Pyrex-Nitride AFM probes

Unravelling the Molecular Network Structure of Biohybrid Hydrogels

Glycosaminoglycan-based biohybrid hydrogels are highly promising materials for tissue engineering and regenerative medicine due to their ability to provide cell-instructive environments. In this article, Jana Sievers-Liebschner, Ron Dockhorn, Jens Friedrichs, Thomas Kurth, Peter Fratzl, Jens-Uwe Sommer, Carsten Werner, and Uwe Freudenberg investigate the nanoscale molecular network structure of these hydrogels using an integrated analytical approach.

The study combines transmission electron microscopy, X-ray scattering, computer simulations, and AFM-based nanoindentation to quantitatively characterize nanoscale polymer network connectivity and structural inhomogeneities. These parameters are essential for understanding hydrogel mechanics, growth factor delivery, and cell–material interactions relevant to regenerative therapies and organoid culture systems.

Atomic force microscopy (AFM)-based nanoindentation measurements were performed to determine the mechanical stiffness of the hydrogels in both PBS and ethanol environments. Measurements were conducted using a modified NanoWorld tipless Pyrex-Nitride PNP-TR-TL-Au AFM probe equipped with a 10 μm silica bead for colloidal probe nanoindentation.

Nanoindentation experiments were carried out using a set point of 6 nN and an approach/retract velocity of 5 μm/s. At least 70 force–distance curves were recorded for each sample at different positions across the hydrogel surface. Young’s modulus values were extracted using the Hertz model, enabling quantitative evaluation of hydrogel nanomechanical properties.

This work demonstrates how AFM-based nanoindentation with a NanoWorld AFM probe contributes to the detailed characterization of biohybrid hydrogel networks and supports the development of engineered matrices for biomedical applications.

Fig. 5. Computational modelling of starPEG-heparin hydrogel networks. A: Simulation snapshots of hydrated and dehydrated starPEG-heparin hydrogels. For the hydrated network (A1), a good solvent (equivalent to PBS) was assumed. To model the dehydrated state (A2), parameters were adjusted to promote the self-aggregation of starPEG molecules in a poor solvent (e.g., ethanol). Networks with different effective molar ratios after crosslinking (γBMC, where BMC is the Biggest Molecule Cluster) are shown, assuming a 90 % extent of reaction between starPEG (grey) and heparin (yellow). Cube size: L = 150 nm. B: Molar ratio of the BMC γBMC after crosslinking at an extent of reaction p = 0.9, plotted as a function of the initial molar ratio. The dotted line represents the theoretical ideal value, while the blue line shows the experimentally determined Young’s moduli as a function of crosslinking degree. C: Incorporation efficiency of starPEG and heparin within the BMC, calculated as the number of starPEG or heparin molecules in the BMC divided by the number in the reaction mixture (ideal network). Insert: Total number of starPEG or heparin molecules in the reaction mixture (initial) or within the BMC. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Full citation:
Sievers-Liebschner, J.; Dockhorn, R.; Friedrichs, J.; Kurth, T.; Fratzl, P.; Sommer, J.-U.; Werner, C.; Freudenberg, U.
Unravelling the molecular network structure of biohybrid hydrogels.
Materials Today Bio 2025, 34, 102249.
https://doi.org/10.1016/j.mtbio.2025.102249

Open Access The article “ Unravelling the molecular network structure of biohybrid hydrogels” is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Does the Hfq Protein Contribute to RNA Cargo Translocation into Bacterial Outer Membrane Vesicles?

Gram-negative bacteria release outer membrane vesicles (OMVs) that play a central role in host–pathogen interactions by transporting biomolecules, including proteins and nucleic acids. In this article, Marisela Velez and Véronique Arluison investigate the role of the RNA chaperone Hfq in mediating the interaction of small regulatory RNAs (sRNAs) with bacterial membranes.

In this article, it is shown that RNA binding to the inner membrane of Escherichia coli occurs in an Hfq-dependent manner. The study further demonstrates that membrane composition is a key factor in this process, with cardiolipin-rich lipid domains significantly enhancing RNA–membrane interactions. These findings provide new insight into the mechanism of RNA translocation from the cytoplasm to the periplasm, supporting its subsequent incorporation into OMVs.

Atomic force microscopy (AFM) was used to verify the formation and integrity of supported lipid bilayers and to monitor peptide–membrane interactions. Imaging was performed in tapping mode using a NanoWorld Pyrex-Nitride PNP-DB AFM probe with a resonance frequency of 15 kHz and a spring constant of 0.48 N/m. Measurements were carried out in liquid environment, enabling high-resolution characterization of biologically relevant membrane structures.

This work highlights the importance of AFM-based analysis for studying lipid–protein interactions and provides new understanding of RNA transport mechanisms in bacterial systems.

Figure 1
E. coli lipid bilayer incubated in the absence (A) or presence (B) of Hfq-CTR. Panel (A) shows the E. coli lipids bilayer. The height profile under the line shown on the upper image indicates that the domains are 0.8 nm higher than the rest of the membrane. The lower panel shows a three-dimensional representation of a small region. Panel (B) shows the E. coli lipid bilayer incubated in the presence of Hfq-CTR. The peptide accumulated on top of some of the domains, generating 1 nm high regions in some of them, as shown on the height profile. The arrows point the regions where the change in height occurs.

Full citation:
Velez, M.; Arluison, V.
Does the Hfq Protein Contribute to RNA Cargo Translocation into Bacterial Outer Membrane Vesicles?
Pathogens 2025, 14(4), 399.
https://doi.org/10.3390/pathogens14040399

License: CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/)

Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells

Gastric cancer has a high rate of recurrence, and as such, immunotherapy strategies are being investigated as a potential therapeutic strategy. *

Although the involvement of immune checkpoints in immunotherapy is well studied, biomechanical cues, such as target cell stiffness, have not yet been subject to the same level of investigation. *

Changes in the cholesterol content of the cell membrane directly influence tumor cell stiffness. *

In the article “Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells” Lijuan Zhu and Hongjin Wang investigate the effect of cholesterol on NK cell-mediated killing of gastric cancer stem-like cells. *

They report that surviving tumor cells with stem-like properties elevated cholesterol metabolism to evade NK cell cytotoxicity. *

Inhibition of cholesterol metabolism enhances NK cell-mediated killing of gastric cancer stem-like cells, highlighting a potential avenue for improving immunotherapy efficacy. *

This study suggests a possible effect of cancer cell stiffness on immune evasion and offers insights into enhancing immunotherapeutic strategies against tumors. *

Measurement of cell stiffness by AFM:

A customized commercially available atomic force microscope (AFM) and NanoWorld Pyrex-Nitride PNP-TR AFM probes were used for the measurement of cell stiffness by AFM. *

Atomic force microscopy cell stiffness was measured according to standard methodology. *

AFM force curves were captured with a customized AFM placed atop an inverted optical microscope that had a heating stage for live-cell imaging and a ×20 objective. Using an XY stage, the materials were moved until the desired cell, which could be observed under an optical microscope, was positioned beneath the AFM tip. Using a NanoWorld PNP-TR-B AFM cantilever (NanoWorld), the force curves on the cell were collected at a rate of ~ 5 μm·s−1 in the relative trigger mode (15 nm trigger threshold). *

By utilizing a thermal tuning and the deflection sensitivity of 170 nm·V−1, the AFM cantilever spring constant was determined to be 0.08 N·m−1. *

Single cells were measured both before and after treatment at 37 °C. The force curves were processed using the AFM’s analysis software, which also computed the Young’s modulus of the sample. This was accomplished by fitting the approach curve to an indentation of less than 500 nm (to account for stiffness) and assuming a cortical Poisson’s ratio of 0.3.*

NanoWorld Pyrex-Nitride PNP AFM probe - silicon nitride AFM cantilever and silicon nitride AFM tip — NanoWorld Pyrex-Nitride AFM probe series – AFM tip and AFM cantilever made of silicon nitride

*Lijuan Zhu and Hongjin Wang
Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells
FEBS Open Bio, Volume 14, Issue 5, May 2024, Pages 855-866
DOI: https://doi.org/10.1002/2211-5463.13793

Open Access The article “Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells” by Lijuan Zhu and Hongjin Wang is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.