AVS2017 Session BI+AS+MI+SA-TuA: Bio from 2D to 3D: Challenges in Fabrication and Characterization & Flash Presentations

Sulphated and non-sulphated glycosaminoglycans (GAGs) can be instrumental in biomedical technologies beyond. In particular, incorporation of GAGs into biomaterials has been demonstrated to allow for the biomimetic modulation of growth factor signaling, providing control over therapeutically relevant cell fate decisions in various different settings. In an attempt to systematically explore the related options, we have introduced a rational design strategy for biology-inspired hydrogels based on multi-armed poly(ethylene glycol), GAGs and peptides (1,2,3). The theoretically predicted decoupling of biochemical and mechanical gel properties was confirmed experimentally and applied for implementing GAG-based biofunctionalization schemes to afford cell adhesiveness and morphogen presentation. A number of applications of customized GAG-based materials will be given, including inflammation-modulating wound dressings (3), cryogel particles to support cell replacement in Parkinson’s disease (4) and gel matrices to enable tissue and disease in vitro models for cancer biology (5,6) and nephrotoxicity studies. In sum, our reported approach demonstrates the power of joint theoretical and experimental efforts in creating bioactive materials with specifically and independently controllable characteristics (7).

References

(1) U. Freudenberg, J.-U. Sommer, K. Levental, P. Welzel, A. Zieris, K. Chwalek, , K. Schneider, S. Prokoph, M. Prewitz, R. Dockhorn, C. Werner (2012) Adv Funct Mater 22:1391

(2) M. Tsurkan, K. Chwalek, S. Prokoph, A. Zieris, K. Levental, U. Freudenberg, C. Werner (2013) Adv Mater 25:2606

(3) U. Freudenberg, A. Zieris, K. Chwalek, M.V. Tsurkan, M.F. Maitz, P. Atallah, K.R. Levental, S.A. Eming, C.Werner (2015) J Control Release 220:79

(4) B. Newland, P.B. Welzel, H. Newland, C. Renneberg, P. Kolar, M. Tsurkan, A. Rosser, U. Freudenberg, C. Werner (2015) Small 11:5047

(5) K. Chwalek, M.V. Tsurkan, U. Freudenberg, C. Werner (2014) Sci Rep 4:4414

(6) L.J. Bray, M. Binner, A. Holzheu, J. Friedrichs, U. Freudenberg, D.W. Hutmacher, C. Werner (2015) Biomaterials 53:609

Biohybrid materials consist of biological entities and artificial, often inorganic materials. These biohybrids may be used in many fields of applications, ranging from biosensors to implant materials. In this context, bottom-up approaches, in which small elementary building blocks of matter are used to form larger elements through self-assembly have gained a lot of interest.

Plant viruses are promising candidates for such building blocks. Because of their simple structure and pre-defined size and form they have a high potential for self-assembly. Furthermore they can be genetically manipulated to create new functionalities by extending the capsid with different side chains.

We could show that unspecific electrostatic interactions govern the formation of large ordered 2D structures of self-assembled icosahedral tomato bushy stunt virus (TBSV) particles. By adding amino acid side chains to the capsid subunit the isoelectric point of the virus is changed. Thus by the right combination of virus modification, substrate and pH (and as a minor effect ionic strength) one can control the dimensions of 2D virus islands which may form layers with macroscopic dimensions. Specific structures in these 2D layers may be introduced by substrates which are pre-structured, e.g. by nano imprint lithography.

In addition to the electrostatic control the amino acid side chains allow also more specific interactions. Examples are histidine side chains interacting with Ni ions or gold binding peptide side chains with Au. With these specific interactions, also the third dimension is accessible. This opens the possibility to play with viruses in a kind of nano Lego which will soon become reality.

In this contribution we will show a scanning force and scanning electron microscopy study of the self-assembly of 2D and 3D structures of TBSV on Si and mica surfaces. The three dimensional structure is based on a homogeneous layer consisting of virus-particles carrying additional 4xAsp6xHis side chains (lowest stack). For the following second stack the chemical selectivity of these side chains to Ni ions (here: Ni-nitrilotriacetic acid (Ni-NTA) carrying a 5 nm Au nanoparticle was utilized. Au-binding virus-particles interact with these Au particles and create the third stack of this 3D virus architecture. The success of this strategy could be proven by SFM height measurements which reveal a height in the range of 66 nm, which corresponds to two layers of virus particles (30 nm each) coupled by Ni-NTA.

Lüders et al. (2012). Tomato bushy stunt viruses (TBSV) in nanotechnology investigated by scanning force and scanning electron microscopy. Colloids Surf. B91, 154

The advent of cluster ion beams for time-of-flight secondary ion mass spectrometry (ToF-SIMS) instrumentation has opened up many opportunities for depth profiling organic samples. Combined with its high lateral resolution imaging capabilities, SIMS can provide 3D imaging information from a wide range of organic materials including cells and tissues. The ability to track chemical changes both across and throughout tissue sections could help identify molecular changes related to targeted drug delivery or disease states in the cellular micro-environment. While there have been many studies showing the utility of ToF-SIMS depth profiling for polymer materials, similar studies with cells and tissues have been limited. This has likely been due to the challenges encountered when working with biological samples. It has been shown that one can depth profile cells as long as the levels of buffer salts and other inorganic components is minimized. Similar work with depth profiling tissues has been limited. Herein we will present our findings on the challenges of depth profiling tissues and discuss ways these challenges may be avoided. Examples will be shown using both single beam argon cluster depth profiling and dual beam depth profiling using Bi3+ for analysis and argon clusters for sputtering. In general a significant loss in signal is seen after the first few layers of a tissue depth profile. This could be due to migration of components to the surface, ion beam damage, or ion suppression due to salts. In spite of these issues, tissue depth profiles can be acquired in most cases. The challenge then becomes processing and interpreting these large data sets. Ideas on how to overcome these challenges will be presented.

With the potential of high-throughput, high-resolution and high-sensitivity label-free imaging in 3D, secondary ion mass spectrometry imaging methods are, arguably, ones of the most powerful techniques for high-resolution chemical imaging of biological samples. However, there are some critical limitations for these analyses. As the high-performance SIMS instruments require high vacuum, a careful consideration of sample preparations is often needed. For example, advanced methods are necessary to prepare and measure complex hydrated bacterial biofilm structures. Also, in the pharmacological imaging of potential drug candidates at their targets, the positioning of water soluble drug compounds within cells or tissues can be altered by pre-treatment processes such as drying, resin-embedding or histological fixation. Advanced cryo-preparation methods are necessary for immobilisation of water in these samples to prevent the ultrastructural reorganisation and the loss or translocation of water-soluble molecules, to circumvent the use of chemical fixation and to enable their analysis in high-vacuum of mass spectrometry instruments.

The UK’s National Centre of Excellence in Mass Spectrometry Imaging (NiCE MSI) at NPL has a special focus on the development of advanced solutions to challenging measurements. Our recently innovated 3D OrbiSIMS instrument has the capability to handle and measure cryogenically-prepared samples. The instrument is equipped with a vacuum cryo transfer system that is compatible with cryo-SEM and cryo-TEM. A shuttle chamber allows the interchange of samples, in vacuum and cryogenically, between cryo-preparative equipment and the 3D OrbiSIMS instrument.

Nanoprobe X-ray fluorescence (nano-XRF) analysis allows spatially resolved imaging with chemical sensitivity. Approaching the diffraction limit at the next generation of storage rings, both, spatial resolution and brilliance are going to be strongly enhanced for nano-XRF experiments. For biological samples, the combination of nano-XRF with cryogenic sample environments allows to understand elemental distributions in cells with minimum preparation artefacts. In addition, the cryo-protected samples provide enhanced resistance against radiation damage, which is particularly important for the high photon densities at modern synchrotron sources. Three different applications of cryo-nano-XRF will be presented. For single melanosomes, the technique enabled us to prove the core-shell organization of the organelles using metals as surrogate markers. As second application, the distribution of metals in single, adherent cells was directly imaged without the requirement of additional markers. Finally, marine adhesives of diatoms were analyzed and the occurrence of metals are linked with the known organic constituents in the EPS of diatoms. In all three cases, the detection of metal distribution has provided a new view on the investigated samples. The cryogenic sample environments proved to be the key to apply synchrotron radiation to all three types of biological samples. The data will also be discussed in relation to the perspectives of new implementations that will enable fast cryo-3D imaging in the future.