Table 1.

Adjuvanticity of Al-NPs compared to microadjuvants and vaccines.

Adjuvant (Particle Size)	Vaccine Formulation	Principal Findings	Ref
Al(OH)3-NPs (~112 nm) vs. Al(OH)3-MPs (~9.3 μm)	Ovalbumin (OVA) and Bacillus anthracis protective antigen (PA) were adsorbed on the adjuvants.	OVA had more affinity to bind to the NPs than the MPs due to larger total surface area and more positive zeta potential of the NP. At equal OVA levels adsorbed on the particles, the NP induced higher anti-OVA IgG levels than the MP. The NP also induced higher anti-PA IgG levels than the MP 4 weeks after the second immunization. APCs internalized significantly higher levels of OVA adsorbed on the NP than the MP.	[15]
Al hydroxyphosphate NPs (<100 nm) vs. Al hydroxyphosphate MPs (~8–13 μm)	Egg lysozyme was adsorbed on the adjuvants.	The NP induced significantly higher antigen-specific IgG levels than the MP.	[28]
The sizes of Al(OH)3 (~0.99–1.96 μm) and AlPO4 (~1 μm) particles were reduced by applying high shear forces, then compared to Alhydrogel® and a commercially available vaccine (TETAVAX).	Diphtheria toxoid was adsorbed on the adjuvants.	The size reduction improved protein loading capacity, boosted antidiphtheria antibody titration, and induced stronger Th2 antibody isotypes (IgG1 and IgA). Size-reduced Al(OH)3 adjuvant also induced stronger Th2 cytokines (IL-5, IL-6, IL-10 and IL-13).	[29]
Al(OH)3-NPs (~141.1 nm) vs. Bacillus Calmette–Guerin (BCG) vaccine	Mycobacterium tuberculosis ESAT-6-like protein EsxV was adsorbed on the Al(OH)3-NPs.	The NP stimulated secretion of Th1 cytokines, e.g., IFN-γ comparable to BCG.	[30]
Amorphous and crystalline forms of Al(OH)3-NPs (150–200 nm) vs. Alhydrogel®	B. anthracis protective antigen domain 4 (D4) was adsorbed on the adjuvants.	The NPs enhanced antigen uptake by THP-1 cells, induced more robust and durable Th1/Th2 responses evidenced by higher IgG1 and IgG2a levels compared to Alhydrogel®, and induced higher Th1 cytokine levels (IL-2 and IFN-γ). Conversely, Alhydrogel® induced comparable or higher Th2 cytokine levels (IL-4 and IL-10). NPs prolonged survival of anthrax spore-challenged mice. The crystalline NP had moderate binding affinity compared to its amorphous counterpart, resulting in moderate antigen release (almost equal to Alhydrogel®).	[31]
Crystalline Al(OH)3-NPs (150–200 nm) vs. Alhydrogel®	D4 was encapsulated by non-ionic surfactant-based vesicles and adsorbed on the adjuvants.	The NP induced higher antigen-specific antibody titres (anti-D4 IgG) and IgG isotypes (IgG1 and IgG2a) than Alhydrogel®. It also stimulated splenocytes to produce both Th1 (IL-2, IFN-γ, and TNF-α) and Th2 (IL-4, IL-6, and IL-10) cytokines. The NP induced superior protection against anthrax spore challenge.	[32]
Al2O3-NPs (~30 nm) as a pulmonary vaccine adjuvant-delivery system vs. AlPO4-MP (2 μm)	OVA was adsorbed on the adjuvants.	NPs had significantly higher uptake by bone-marrow-derived dendritic cells (BMDCs) and promoted DC maturation to a higher degree, measured as CD40, CD80, and CD86 surface expression. NPs did not influence Raw264.7 (macrophage) cell viability at concentrations as high as 200 µg/mL. The NP induced more balanced Th1/Th2 responses, measured as anti-OVA IgG, mucosal IgA, and cytokine secretion (IFN-γ and IL-4), with only mild pulmonary inflammation.	[33]
Rod-shaped Al(OH)3-NPs stabilized with PAA (~60 nm) vs. Alhydrogel®	ID93 (M. tuberculosis vaccine antigen) or recombinant rHA (seasonal influenza hemagglutinin) were adsorbed on the adjuvants.	Unlike Alhydrogel®, the NP increased splenic IFN-γ-secreting CD4+ T cells and levels of Th1 cytokines, IL-18, and IL-12p70. The NP induced more robust and durable ID93-specific IgG1 and IgG2c antibodies, whereas Alhydrogel® induced IgG1 antibody and was biased toward a Th2 response. The NP induced superior protection against lethal influenza challenge.	[17]
Al2O3-NPs (~28 nm), phospholipid bilayer-coated Al2O3-NPs (PLANs, ~33 nm) and the PEGylated PLANs (PEG-PLANs, ~31 nm) vs. AlPO4-MPs (~2 μm)	OVA was adsorbed on the adjuvants.	BMDC uptake of formulations ranked in the order of AlPO4-MPs<Al2O3-NPs<PEG- PLANs<PLANs. The microparticle reduced cell (Raw264.7) viability. NPs did not show cytotoxicity and promoted cell growth. NPs and, more specifically, PLANs promoted DC maturation, measured as CD40, CD80, and CD86 surface expression. PEG-PLANs accumulated in draining lymph nodes at significantly higher levels. Whereas PLANs and PEG-PLANs elicited stronger humoral responses than AlPO4-MPs, Al2O3-NPs did not. NPs induced Th1 responses (IgG2a> IgG1), and conversely, the MP induced Th2 responses. NPs increase IL-4 and IFN-γ levels, as well as CD8+ T cells, in spleen compared to the MP. PEG-PLANs were the most effective adjuvant.	[34]
AlOOH nanorods (ALNRs) functionalized with (3-aminopropyl) triethoxysilane (ALNR-NH2) or 3-(trihydroxysilyl)- 1-propanesulfonic acid (ALNR-SO3H) (diameter: 20 nm, length: 150–200 nm) vs. Imject®	OVA was adsorbed on the adjuvants.	THP-1 cell uptake of formulations ranked in the order of ALNR-NH2 = ALNR-SO3H< Imject®. Moreover, IL-1β secretion by THP-1 cells ranked in the order of ALNR-SO3H≤Imject®<ALNR-NH2. Cellular oxidative stress (measured as glutathione level) of formulations ranked in the order of ALNR-SO3H <alum<ALNR-NH2. ALNR-SO3H and Imject® had the same capacity to induce anti-OVA IgG1 and IgE, whereas ALNR-NH2 induced significantly higher levels of the antibodies.	[35]
Amorphous AlOOH nanosticks (diameter: ~8 nm, length: ~80 nm) vs. Alhydrogel® (~900 nm)	OVA was adsorbed on the adjuvants.	J774A.1 macrophage uptake of NPs was higher than that of Alhydrogel®. THP-1 cells treated with NPs released higher levels of IL-1β than Alhydrogel®. NPs induced higher levels of serum anti-OVA IgG and IgG1 than Alhydrogel®. Al nanosticks and Alhydrogel® induced local subcutaneous nodule and granuloma formation, although the site injected with the Al nanosticks had a relatively lower cellularity.	[36]
Al(OH)3-NPs (~40 nm), phospholipid bilayer-coated Al(OH)3-NPs (PLAlOH3: ~50 nm) vs. Al(OH)3-MPs (~10 μm)	OVA was adsorbed on the adjuvants.	BMDC uptake of formulations ranked in the order of Al(OH)3-MPs<Al(OH)3-NPs<PLAl(OH)3. NPs induced more durable and higher anti-OVA IgG and IgA than the MP. PLAl(OH)3 induced balanced IgG2a>IgG1, contrary to the MP which induced biased Th2 response (very high level of IgG1). Whereas PLAl(OH)3 elevated both IL-4 and IFN-γ in serum and supernatant of splenocytes, the MP increased only the IL-4 level. The MP increased only CD4+ T-cell populations in the spleen, but the PLAl(OH)3 elevated both CD4+ T and CD8+ T-cell populations. The stimulation index for splenocyte proliferation was ~2-fold higher for PLAl(OH)3 than the MP. Following subcutaneous injection into a forelimb, PLAl(OH)3 was accumulated in axillary lymph nodes and taken up by DCs. Following intramuscular injection, neither NP induced local inflammation, but the MP induced severe inflammatory reactions.	[37]
Al2O3-nanowire (diameter: ~20–40 nm, length: ~20–60 µm) vs. Al2O3-MPs (20 µm scale) and Alhydrogel® (2 µm)	OVA was adsorbed on the adjuvants.	All formulations were non-toxic to HeLa and THP-1 cells (up to 200 µg/mL); however, the nanowire was slightly toxic to U87MG cells (viability: ~70%) compared to Al2O3-MPs (viability: ~80%) and Alhydrogel® (viability: ~85%) at the mentioned concentration. The nanowire induced higher levels of anti-OVA IgG than MPs 11 days after the second immunization. Cellular immune response, measured as delayed-type hypersensitivity, was stronger in nanowire-treated mice than the Al2O3-MP-injected cohort at 6–24 h after antigen exposure. Following injection into an air sac in the flank, the nanowire induced a lower degree of microvascular damage and oedema than Alhydrogel®.	[38]