis, necroptosis, and ferroptosis in murine cancer cells. iScience 2020, 23, 101816. Doi: 10.1016/j.isci.2020.101816.

5. Mihaela Delcea, Stephan Schmidt, Raghavendra Palankar, Paulo AL Fernandes, Andreas Fery, Helmuth Möhwald, André G Skirtach. Mechanobiology: correlation between mechanical stability of microcapsules studied by AFM and impact of cell‐induced stresses. Small 2010, 6, 2858-2862.

6. Jie Li, Dmitry Khalenkow, Dmitry Volodkin, Ales Lapanje, Andre G Skirtach, Bogdan V Parakhonskiy. Surface enhanced Raman scattering (SERS)-active bacterial detection by Layer-by-Layer (LbL) assembly all-nanoparticle microcapsules. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2022, 650, 129547. Doi: 10.1016/j.colsurfa.2022.129547.

Dr. Stefan Schymura (HZDR)

The use of radioactive tracers for (nano-)colloid tracing in the environmental sciences is introduced. Radiotracers are composed of atoms with an unstable nucleus, so-called radionuclides, due to an excess in protons or neutrons, mass or energy. These atoms decay to stable atoms by sending out ionizing radiation. Of the different radiation types - alpha, beta, gamma – only beta and gamma radiation can leave the sample easily. This renders radionuclides that undergo beta or gamma decays useful as tracers, as they can easily be quantified from the outside of the sample. This is especially useful when tracing substances such as manufactured nanomaterials at low environmentally relevant concentrations in complex environmental compartments, as the detection is very sensitive and almost independent of the sample matrix. For scientific usage radiotracers are obtained from suitable target materials via nuclear reactions using for example proton irradiation at a cyclotron or neutron irradiation at a nuclear reactor, as well as the isolation from fission products produced in a nuclear reactor. The radiotracers can then be introduced into the nanoparticles to-be-traced via the synthesis of the nanoparticles using radioactive starting materials, the binding of the radiotracer to the nanoparticles, the activation of the nanoparticles using proton/neutron irradiation, the recoil labeling utilizing the recoil of a nuclear reaction to implant a radiotracer into the nanoparticle, and the in-diffusion of radiotracers into the nanoparticles at elevated temperatures. After such a labeling process the nanoparticles can be detected in 1 – 4D using various techniques, even against high elemental and particle backgrounds. Safe usage of radiotracers is ensured by following basic rules of radiation protection in a controlled lab zone.

Literature:

Kratz: Nuclear and Radiochemistry Fundamentals and Applications, 2021, Wiley.

Loveland et al.: Modern Nuclear Chemistry, 2017, Wiley.

Hildebrand et al. 2015 J Nanopart. Res 17: 278. DOI: 10.1007/s11051-015-3080-8

Yin et al. 2017 Chem. Rev. 117(5) 4462-4487. DOI: 10.1021/acs.chemrev.6b00693

Schymura et al. 2017 Angew. Chem. Int. Ed. 56(26) 7411-7414 DOI: 10.1002/anie.201702421

Gibson et al. 2011 Arch. Toxicol. 85:751–773. DOI 10.1007/s00204-011-0701-6

SurfBio WP1 Webinar Series: SurfBio website

Dr. Stefan Schymura (HZDR)

The use of radiotracing of (nano-)colloids in the environmental sciences is illustrated using example studies concerning the environmental fate of ceria nanoparticles. Radiolabeling of nanoparticles can be achieved by the synthesis of the nanoparticles using radioactive starting materials, the binding of the radiotracer to the nanoparticles, the activation of the nanoparticles using proton/neutron irradiation, the recoil labeling utilizing the recoil of a nuclear reaction to implant a radiotracer into the nanoparticle, and the in-diffusion of radiotracers into the nanoparticles at elevated temperatures. Depending on the specific activity of the radiolabeled nanoparticles they can be detected well into the ng/L range. Radiolabeling procedures exist for many of the most common nanoparticles.

The activation of CeO2 nanoparticles using proton or neutron activation or the production of a Ce from Lanthanum at a cyclotron and a subsequent in-diffusion labelling can be used to produce radiolabeled CeO2 nanoparticles. The In-Diffusion technique produces [Ce]CeO2 nanoparticles with the radiolabel situated close to the particle surface while the activation produces an equally distributed radiolabel. This leads to different release rates upon dissolution of the particles which can be used to track the role of dissolution processes in CeO2 NP fate. Using different batches of [Ce]CeO2 and a batch of dual-labeled [Ce]CeO2 NP we could prove the particulate uptake of anthropogenic cerium by plants and the ionic uptake by freshwater shrimp. Gamma spectroscopy allows the sensitive detection of the radionuclides in the plants and animals without excessive sample preparation and autoradiographic imaging provides 2D spatial information about the tracer distribution.

Literature:

Schymura et al. 2017 Angew. Chem. Int. Ed. 56(26) 7411-7414 DOI: 10.1002/anie.201702421

Schymura et al. 2021 Environ. Sci.: Nano 8, 1934-1944 DOI: 10.1039/D1EN00264C

Hildebrand et al. 2015 J Nanopart. Res 17: 278. DOI: 10.1007/s11051-015-3080-8

Hildebrand et al. 2012 J Nanopart. Res 14: 1142. DOI: 10.1007/s11051-012-1142-8

Gibson et al. 2011 Arch. Toxicol. 85:751–773. DOI 10.1007/s00204-011-0701-6

Deng et al. 2008 Nanotechnology 19 075101. DOI: 10.1088/0957-4484/19/7/075101

Yin et al. 2017 Chem. Rev. 117(5) 4462-4487. DOI: 10.1021/acs.chemrev.6b00693

Cydzik et al. 2012 J Nanopart 14:1185. DOI: 10.1007/s11051-012-1185-x

Ichedef et al. 2013 J Nanopart Res 15:2073. DOI: 10.1007/s11051-013-2073

Holzwarth et al. 2014 J Nanopart Res 16, 2574. DOI: 10.1007/s11051-014-2574-0

SurfBio WP1 Webinar Series

Dr. Stephan Hilpmann, from Helmholtz-Zentrum Dresden-Rossendorf (HZDR)

Microbial uranium(VI) reduction processes play an important role for the safe disposal of high-level radioactive waste, as well as for new bioremediation strategies of radionuclide-contaminated surroundings. In this chapter, you will learn about different spectroscopic and microscopic techniques that can be used to investigate microbial uranium(VI) reduction processes on a molecular level. Decreasing uranium concentration during reduction experiments can provide a first hint for an ongoing formation of uranium(IV) due to its lower solubility. Luminescence spectroscopic techniques can be used to investigate the speciation of uranium(VI) in the initial solution and possible changes over time. UV/Vis spectroscopy can verify the formation of uranium(IV). Furthermore, the recorded spectra allow calculating the proportions of both oxidation states in the samples. Uranium can be localized in the samples using transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy. Moreover, this method is able to show a co-localization of the radionuclide with other elements. A complementary application of these different methods can be used to get a deeper inside into the ongoing processes during microbial uranium(VI) reduction.

Literature:

Wall JD, Krumholz LR. Uranium reduction. Annu Rev Microbiol. 2006;60: 149–166. doi:10.1146/annurev.micro.59.030804.121357

Lloyd JR. Microbial reduction of metals and radionuclides. FEMS Microbiol Rev. 2003;27: 411–425. doi:10.1016/S0168-6445(03)00044-5

Lovley DR, Phillips EJP, Gorby YA, Landa ER. Microbial reduction of uranium. Nature. 1991;350: 413–416. doi:10.1038/350413a0

Vettese GF, Morris K, Natrajan LS, Shaw S, Vitova T, Galanzew J, et al. Multiple Lines of Evidence Identify U(V) as a Key Intermediate during U(VI) Reduction by Shewanella oneidensis MR1 . Environ Sci Technol. 2020;54: 2268–2276. doi:10.1021/acs.est.9b05285

Hilpmann S, Rossberg A, Steudtner R, Drobot B, Hübner R, Bok F, et al. Presence of uranium (V) during uranium (VI) reduction by Desulfosporosinus. Sci Total Environ. 2023;875: 162593. doi:10.1016/j.scitotenv.2023.162593