Benchtop LVEM5 electron microscope helps development of novel antimicrobial agents

Antimicrobial peptides (AMP) proposed as antibiotic alternative

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Chitosan nanocrystal scaffolds

In their 2022 publication, "Design of chitosan nanocrystals decorated with amino acids and peptides", the National Research Council of Canada has proposed a new methodology to functionalize chitosan nanocrystals (ChsNC) with amino acids and/or peptides, applying fundamentals of Solid-phase peptide synthesis (Merrifield,1963) with the purpose of creating new nanomaterials-based antimicrobials which can target specific cells based on their functionalization.

According to the authors, owing to chitosan’s structure and ease in functionalization, the material shows potential as a scaffold material for targeted therapeutic peptides. With primary amino groups naturally present of the surface of chitosan and chitosan nanoparticles, amino acids and subsequent peptides can be grafted to its surface.

While in the scope of this work generic peptides were attached to ChsNCs, these efforts offer a proof-of-concept for target-specific AMP-grafted ChsNCs.

TEM imaging used to assess grafting methodology  

In addition to DLS, ELS and NMR characterization, TEM images provided by a LVEM5 benchtop TEM operating at 5kV were collected at each step of the grafting and functionalization of the particle to assess impact on size and morphology of the rod-shaped nanoparticles. Examples shown below are unmodified, Lysine-modified and Lysine-Arginine-Lysine modified ChsNCs synthesized by the group.

Fig. 1. TEM images of A) unmodified ChsNCs, dispersed in water, B) ChsNC-Lys, C) ChsNC-Lys-Arg-Lys. (Adapted from Hrapovic, 2022)

Subsequently to imaging the unmodified nanocrystalline chitosan rods, the TEM analysis conducted by the authors confirmed that the general dimensions and morphology of the nanocrystals were not affected by the multiple grafting steps, which involved a variety of harsh reactive chemicals and wash solvents.

Conclusion

Researchers from the National Research Council of Canada demonstrated a novel methodology for producing amino acid and peptide decorated chitosan nanocrystals, termed ChsNCs, allowing for potential applications in the global fight against antimicrobial resistance.  The LVEM 5’s low-voltage operation allows enhanced contrast imaging in low atomic weight materials. In the case of the studied ChsNC, heavy metal contrast staining was not required for imaging, saving preparation time and resources while avoiding potential structural alterations that can be attributable to common staining protocols.

 

About the author:

Marie Beaulieu, B.Eng, has many years of experience working with materials ranging from pharmaceuticals, to industrial biocides, to nanomaterials. As part of the Applications team at Delong America she enjoys supporting LVEM users from industry and academia alike.

 

References:

• Merrifield, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society, 85(14), 2149–2154. https://doi.org/10.1021/ja00897a025  
• Rotem, S., & Mor, A. (2009). Antimicrobial peptide mimics for improved therapeutic properties. Biochimica et Biophysica Acta (BBA) -Biomembranes, 1788(8), 1582–1592. https://doi.org/10.1016/j.bbamem.2008.10.020
• Sabahudin Hrapovic, Camilo F. Martinez-Farina, Jessie Sui, Jean-Danick Lavertu, Usha D. Hemraz, Design of chitosan nanocrystals decorated with amino acids and peptides. Carbohydrate Polymers, Volume 298, 2022, 120108, ISSN 0144-8617, https://doi.org/10.1016/j.carbpol.2022.120108.
• Saravanan, M., Barabadi, H., Mostafavi, E., & Webster, T. J. (2022). In Emerging nanomaterials and nano-based drug delivery approaches to combat antimicrobial resistance (p. 53). Elsevier.

Application Notes