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. 2022 Feb 11;12(4):608.
doi: 10.3390/nano12040608.

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers

Md Arif Khan  1 , Ramy W Ghanim  1 , Maelyn R Kiser  1 , Mahsa Moradipour  1 , Dennis T Rogers  2 , John M Littleton  2 , Luke H Bradley  3 , Bert C Lynn  4 , Stephen E Rankin  1 , Barbara L Knutson  1
Affiliations

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers

Md Arif Khan et al. Nanomaterials (Basel). .

Abstract

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4'-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Keywords: conjugation; diazirine; heterobifunctional linker; mesoporous silica; nanoparticle; oligopeptide.

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Conflict of interest statement

J.M.L. and D.T.R. are employees of Naprogenix Inc. and J.M.L. owns stock in the company. The funders of this work had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic diagram of the peptide attachment strategies using hetero-bifunctional cross-linker Sulfo-NHS-LC-Diazirine (SNLD): (a) Type-1 attachment of the linker to the particle amine group first using the NHS group and then attaching to the peptide amine group using the UV-reactive diazirine group and (b) Type-2 attachment of the linker to the peptide amine group first using the NHS group and then attaching to the particle amine group using the UV-reactive diazirine group.
Figure 2
Figure 2
Representative (a) transmission electron micrograph and (b) scanning electron micrograph of bare MSNPs showing spherical particles with average particle diameter 146 ± 27 nm and radially oriented mesopores with an average pore diameter of 8 nm.
Figure 3
Figure 3
UV–Vis absorbance (left) and fluorescence intensity (right) of (a,b) particles re-suspended in solution and (c,d) supernatant after fluorescein isothiocyanate (FITC)-labeled RSSV (Arg-Ser-Ser-Val tetrapeptide) attachment to the particles using Type-2 conjugation. Solid red lines and dashed blue lines represent results with or without UV treatment, respectively. Both UV absorbance and fluorescence intensity of the particles increases after UV treatment, whereas supernatant intensity decreases, suggesting successful binding of peptide.
Figure 4
Figure 4
FTIR spectra of peptide functionalized particles relative to bare MSNPs and MSNPAs, as well as fresh linker (sulfo-NHS-LC-diazirine) and peptides with peaks corresponding to major functional groups labeled. In the figure, 4RSSV is a peptide made of 4 sequential RSSV units and 4G is a tetraglycine.
Figure 5
Figure 5
Thermogravimetric analysis (TGA) profiles of particles showing relative mass loss with temperature increase for (a) MSNP, (b) MSNPA, (c) MSNPA-4G, (d) MSNPA-RSSV and (e) MSNPA-4RSSV.
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