Miranda MCR et al. (2025), Adjuvant Effect of Mesoporous Silica SBA-15 of ...

ECB-ART-54098

ACS Omega 2025 Jun 01;1025:27534-27549. doi: 10.1021/acsomega.5c03459.

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Adjuvant Effect of Mesoporous Silica SBA-15 of Different Morphologies on Antidiphtheria Immune Response.

Miranda MCR , Nunes CM , Losito DW , Rocha FM , Pedro JAF , Favoretto BC , Teobaldo GBM , Cides da Silva LC , Lopes JLS , Oliveira CLP , Fantini MCA , Ribeiro OG , Sant'anna OA , Martins TS .

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Ordered mesoporous silica SBA-15 improves the humoral response as a vaccine adjuvant because of its structural properties. Its morphology is dependent on synthesis conditions and can alter antigen encapsulation and immune response; all tested variants were safe and able to immunize against diphtheria. One of the additional advantages of SBA-15 is that its morphology can be modulated by adjusting the synthesis conditions like temperature, stirring speed, and solvent addition. In this study, SBA-15 was selected as a vaccine adjuvant in immunization against diphtheria by varying four modifications of the synthesis parameters for preparing SBA-15 particles with different morphologies. SEM analyses confirmed that different morphologies were obtained including rope-shaped aggregated rods (S1), filiform rods (S2), hexagons (S3), and nanospheres (S4). All synthesized SBA-15 samples presented an ordered mesoporous structure, with the characteristic reflections of a two-dimensional hexagonal structure and lattice parameter values with small differences (a(hkl) = ∼11 nm at 12), indicating that the silica mesostructure was preserved after incorporation from diphtheria anatoxin (dANA). The SAXS and NAI results indicate that mainly in samples S1 and S4, the dANA is encapsulated in the mesopores as well as in the SBA-15 macropores. Fluorescence analyses revealed the preservation of the aromatic microenvironment of tryptophan, similar to pure protein, except for sample S3, which showed a shift in emission wavelengths to 356 and 372 nm, indicating exposure of tryptophan to the more polar microenvironment. SRCD analyses confirmed the maintenance of dANA's secondary structure in all samples. In the immunogenic assay, the S3dANA sample stood out, presenting a significantly higher primary immune response. However, the immunogenic responses increased and became equal in the secondary response without any variation between different silica morphologies. It is concluded that all SBA-15-based adjuvants with different morphologies are biocompatible and present a good immunogenic response when they are applied as vaccine adjuvants.

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	1. SEM micrographs of S1, S2, S3, and S4 samples: low- and high-magnification images revealing the different morphologies of SBA-15 samples affected by synthesis parameters. The upper part scales: S1, S2, and S3 are 10 μm and S4 is 1 μm. Lower part scales: S1, S2, and S3 are 1 μm and S4 is 0.5 μm.
	2. SAXS curves of the SBA-15 and SBA-15:dANA samples: A) S1 and S1dANA; B) S2 and S2dANA; C) S3 and S3dANA; and D) S4 and S4dANA.
	3. Advanced modeling was performed for the samples investigated. Symbols: experimental data. Red solid lines: smeared data. Blue solid lines: desmeared data. For sample S4 and S4dANA the results for the two phases are shown.
	4. Nitrogen adsorption–desorption isotherms (A,B) and pore size distributions (C,D) of the pure SBA-15 and SBA-15:dANA samples, respectively.
	5. TG and DSC curves of pure SBA-15 samples (S1, S2, S3, and S4), and SBA-15:dANA composites (S1dANA, S2dANA, S3dANA and S4dANA).
	6. FTIR spectra of pure SBA-15 samples (S1, S2, S3, and S4) (A, B) and S1dANA, S2dANA, S3dANA, and S4dANA composites (C, D).
	7. Fluorescence spectra of dANA in solution and SBA-15:dANA composites, excitation was performed at 280 nm.
	8. SRCD spectra of dANA in solution and SBA-15 incorporated with dANA.
	9. Hemolytic activity of pure SBA-15 samples (S1, S2, S3, and S4) and SBA-15:dANA composites (S1dANA, S2dANA, S3dANA, and S4dANA). Note: The circles at the top of the bars are the wells of the plate with the solutions tested, showing that the samples did not show a red color that would represent hemolysis and toxicity.
	10. Anti-dANA (diphtheria anatoxin) antibody response (log2 titers) of male and female animals of the HIII strain immunized subcutaneously with SBA-15:dANA composites (S1dANA, S2dANA, S3dANA, and S4dANA). The results are presented as mean ± SD (N = 5). * visualization × primary response; δ secondary response × other groups. Considered significant at p < 0.01.