Mesoporous Silica Nanoparticles as a Potential Platform for Vaccine Development against Tuberculosis

The increasing emergence of new strains of Mycobacterium tuberculosis (Mtb) highly resistant to antibiotics constitute a public health issue, since tuberculosis still constitutes the primary cause of death in the world due to bacterial infection. Mtb has been shown to produce membrane-derived extracellular vesicles (EVs) containing proteins responsible for modulating the pathological immune response after infection. These natural vesicles were considered a promising alternative to the development of novel vaccines. However, their use was compromised by the observed lack of reproducibility between preparations. In this work, with the aim of developing nanosystems mimicking the extracellular vesicles produced by Mtb, mesoporous silica nanoparticles (MSNs) have been used as nanocarriers of immunomodulatory and vesicle-associated proteins (Ag85B, LprG and LprA). These novel nanosystems have been designed and extensively characterized, demonstrating the effectiveness of the covalent anchorage of the immunomodulatory proteins to the surface of the MSNs. The immunostimulatory capacity of the designed nanosystems has been demonstrated by measuring the levels of pro- (TNF) and anti-inflammatory (IL-10) cytokines in exposed macrophages. These results open a new possibility for the development of more complex nanosystems, including additional vesicle components or even antitubercular drugs, thus allowing for the combination of immunomodulatory and bactericidal effects against Mtb.

"red" laser. For this purpose, 1 mg of nanoparticles was added to 10 mL of water followed by vortex and ultrasound to get a homogeneous suspension. Measurements were recorded by placing ca. 1 mL of suspension in DTS1070 disposable folded capillary cells (Malvern Instruments). The hydrodynamic size of nanoparticles was measured by dynamic light scattering (DLS) with the same Malvern instrument. Values presented are mean ±SD from triplicate measurements.
Surface morphology was analysed by scanning electron microscopy (SEM) on a JEOL JSM 6335F microscope (Tokyo, Japan). For this purpose, samples were mounted onto a copper stud, dried at 70 °C for 48 h under vacuum and coated with a film of Au previous to observation.
Transmission Electron Microscopy (TEM) was carried out with a JEOL JEM 2100 instrument operated at 200 kV (JEOL Ltd., Tokyo, Japan). Sample preparation was performed by dispersing ca. 1 mg of sample in 1 mL of 1-butanol followed by sonication in a low power bath sonicator (Selecta, Spain) for 5 min and then depositing one drop of the suspension onto carbon-coated copper grids.

Synthesis and characterization of MSNs
MSNs. Fluorescent MSNs were prepared following a modified Stöber method in which the fluorescent dye was covalently linked to the silica network [1,2,3]. APTS (5 μL, 0.023 mmol) was added over a stirred FITC (2 mg, 0.005 mmol) solution in EtOH (0.25 mL), under N2 atmosphere. The reaction was stirred for 2 h in the dark at RT and then added to a solution containing TEOS (5 mL, 0.023 mol) and EtOH (1 mL). The resulting solution was subsequently placed on a srynge dispenser to be transferred to the next reaction. The structure directing agent CTAB (1 g, 2.74 mmol) was dissolved in 480 mL of water and 3.75 mL of NaOH 2 M and the solution heated to 80 °C under gently stirring. At that point, the solution containing TEOS and the silane-modified fluorescein was added dropwise at a constant rate of 0.43 mL/min under vigorous stirring. The reaction was vigorously stirred for 1 h at 80 °C in the dark and then the suspension was cooled to room temperature, centrifuged and the particles washed with water, EtOH and finally dried.
The SEM analysis revealed that the obtained MSNs had uniform spherical shape with an average particle diameter of ca. 150 nm, and TEM images showed a highly ordered mesopore network of the MSNs ( Figure S2A and S2B, respectively). Low-angle XRD measurement showed a well-resolved characteristic pattern indexed to a p6mm symmetry of 2D hexagonal MCM-41 materials with a unit cell parameter of 4.46 nm (a0 = 2/3 √3 d10) ( Figure S2C). The well-ordered mesoporous structure was also confirmed by N2 adsorption porosimetry, exhibiting type IV isotherms which were analysed by BET and BJH methods to determine a surface area of 1368.4 m 2 /g and average pore diameter of 2.73 nm, respectively (see Figure 4 and Table 4 in manuscript).
MSNs-COOHext. The external surface functionalization of MSNs with carboxylic acids was performed onto the pore surfactant containing material (34.6% wt.). Approximately a quarter of the specific surface area of the free-surfactant material (1368.4 m2/g) was considered to be functionalized [1]. First, 1 g of CTAB-containing MSNs (0.654 g MSNs) was dehydrated at 80 °C under vacuum for 3 h in the dark. Subsequently, TESPSA (0.2032 g, 10% exc.) was dissolved in 15 mL of dry toluene and added to a vigorously stirred suspension of the CTABcontaining MSNs dispersed in dry toluene (60 mL), under N2 atmosphere. The reaction mixture was heated to 110 °C overnight in the dark, afterwards it was centrifuged and the modified MSNs were exhaustively washed with toluene and acetone and finally dried. The surfactant extraction was carried out by heating a well dispersed suspension of obtained solid in EtOH (360 mL), water (40 mL) and HCl (10 mL) overnight at 60 °C and then the solid was washed with water and EtOH. This process was repeated for 2 h and the solid dried under vacuum. The experimental value of -COOH groups in this material is 6.74 × 10 −4 mol/g MSN-COOHext, calculated from the organic content (5.4%) due to the functionalization, as determined by termogravimetry.
To provide anchoring points for the proteins, the external surface of the MSNs was functionalized with carboxylic acid groups in a first step, using a post-synthesis method. The condensation of the succinic anhydride alkoxysilane derivative TESPSA with the silanol groups of the outer silica surface, under water free conditions [4], was performed using the assynthesized MSN material containing the organic template filling the pores. With this methodology, a favoured functionalization of the outer surface of MSNs is foreseeable [5]. The surfactant was removed afterwards using acidified ethanol as extracting solution, affording in the same stage the anhydride ring opening. The required amount of alkoxysilane derivative was calculated to achieve a maximum coverage of the external nanoparticle surface (a 100% nominal degree of functionalization). For this, approximately a quarter of the specific surface area was estimated to correspond to the external surface and a molar ratio of three Si-OH groups with one R-Si(OEt)3 molecule was the stoichiometry used. In addition, it was presumed that the average surface concentration of Si-OH in silica materials is 4.9 OH/nm 2 [6].