Silica-based cationic bilayers as immunoadjuvants
© Lincopan et al; licensee BioMed Central Ltd. 2009
Received: 01 August 2008
Accepted: 19 January 2009
Published: 19 January 2009
Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design.
Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris.HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001–1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant.
Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.
Over the last two decades novel assemblies obtained from particles and lipids have been introduced as important tools to novel applications in drug and vaccine delivery [1–4]. Particulates such as silica, latex or hydrophobic drugs have been coated by lipids and successfully employed in biomolecular recognition [5, 6] drug delivery [7, 8] and antigen presentation [9, 10]. The systematic and quantitative evaluation of particle-lipid interaction has been realized by means of adsorption isotherms of lipids on particles, effects of lipids on particle size and zeta-potential from dynamic light scattering methods and determination of colloid stability from turbidity kinetics or particle sedimentation over time [1–10]. Cationic lipids, in particular, are especially interesting to cover particles, since cationic particles may electrostatically combine with a vast variety of oppositely charged biomolecules, cells or other biological structures. Cationization, in general, has often been explored as a convenient approach to target active biomolecules into cells .
The control of lipid assembly on particles turned out to be dependent on properties of the intervening medium, eg ionic strength, and on the proportion of surface areas for bilayer vesicles and particles in dispersion [12–15]. From equivalence of total surface areas for particles and cationic lipid bilayers, over a range of low ionic strength, a good colloid stability was reported for the bilayer-covered cationic particles [3, 4, 12, 15].
In this work, the interaction between silica previously coated with cationic bilayers of dioctadecyldimethylammonium bromide (DODAB)  and the model protein bovine serum albumin (BSA) is investigated aiming at antigen presentation to the immune system by silica-based cationized particles. BSA choice was due to various reasons: its extensively studied adsorption behaviour at interfaces [16, 17]; its utility to prevent nonspecific binding in biosensing and proteomics applications [17–19], its conformational adaptability as a "soft" globular protein , its thoroughly investigated adsorption onto hydrophobic or hydrophilic particles sometimes resulting in exchange between the adsorbed and dissolved states [21–24] and its substantial adsorption onto cationic and large DODAB vesicles .
The 18 kDa-hsp protein belongs to a conserved protein family of M. leprae heat-shock proteins that display pronounced immunogenicity and are considered important targets of the immunoresponse to mycobacteria and, as such, relevant to subunit vaccine design. Peripheral blood mononuclear cells and T-cell lines from M. leprae vaccinated subjects proliferated in response to this protein . Furthermore, overexpression and scaling-up of 18 kDa-hsp production in Saccharomyces cerevisae has already been described so that this protein is available in sufficient amount for a complete physico-chemical study of the adjuvant-antigen interaction [27–29].
The DODAB cationic lipid and its assemblies in water dispersion have been established as effective immunoadjuvants able to stimulate dendritic cells and often employed to present antigens [29–34]. Silica particles are biocompatible, represent a reference adsorbent, offer a chemically well defined surface and are widely used as a chromatographic stationary phase [34, 35].
We have recently combined the typical property of particles that stimulate dendritic cells uptake with the adjuvant effect of DODAB by using supported DODAB bilayers on latex to present antigens . Here we take advantage of the biocompatible character of silica [35, 36] to produce DODAB-covered silica particles for further immobilization and presentation of two different model antigens: BSA and 18 kDa-hsp protein.
Results and discussion
Coverage of silica particles with a cationic bilayer and BSA adsorption
Charge density on silica particles increases with pH and ionic strength  so that electrostatic attraction between DODAB bilayer and silica is substantial over the 1–10 mM range of monovalent salt concentration, and leads to DODAB bilayer deposition onto particles . One should notice that poor or none DODAB adsorption on silica was previously reported for pure water as intervening media [13–15, 38]. Therefore, the experiments in this work were designed either at 1 or at 5 mM monovalent salt.
The DODAB bilayer in closed vesicles is in the rigid gel state at room temperature. This represents an important limitation regarding deposition of bilayers onto particles hampering the occurrence of vesicle disruption which is essential for bilayer deposition. We have previously shown that closed vesicles with bilayers in the gel state do not disrupt upon contact with silica particles , thus, in this work, bilayer deposition on silica is obtained by employing disrupted vesicles or bilayer fragments.
The stability of the silica/DODAB system was sistematically described in a previous work . The adjuvant system is very reproducible yielding always the same mean Dz and zeta-potential for the same final silica and DODAB concentrations. However, upon antigen addition, zeta-potential may decrease to values close to zero so that colloid stability will be low.
Physical properties of SiO2, DODAB BF dispersion, BSA, 18 kDa-hsp leprae and their mixtures in 1 mM NaCl (pH 6.3) or in 5 mM TrisHCl (pH 7.4)
328 ± 7
-40 ± 2
0.240 ± 0.01
67 ± 1
41 ± 3
0.120 ± 0.04
25 ± 4
-28 ± 1
0.450 ± 0.09
72 ± 1
3 ± 1
0.245 ± 0.01
304 ± 2
32 ± 1
0.260 ± 0.02
324 ± 8
24 ± 1
0.301 ± 0.01
294 ± 2
-47 ± 2
0.180 ± 0.02
74 ± 1
33 ± 5
0.270 ± 0.04
24 ± 1
-58 ± 18
0.397 ± 0.01
373 ± 7
33 ± 2
0.260 ± 0.02
851 ± 36
-1.3 ± 4
0.396 ± 0.03
18 kDa-hsp M. leprae(i)
732 ± 188
-29 ± 2
0.35 ± 0.05
208 ± 8
39 ± 5
0.315 ± 0.01
18 kDa-hsp M. leprae(ii)
360 ± 16
-43 ± 18
0.180 ± 0.09
2132 ± 370
-2 ± 2
0.492 ± 0.04
458 ± 3
28 ± 3
0.191 ± 0.04
702 ± 17
14 ± 2
0.317 ± 0.01
4888 ± 665
0.272 ± 0.07
10607 ± 2998
0.480 ± 0.03
Maximal adsorption, affinity constant (K) and area per adsorbed BSA molecule on silica (0.1 mg/ml) or on DODAB covered silica particles in two different media
Area per adsorbed molecule
4.14 × 1016
1.19 × 109
4.75 × 1016
8.19 × 107
7.20 × 1016
5.18 × 107
3.22 × 1017
8.98 × 108
1.12 × 1017
10.00 × 1017
Silica-based cationic bilayers for induction of humoral immune response
Percentage footpad swelling (%fs) (delayed-type hypersensitivity reaction) to BSA or 18 kDa-hsp M. leprae antigens supported on DODAB-covered silica particles, or complexed with Al(OH3).
Systems for sensitization
Ag/dose for sensitization (μg/mouse)
18 kDa-hsp M. leprae
% footpad swelling ± SEM
2 ± 2
2 ± 2
2 ± 2
2 ± 2
3 ± 2
2 ± 2
3 ± 2
2 ± 2
11 ± 2(iv)
14 ± 4(iv)
18 kDa-hsp M. leprae
4 ± 2
10 ± 2
DODAB BF(iii)/18 kDa-hsp
56 ± 8 (iv), (v)
95 ± 16 (iv), (v)
60 ± 7 (iv), (v)
89 ± 8 (iv), (v)
18 ± 4 (iv)
33 ± 1 (iv)
Silica-based cationic bilayers for induction of cellular immune response
Supported cationic bilayers built on silica can effectively adsorb antigens to elicit superior immune responses in vivo. They can be prepared from a tiny amount of cationic and inexpensive synthetic lipid, just enough for covering silica particles with a cationic layer. The main advantage of this adjuvant system is precisely this low amount of cytotoxic cationic lipid employed in comparison to cationic liposomes usually used over a range of millimolar concentrations. Regarding physical properties, silica/DODAB particulates are less polydisperse than alum allowing better antigen presentation and eliciting superior cellular immune responses. Therefore, cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum and better performance regarding cellular immune responses.
Lipids, silica particles and antigen
Dioctadecyldimethylammonium bromide (DODAB) 99.9% pure was obtained from Sigma-Aldrich (St Louis, MO, USA). Silica (Aerosil OX-50) with a 50 nm mean diameter from transmission electron microscopy and nominally, 26 m2/g specific surface area was a gift from Degussa (Degussa Co.). A stock silica dispersion at 4 mg/ml was prepared in 1 mM NaCl (pH 6.3) or 5 mM Tris.HCl (pH 7.4) solutions, which provide adequate ionic strengths to assemble DODAB as a single bilayer onto particles . Bovine serum albumin (BSA) was purchased from Sigma-Aldrich, and prepared as a 1 mg/ml stock solution in 1 mM NaCl (pH 6.3) or 5 mM Tris.HCl 5 mM (pH 7.4) and stored in a freezer in 1 ml aliquots for quick use. Recombinant 18 kDa-hsp Mycobacterium leprae protein (18 kDa-hsp) was prepared as previously described  and diluted in 1 mM NaCl (pH 6.3) or 5 mM Tris.HCl (pH 7.3) to obtain a stock solution at 2 mg/ml. 18 kDa-hsp concentration was determined spectrophotometrically measuring the absorbance at λ = 230 nm, using a standard curve (5 – 160 μg/ml) of 18 kDa-hsp, as previously described . BSA concentration was determined by a protein microassay, based on the method of Lowry , using a standard curve (10 – 100 μg/ml) of BSA. Aluminium hydroxide adjuvant Al(OH3) was obtained from Merial do Brasil (Merial Ltda.). NaCl, Trizma base, and all other reagents were analytical grade. Water was Milli-Q quality.
Preparation of lipidic dispersions and analytical determination of lipid concentration
Small DODAB bilayer fragments were prepared by sonication with titanium macrotip probe in 1 mM NaCl (pH 6.3) or 5 mM Tris.HCl (pH 7.4) water solution at ca. 2.0 mM DODAB (nominal potency, 80 W/15 min of sonication time) as previously described . Following sonication, the solutions were centrifuged (10.000 g/15°C/40 min) to eliminate the titanium ejected from the tip. Mean size and ζ-potential for the DODAB dispersions are shown in Table 1. The DODAB concentration was determined spectrophotometrically from Orange G/DODAB solubilization in neutral micelles  or from halide microtitration . The silica powder was routinely dispersed by sonication with a titanium tip (85 W/10 min) in 1 mM NaCl (pH 6.5) or 5 mM Tris.HCl (pH 7.5). Titanium particles ejected from the tip were allowed to pellet for 1 h before the silica dispersion was withdrawn from the supernatant for further use.
Preparation of silica/DODAB and silica/DODAB/protein assemblies
Stock dispersions of silica particles at 4 mg/ml and stock DODAB bilayer fragments BF dispersions at 2.0 mM DODAB were dispersed either in 1 mM NaCl (pH 6.3) or in 5 mM Tris.HCl (pH 7.4) and diluted to the final desired concentration using this same solution of NaCl or Tris.HCl. First, to obtain lipid-covered silica particles, silica, at 0.1 mg/ml final concentration, and oppositely charged DODAB BF solutions ranging from 0.1 μM to 1 mM, interacted for 1 h/25°C. DODAB final co ncentration for producing the assemblies was selected as 50 μM at 0.1 mg/ml of silica since this concentration is the one required to cover each silica particle with a DODAB bilayer . In fact, experimentally it is shown in Figure 1 that from this concentration cationic particles are indeed obtained. In a second experimental step, the stock BSA or 18 kDa-hsp solutions were used to obtain final protein concentrations ranging from 5 to 50 μg/ml after addition to the silica/DODAB mixture, for 1 h/25°C interaction. Thereafter, sizes, zeta-potentials, and polydispersities were determined. Considering the total surface area of 2.6 × 10-3 m2 the selected DODAB concentration of 0.05 mM was more than sufficient to produce bilayer-covered particles.
Determination of average zeta-diameter and zeta-potential for particles, bilayer fragments, or mixtures of both
Particle size (mean diameter D z ), size distribution, polydispersity and zeta-potential (ζ) in the presence or absence of silica, DODAB and BSA or 18 kDa-hsp were determined using the ZetaPlus-ZetaPotential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY), which was equipped with a 677 nm laser and dynamic light scattering (PCS) at 90° for particle sizing. Mean d iameters were obtained by fitting data to log-normal size distributions which do not discriminate between one, two, or more different populations and considers always all scattering particles as belonging to one single Gaussian population. On the other hand, for the size distribution data, fitting was performed by the apparatus software using the non-negatively constrained least squares (NNLS) algorithm, which is a model independent technique allowing to achieve multimodal distributions . ζ was determined from electrophoretic mobility μ in 1 mM NaCl and the Smoluchowski's equation: ζ = μη/ε, where η is the medium viscosity and ε the medium dielectric constant.
Determination of BSA and 18 kDa-hsp adsorption isotherms
BSA adsorption isotherms on silica alone, in 1 mM NaCl (pH 6.3) or 5 mM Tris.HCl (pH 7.4), were obtained by mixing 0.05 ml of silica solution (0.4 mg/ml) with 0.15 ml of the appropriate BSA dilution in 1 mM NaCl or 5 mM Tris.HCl. For BSA adsorption onto silica/DODAB particles, prior to protein addition, 0.05 ml of silica solution was allowed to interact for 1 hour with 0.05 ml of a 0.2 mM DODAB BF dispersion; thereafter, BSA solution was added to yield a final volume of 0.2 ml. Final concentration of BSA in the assays ranged from 0 – 150 μg/ml, at a fixed concentration of 0.1 mg silica/ml and 0.05 mM DODAB BF. After 1 h silica/BSA or silica/DODAB/BSA interaction at 25°C, a clear supernatant was obtained by centrifugation at 15,000 rpm for 1.5 h. The concentration of protein in the supernatant was determined by Lowry microassay using a standard curve prepared from 10 – 100 μg/ml BSA . The method is sensitive over a protein concentration range of 0.005–0.100 mg/ml. A microplate reader equipped with a 655 nm filter (Ultramark, Model 550 Bio-Rad, Hercules, CA, USA) was used for absorbance measurement.
18 kDa-hsp adsorption isotherms on silica alone, in 5 mM Tris.HCl (pH 7.4), were obtained by mixing 0.025 ml of stock silica dispersion (4 mg/ml) with 0.975 ml of the appropriate 18 kDa-hsp dilution in 5 mM Tris.HCl. For 18 kDa-hsp adsorption onto silica/DODAB particles, prior to protein addition, 0.025 ml of stock silica dispersion was allowed to interact for 1 hour with 0.025 ml of a 2 mM DODAB BF dispersion; thereafter, 18 kDa-hsp solution was added to yield a final volume of 1 ml. Final concentration of 18 kDa-hsp in the assays ranged from 0 – 120 μg/ml, at a fixed concentration of 0.1 mg silica/ml and 0.05 mM DODAB BF. After 1 h silica/18 kDa-hsp or silica/DODAB/18 kDa-hsp interaction at 25°C, a clear supernatant was obtained by centrifugation at 15,000 rpm for 1.5 h. The concentration of protein in the supernatant was determined spectrophotometrically by measuring the absorbance at λ = 230 nm, using a standard curve (5 – 160 μg/ml) of 18 kDa-hsp, as previously described . A spectrophotometer Hitachi U-2000 was used for absorbance measurement.
For both proteins, the amount of adsorbed protein was determined by the difference between the total protein added and the amount of protein recovered in the supernatant.
Adsorption was expressed as the number of molecules adsorbed per square meter silica. Curves were fitted using cubic polynomial regression. Wherever possible, the Langmuir model was employed for isotherms linearization and determination of adsorption constants such as affinity constant (K, in M-1) and maximal adsorption (in number of molecules per m2 silica) .
Subcutaneous immunization, assay for delayed-type hypersensitivity (DTH) and antigen-specific ELISA
BALB/c female mice 8–12-week old were purchased from the University of São Paulo, São Paulo, Brazil. Six groups of 8 – 10 female mice were challenged subcutaneously (s. c.) in the abdomen at two separate sites. Total volume injected in each site was 0.1 or 0.15 mL for BSA or 18 kDa-hsp, respectively. The dispersion injected contained either: (a) 10 μg BSA or 15 μg 18 kDa-hsp in 5 mM TrisHCl (pH 7.4); or (b) 10 μg BSA or 15 μg 18 kDa-hsp in 1 mM NaCl and 0.1 mM DODAB; or (c) 10 μg BSA or 15 μg 18 kDa-hsp in 0.1 mg silica/mL, 5 mM Tris.HCl; or (d) 10 μg BSA or 15 μg 18 kDa-hsp in silica/DODAB (0.1 mg mL-1/0.05 mM) in 5 mM Tris.HCl; or (e) 0.1 mg silica/mL in 5 mM Tris.HCl. For DTH evaluation, the footpad swelling test was carried out essentially as previously described [9, 26, 27]. On the fifth day post subcutaneous immunization, pre-immunized mice with BSA, 18 kDa-hsp, DODAB/BSA, DODAB/18 kDa-hsp, silica/BSA, silica/18 kDa-hsp, silica/DODAB/BSA, silica/DODAB/18 kDa-hsp or silica alone, were challenged in the left-hind footpad with a total elicitation dose of 4 or 40 μg BSA or 3 or 30 μg 18 kDa-hsp in 50 μL Tris.HCl 5 mM, respectively. Footpad swelling was measured 24 h later with a Mitutoyo engineering micrometer. Depending on the age of the animals, the thickness of uninjected hind footpad varied from 1.60 to 1.70 mm. Percentage footpad swelling (%fs) is calculated according to the formula below with results expressed as %fs ± standard error of the mean (SEM).
%fs = 100 [(left hind footpad thickness) - (right hind footpad thickness)]/(mean thickness uninjected left hind footpad).
For evaluation of humoral immune response, the same groups previously immunized and challenged with BSA above were bled through the ophthalmic plexus in days 24 and 36, after immunization. The sera obtained were analyzed by ELISA. Each well of 96-well ELISA polystyrene high binding plates (Costar Corning Inc., Cambridge, Mass.) was coated with 100 μL of BSA (final concentration of 0.05 μg/well) in 0.5 M carbonate-bicarbonate buffer (pH 9.6) for 18 h in an humidified chamber at 4°C. The wells were blocked for 1 h with 5% milk in PBS containing 0.05% Tween 20 (PBS/T), and then incubated for one hour with serum samples diluted 1:50 or 1:200, for specific IgG antibody quantitation. In each well, 100 μL of goat anti-mouse IgG peroxidase-conjugate (Sigma) diluted 1:3000 was added and plates were incubated for 1 h. After each incubation step, the plates were washed using an automatic washer, with four cycles of PBS/T. Ortho-phenylenediamine (1 mg/mL) (Sigma) and H2O2 (1 μL/mL) diluted in 0.2 M citrate buffer (pH 5.0) were added (in the dark) as chromogenic substrate and plates were incubated for 10 min. The reactions were stopped by adding 100 μL of 2 M H2SO4. Color intensity was quantified using an ELISA plate reader (Diagnostics Pasteur, Strassburg-Schiltigheim, France) at 492 nm. All incubations were carried out at 37°C. Antibody titers remained below 1/100 dilution, meaning that the dilution is not high enough to discriminate specific and non specific antibodies against BSA. Serum titration included a serum from naïve mice and a serum from mice immunised with a non relevant antigen towards induction of humoral immune response, namely, hsp-18 kDa protein itself.
For evaluation of humoral immune response against 18 kDa-hsp, the same groups previously immunized and challenged with 18 kDa-hsp above were bled through the ophthalmic plexus in days 14, 28, and 84 after immunization. The sera obtained were analyzed by ELISA. Each well of 96-well ELISA polystyrene maxisorpt plates (Maxisorp, Nunc) was coated with 100 μL of 18 kDa-hsp (final concentration of 40 μg/well) in 0.5 M carbonate-bicarbonate buffer (pH 9.6) for 18 h in an humidified chamber at 4°C. The wells were blocked for 1 h with 5% milk in PBS containing 0.05% Tween 20 (PBS/T), and then incubated for one hour with serum samples diluted 1:2, for specific IgG antibody quantitation. In each well, 100 μL of goat anti-mouse IgG peroxidase-conjugate (Sigma) diluted 1:1000 was added and plates were incubated for 1 h. After each incubation step, the plates were washed using an automatic washer, with four cycles of PBS/T. Ortho-phenylenediamine (1 mg/mL) (Sigma) and H2O2 (1 μL/mL) diluted in 0.2 M citrate buffer (pH 5.0) were added (in the dark) as chromogenic substrate and plates were incubated for 10 min. The reactions were stopped by adding 100 μL of 2 M H2SO4. Color intensity was quantified using an ELISA plate reader A microplate reader equipped with a 405 nm filter (Ultramark, Model 550 Bio-Rad, Hercules, CA, USA) was used for absorbance measurement. All incubations were carried out at 37°C.
Cell culture and cytokine analysis
Six days after immunization, cell suspensions from inguinal and periaortic lymph nodes (LN) of 5 mice were prepared in RPMI 1640 (GIBCO) supplemented with 10 mM HEPES, 50 μM 2-Mercaptoethanol, 216 mg L-glutamine/L and 5 % FCS (GIBCO). The cell suspensions (8 × 106 cells) were distributed into tissue culture 24-wells plates (Costar) and incubated with medium containing 250 μg/mL of 18 kDa-hsp M. leprae in a humidified CO2 incubator for 48 hours. Wells containing medium only or 2.5 μg/mL of ConA (Sigma Aldrich) were included in all experiments as negative and positive controls, respectively. After this time, the plates were centrifuged for 8 minutes at 500 × g and the supernatants collected for cytokine content. The levels of cytokines (IL-10, IL12, IL-13 and IFN-γ) in the supernatants were assayed by sandwich kit enzyme-linked immunosorbent assay (ELISA), using the following monoclonal antibodies: MAB417 and biotinylated-BAF417 for IL-10; MAB419 and biotinylated-BAF419 for IL-12; MAB413 and biotinylated-BAF413 for IL-13; and XMG 1.2 and biotinylated-AN18 for IFN-γ, according to the manufacturer's suggestion (R&D Systems – Minneapolis, MN). Binding of the biotinylated antibodies was determined using the streptavidin-peroxidase conjugate (Sigma) and TMB (3,3',5,5'-Tetramethylbenzidine; Sigma) solution in citrate buffer plus Hydrogen peroxide. The plates were read (450 nm) in an automated ELISA reader (Dynatech MR5000). Samples were quantified by comparison with standard curves of purified recombinant cytokines and the values expressed as ng/mL. The limit of detection was 156 pg/mL for IL-10 and IL-12, and 78 pg/mL for IL-13 and IFN-γ respectively.
ANOVA one-way multiple comparison tests and the Kruskal-Wallis non-parametric test were used when needed. A P value of ≤ 0.05 was considered significant.
FAPESP and CNPq are gratefully acknowledged for financial support. NL and MRAS were recipients of a FAPESP postdoctoral and CNPq trainee technician fellowships, respectively.
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