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Research Article

Intraperitoneal Acute Toxicity of Aluminum Hydroxide Nanoparticle as an Adjuvant Vaccine Candidate in Mice

Sjaikhurrizal El Muttaqien, Etik Mardliyati, Silmi Rahmani, Sabar Pambudi, Siti Maimunah, Ziah Izati Azkia, Nurmeilis , Asri Sulfianti, Julham Efendi, Damai Ria Setyawati, Asutiati Nurhasanah and Irvan Faizal
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Background and Objective: Aluminum-containing adjuvant, such as aluminum hydroxide Al(OH)3), has been widely used and is generally regarded as safe to use in human vaccines. It is known that particle size of adjuvant plays critical role in dictating the adsorption of antigen. Numerous researches reported that down-sizing of Al(OH)3 into nano-sized particles could improve its immunopotentiation character. However, the safety of Al(OH)3 nanoparticle needs to be evaluated through acute toxicity study. This study aimed to assess biological character of nano-sized Al(OH)3, particularly its in vivo acute toxicity. Materials and Methods: Three types of samples including commercial Al(OH)3 of Alhydrogel®, Al(OH)3 microparticle (Mp) and Al(OH)3 nanoparticle (Np) were prepared and then peritoneally injected to BALB/c mice with 4 different doses (0.16, 1.28, 10.84 and 81.92 mg kg1 b.wt.). Post-administration, mice’s behavior and their body weight were recorded. After 14 days, mice were sacrificed and major organs were taken out for preparing hematoxylin-eosin (H and E) stains. Results: The average particle size of Alhydrogel®, Al(OH)3 Mp and Al(OH)3 Np were 4.02, 4.21 and 0.162 μm, respectively. The acute toxicity study showed that there is no lethal effect, clinical sign related complications and behavioral alteration even in highest administered dose for 14 days of observation period. In general, histological parameter of H and E stained tissue section among the treated groups was also comparable to those of control group, revealing relatively safe character of Al(OH)3 Np after peritoneal administration. Conclusion: Therefore, these results confirmed the potency and safety of Al(OH)3 Np as an adjuvant vaccine candidate.

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Sjaikhurrizal El Muttaqien, Etik Mardliyati, Silmi Rahmani, Sabar Pambudi, Siti Maimunah, Ziah Izati Azkia, Nurmeilis , Asri Sulfianti, Julham Efendi, Damai Ria Setyawati, Asutiati Nurhasanah and Irvan Faizal, 2020. Intraperitoneal Acute Toxicity of Aluminum Hydroxide Nanoparticle as an Adjuvant Vaccine Candidate in Mice. Journal of Pharmacology and Toxicology, 15: 22-35.

DOI: 10.3923/jpt.2020.22.35



Aluminum salts, including Al(OH)3, have been extensively used as an adjuvant since past decades to amplify the humoral immunogenicity of the administered vaccine as was first described by Glenny et al.1, Gupta et al.2 and Gupta3. This type of adjuvant hypothetically works by providing an antigen local reservoir that could prolong the exposure of antigen to the antigen-presenting cells (APCs) and thus stimulating the activation of immune competent component4-6. This depot theory, however, has been questioned as the direct correlation of antigen-Al(OH)3 complex retention at the administration site and the resulting antibody response has never been demonstrated5,7-9. Despite the persisted controversy of molecular mechanism of alum adjuvanticity, various attempts to improve the immune response of Al(OH)3 gel have been done by altering the physicochemical properties of adjuvant that can increase the adsorption of the antigen onto the surface of Al(OH)3 gel. For example, recent studies have shown that the modification of shape, dimensions and surface charge of the adjuvant particles critically affect its immune responses10-14. In our previous work, Al(OH)3 Nps were successfully prepared with the particle size around 200 nm15 and its potency was evaluated as adjuvant for potentiating the immune response of purified DENV3 pre Membrane Envelope (prM-E) recombinant protein as an antigen16. These studies revealed that the complexes of Al(OH)3 Np-prM-E significantly enhanced immune response compared to those of commercial and micro-sized Al(OH)3 through the induction of nitric oxide release from mouse macrophage RAW264.7cells. Nitric oxide was regarded as an effecter molecule that is involved in macrophages activation and become an important parameter of immune stimulatory activity of the adjuvant17-19.

In terms of its toxicity, Al(OH)3 adjuvants are generally well tolerated and display favorable safety profile which was indicated by its relative rarity of side effect20. Also, previous studies concluded that no clear associations between vaccinations using aluminum-containing vaccines and serious adverse events as 66-70% of injected aluminum was excreted from the body21 within 24 h. However, it is common that the down-sizing of particle size may alter not only biological activity but also intrinsic toxicity profile of its bulk material. So, the current study was aimed to evaluate the acute toxicity of Al(OH)3 Np and determine its potential risk.


The animal study was conducted in May-April 2019 in Animal and Pharmacology Laboratories, Center for Pharmaceutical and Medical Technology, BPPT, PUSPIPTEK, Serpong. All animal experiments were approved by the Animal Care and Use Committee of University of Indonesia and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals as stated at University of Indonesia.

Materials: The Al(OH)3 Mps and NPs were prepared by reacting aluminum chloride with sodium hydroxide, as previously reported by Mardliyati et al.15. As a stabilizer, sodium polyphosphate (0.2%) was added to Al(OH)3 Np suspension to avoid particle aggregation. Alhydrogel® was purchased from in vivo gen (Frederikssund, Denmark). Phosphate-buffered saline (PBS) pH 7.4 was prepared by dissolving one PBS Tablet from Merck in 200 mL of deionized water, yielding 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 SM sodium chloride, pH 7.4.The size and morphology of Al(OH)3 was measured by static light scattering (Horiba LA95A)and transmission electron microscopy/TEM (JEOL F200). The isoelectric point (PI) of each sample was evaluated by Malvern Zetasizer Nano ZS90 equipped with autotitrator mpt-2.

Animals: A total of 65 BALB/c mice (female, 4 weeks old) were obtained from Indo Anilab Laboratories Indonesia, Inc. (Bogor, Indonesia). All animal experiments were approved by the Animal Care and Use Committee of University of Indonesia and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals as stated at University of Indonesia. The animals were housed in aluminium cages in a well-ventilated room with maintained temperature (20±1°C) and relative humidity (40-70%). Conventional laboratory diet was provided twice daily with unlimited supply of hygienic drinking water. All animals were acclimatized under observation for 1 week before starting the test.

Acute toxicity protocol: The protocol was adopted from the previous intraperitoneal study of acute toxicity conducted by Tubaro and coworkers with slight modification22. The animals were randomly divided into 4 groups (5 mice each) as follow: PBS control group, Alhydrogel®, Al(OH)3 Mp and Al(OH)3 Np. For the Al(OH)3 treated group, four different doses were used: 0.16, 1.28, 10.24 and 81.92 mg kg1 b.wt. The lowest dose used in this experiment was obtained from the conversion of recommended Al(OH)3 dose on human (0.2-0.8 mg)23. After intraperitoneal administration of the corresponding sample, animal behavior, toxicity sign, body weight and lethal response were observed daily for14 days. At the end of observation period, final body weight of mice was recorded. Mice then were sacrificed after ether anesthesia and their major organs were excised and accurately weighed to measure organs mass index.

Histopathological study: For histological examination, the procedure explained by Slaoui and Fiette24 was used. The collected lung, liver, kidney and spleen were first fixed in 10% neutral buffered formalin for 24 h at room temperature. Following fixation, the collected tissues were dehydrated, embedded in paraffin, sliced using microtome and placed onto glass slides and stained by hematoxylin and eosin (HE) stain. The slides were then observed using an optical microscope.

Statistical analysis: Result was expressed as mean±standard deviation (SD). The statistical significance was determined using one-way analysis of variance (ANOVA) with the alpha level for all tests were set at p<0.05.


Material characterization: The particle size distribution and morphology of Al(OH)3 were evaluated by particle size analyzer and TEM. As can be seen from Table 1, the diameter of Al(OH)3 Mp measured by particle size analyzer ranged from 1.98-15.17 μm, with the average hydrodynamic diameter of 4.21 μm. On the other hand, the dominant diameter of Al(OH)3 Np fell around 0.150 μm with the mean diameter of 0.162 μm and narrow size distribution (0.12-0.58μm), indicating the affectivity of high shear homogenization to reduce the particle size. In comparisons to these two samples, the average particle size of Alhydrogel® measured in this study was 4.02 μm with almost identical particle size range to those of Al(OH)3 Mp.

From morphological observation (Fig. 1), the TEM image of Al(OH)3 Np clearly showed the aggregate formation with the size of single nano structure was in the order of 30 nm. The particle size variance between TEM image and static light scattering measurement was due to the difference of principle used of these two apparatuses. Particle size analyzer measures the size distribution of the particle on aqueous suspension which may have high tendency of particle aggregation to take place. On the other hand, TEM image can directly measures the diameter of single particle and observes its morphological shape on dry state. The TEM photograph also revealed that Al(OH)3 Np exhibited relatively uniform and spherical shape structure. Such similar morphological shape was also identified in the TEM image of Al(OH)3 Mp (data not shown). Also, Al(OH)3 Np had slight lower isoelectric point (PI)and pH compared to those of Al(OH)3 Mp and Alhydrogel® (Table 1), which may attributed to the addition of stabilizer.

Acute toxicity: During the acute toxicity test, Al(OH)3 samples at all doses did not induce any mortality effect to the treated mice (Table 2). Therefore, the LD50 of all type of Al(OH)3 was estimated to be higher than 81.92 mg kg1. Following the sample administration, the morbidity of the animal such as tremor, drowsiness, hypoactivity and piloerection was examined, as resumed in Table 3.

Fig. 1(a-b): (a-b) TEM image of Al(OH)3 Np

Table 1: Characteristics of Al(OH)3 used in this study
aBased on information written on the certificate analysis of the Alhydrogel®, bMeasured by X-Ray fluorescence (XRF) spectrometers, cMeasured by light scattering analysis

Table 2: Mortality data of test animal

Table 3: Behavioral and physical symptoms observation of test animal within 4 h of sample administration
Statistical significance was evaluated using one-way analysis of variance (ANOVA) (*p<0.05)

Table 4:
Extended behavioral and physical symptoms observation of test animal after PBS administration
+: Symptom observed, -: Symptom was not observed

At the 1st day of sample administration, only animal treated with the higher dose showed drowsiness and hypoactivity symptoms with statistically significant difference compared to those of control animal. In the next days, however, the drowsiness and hypoactivity effect was vanished (Table 4-7).

The body weights profile of the tested animals under observation period is shown in Table 8. The obtained result revealed that there was no difference in alteration of body weight of all sample-treated mice compared to those of control group throughout the study period. Although there was a slight attenuation on body weight profile at one day after sample administration which presumably due to the stress caused by sample administration, all mice resumed their body weight development in the next day. Thus, it may indicate that Al(OH)3 has a negligible toxicity on the animal growth.

The postmortem observation of mice organ including liver, kidney, spleen, lung and hearth was also conducted (Table 8). Visually, there is no remarkable change of organ compared to those of control group. In organ index evaluation, Al(OH)3administration did not alter the relative organ weights, except those of kidney and spleen of mice treated with Al(OH)3 Np. The Al(OH)3 Np at all doses induced a significant increase of kidney index. Similarly, Al(OH)3 Np at doses of 1.28 and 10.24 mg kg1 altered spleen index as compared to PBS-administered mice.

Table 5: Extended behavioral and physical symptoms observation of test animal after Alhydrogel® administration
+: Symptom observed, -: Symptom was not observed

Table 6: Extended behavioral and physical symptoms observation of test animal after Al(OH)3 Mp administration
+: Symptom observed, -: Symptom was not observed

The histological specimens of lung, liver, kidney and spleen are shown in Fig. 2-5. In the tissue section of lung (Fig. 2), thickening of alveolar septal walls and pulmonary arterioles dilatation were obviously found, especially in mice treated with the highest dose of Al(OH)3. Also, granuloma and eosinophilic infiltration were identifiable in all groups which indicating lung inflammation. The histological photomicrographs of the liver are shown in Fig. 3. In general, normal hepatocytes arrangement forming trabecular pattern was observed in all tissue sections. Also, mild inflammation and necrosis were noticed in those of PBS-and sample-treated groups. The notable morphological change in liver histology of sample-treated mice appeared in the form of granuloma and lymphocytic infiltration, in which the tissue section from Al(OH)3 Np treated mice showed more in ltrating mononuclear cells than those from the other groups. In addition, minor enlargement of hepatocyte and binucleated cells were observed occasionally, both those of control group and sample-treated groups. With regard to kidney histopathological study (Fig. 4), the tissue section of all sample-treated groups was normal, it exhibited preserved glomerular and tubular structure, relatively similar to those of the control group. Also, the H and E staining of splenic sections from control-and sample-treated groups displayed normal spleen architecture without any major histological abnormality, even at high concentrations of sample (Fig. 5).

Fig. 2(a-j):
Representative images of lung histopathology, (a) Control mice, (b-d) Alhydrogel® treated mice with administration dose of (b) 1.28 mg kg1, (c) 10.24 mg kg1, (d) 80.92 mg kg1, (e-g) Al(OH)3 Mp treated mice with administration dose of (e) 1.28 mg kg1, (f) 10.24 mg kg1 and (g) 80.92 mg kg1, (h-j) Al(OH)3 Np treated mice with administration dose of (h) 1.28 mg kg1, (i) 10.24 mg kg1 and (j) 80.92 mg kg1
White arrows show pulmonary interstitial thickening, black arrows show pulmonary arterioles dilatation and congestion, tissue section was stained with H and E and examined by light microscopy (40x magnification), scale bar 100 μm

Fig. 3(a-j):
Representative images of liver histopathology, (a) Control mice, (b-d) Alhydrogel® treated mice with administration dose of (b) 1.28 mg kg1, (c) 10.24 mg kg1, (d) 80.92 mg kg1, (e-g) Al(OH)3 Mp treated mice with administration dose of (e) 1.28 mg kg1, (f) 10.24 mg kg1, (g) 80.92 mg kg1, (h-j) Al(OH)3 Np treated mice with administration dose of (h) 1.28 mg kg1, (i) 10.24 mg kg1 and (j) 80.92 mg kg1
White arrows show lymphocytic and eosinophilic infiltrates, black arrows show hepatic cell swelling, tissue section was stained with H and E and examined by light microscopy (40x magnification), scale bar 100 μm

Fig. 4(a-j):
Representative images of kidney histopathology, (a) Control mice, (b-d) Alhydrogel® treated mice with administration dose of (b) 1.28 mg kg1, (c) 10.24 mg kg1, (d) 80.92 mg kg1, (e-g) Al(OH)3 Mp treated mice with administration dose of (e) 1.28 mg kg1, (f) 10.24 mg kg1, (g) 80.92 mg kg1, (h-j) Al(OH)3 Np treated mice with administration dose of (h) 1.28 mg kg1, (i) 10.24 mg kg1 and (j) 80.92 mg kg1
  Tissue section was stained with H and E and examined by light microscopy (40x magnification), scale bar 100 μm

Fig. 5(a-j):
Representative images of spleen histopathology, (a) Control mice, (b-d) Alhydrogel® treated mice with administration dose of (b) 1.28 mg kg1, (c) 10.24 mg kg1, (d) 80.92 mg kg1, (e-g) Al(OH)3 Mp treated mice with administration dose of (e) 1.28 mg kg1, (f) 10.24 mg kg1, (g) 80.92 mg kg1, (h-j) Al(OH)3 Np treated mice with administration dose of (h) 1.28 mg kg1, (i) 10.24 mg kg1 and (j) 80.92 mg kg1
  Tissue section was stained with H and E and examined by light microscopy (40x magnification).N 5, scale bar 100 μm

Table 7: Extended behavioral and physical symptoms observation of test animal after Al(OH)3 Np administration
+: Symptom observed, -: Symptom was not observed

Table 8: Body and organ weight of test animal
Statistical significance was evaluated using one-way analysis of variance (ANOVA) (*p<0.05), PBS: Phosphate-buffered saline


In the present study, Al(OH)3 Np with sub-micrometer size was successfully produced and importantly, its intraperitoneal acute toxicity profile was evaluated. The synthesized Al(OH)3 Np was not only demonstrated superior adjuvant effect compared to its micro-sized counterpart and commercial Al(OH)3 as was proved in the previous studies by Pambudi et al.16 and Sun et al.25, but also exerted a relatively low intraperitoneal acute toxicity. This toxicity profile is a key issue in the development of future nano-sized Al(OH)3 adjuvant.

The application of down-sizing strategy on alum-based adjuvant as we did in this research is a popular alternative to further improve its immunostimulant activity and could contribute to the next-generation vaccine development. To generate Al(OH)3 Nps, various top-down manufacturing method can be used, such as high through high-shear homogenization, ultrasonication and high-pressure microfluidization14,15,26. These strategies have been reported could successfully produce nanoscale Al(OH)3 with the average diameter of less than 200 nm and importantly increase the immune response13,25. Considering the scaling-up production process, we developed simple fabrication method to produce Al(OH)3 NPs by high-shear homogenization. For Alhydrogel®, it was known from previous study that its stock suspension has a relatively big median size (2.67 μm) and large polydispersity index11,26. It displayed a broad and heterogeneous particle size distribution, ranging from 0.5-10 μm, due to it highly tendency to form an aggregate14.

The prepared Al(OH)3 Nps were stabilized by the addition of low-concentration of sodium polyphosphate, resulting in a stable particle size over 2 months when stored at room temperature (data not shown). From our previous study, sodium polyphosphate seems to be most appropriate stabilizer compared to those of trehalose and polyvinyl alcohol27. Also, in drug delivery research, sodium polyphosphate has been intensively used as a crosslink agent as well as a stabilizer for metal oxide nanoparticulate28-30. In contrast, Alhydrogel® and Al(OH)3 Mps without any addition of stabilizer easily formed aggregate during the storage due to the flocculation, yet this micro-suspension could be quickly re-dispersed using physical agitation. However, It worth to note that the addition of sodium polyphosphate in Al(OH)3 Np suspension should be controlled. The excess of phosphate ion may alter the physicochemical character of Al(OH)3 NPs as the ligand exchange adsorption of the phosphate ion with the hydroxyl group of Al(OH)3 could take place31. This phenomena could be observed from the reduced PI value of Al(OH)3 Np. The exposure of phosphate ion from the sodium polyphosphate reduced PI value of Al(OH)3 Np to be around 7.2, while those of Alhydrogel® and Al(OH)3 Mp was 8.18 and 9.22, respectively. This PI alteration was consistent with previous research that reporting the reduced PI of Al(OH)3 into 4.0 due to the substitution of phosphate for hydroxyl32. For some antigens, the PI change due to the ligand exchange may critically affect protein binding capacity33-35 of the Al(OH)3. In morphological evaluation using TEM, Al(OH)3 Np exhibited spherical structure. Meanwhile, it was known that the common Al(OH)3 used for adjuvant including Alhydrogel® displayed fiber-likemicro-crystalline substructure. Such structural variations may be caused by the different preparation procedure and the chemical precursor used for producing36 Al(OH)3.

Besides highlighting the synthesis and physicochemical characterizations of Al(OH)3, the principal purpose in this research was to elucidate the toxicity profile of the nanosized Al(OH)3 as this toxicity study nano-scale Al(OH)3 has been characterized to a much lesser degree. It was well-known that engineered nanomaterials may have higher toxicity compared those in bulk form which may lead to undesirable effects. For example, the LD50 of bulk ZnO in mice after intratracheal instillation was 7950 mg kg1, while ZnO nanoparticle having diameter around 20 nm had increased toxicity36 with LD50 of 5 mg kg1. In another study, the LD50 of 20 and 50 nm silver nanoparticles in mice were estimated at 169 and 354 mg kg1, indicating the critical effect of particle size on toxicity profile of nanomaterial37. For aluminum-based material, the toxicity is highly dependent on its salt form. In general, the acute toxicity of aluminum compounds was relatively low due to the low solubility of these compounds38. The LD50 of bulk form of Al(OH)3 on rat through oral administration was higher than 2000 mg kg1 as only less than 0.01% Al(OH)3 was absorbed into the blood stream39. Lack of toxicity sign of Al(OH)3 in human following acute administration was also demonstrated40. In this study, dose of tested materials up to than 81.92 mg kg1 was clinically safe and did not cause any lethal effect on mice. This finding seems consistent with the previous work that reported no lethal sign on Sprague-Dawley rats after oral administration of higher dose of Al(OH)3 (302 mg kg1/day) for 28 days41. For the future work, sample with higher dose of Al(OH)3 are needed to obtain exact LD50, especially for those of Al(OH)3 Np.

Although the administered samples in this study did not display any lethal effect to the mice, some toxicological examinations, including behavioral alteration, body weight growth, organ index and organ histopathology analysis were carried out to comprehensively observe any undesired effect of Al(OH)3. In this study, the clinical symptoms shared among groups that took place during the sample administration day were drowsiness and hypoactivity and importantly such effects did not remain longer in mice. From body weight profile, all the mice showed positive trend after 14 day-post sample injection, the increase of body weight ranged from 21-35%. It was known that the 10% decrease of body weight from its initial value indicated the occurrence of side effect of the tested material as a body weight is an important marker of gross toxicity42. Hence, the administered samples in present research emphasized a non-toxic character of the tested Al(OH)3. From organ mass index data, mice treated with Al(OH)3 Np increased kidney and spleen mass coefficient which may attribute to the inflammation on these organs. However, the alteration of organ mass index did not necessarily result in change of tissue architecture; the histological examination of kidney and spleen treated with Al(OH)3 Np displayed no significant difference compared to those of control group. It could reveal that spleen and kidney may be the main organ which are responsible for filtering and clearing the accumulation of foreign particle of Al(OH)3 Np. Such spleen alteration may be facilitated by macrophage as was observed by Kwon et al.43 when observing the biodistribution of inhaled fluorescent magnetic nanoparticles.

The macrophage activation in spleen, however, did not cause histological accumulation of megakaryocytes in the red pulp as the spleen microstructure was normal and no pathological change was observed. Although sample was administered through intraperitoneal route, histology of lung tissue-treated with the highest dose of samples displayed noticeable effect. It thus may indicate the absorption of administered sample into systemic circulation before reaching lung.

Tremendous effort has been spent on developing of nanoparticle of aluminum salts adjuvant12,25,44-46 as well as an attempt to understand the exact immunostimulating mechanism underlying their strong adjuvant activity47. However, the in vivo toxicity study of Al(OH)3 Nps, to the best of our knowledge, has not been performed yet. Despite the favorable safety profile of Al(OH)3 in its conventional bulk form, the toxicity of Al(OH)3 NPs as a different entity is an important factor that should be investigated to answer the questioned biosafety of nano materials. Therefore, this study provided knowledge of toxicity profile of Al(OH)3 Nps administered through intraperitoneal administration, that could imply its safety for further development of nano-range particulates adjuvant.


Considering all findings in this study, it can be inferred that the toxicity profile of Al(OH)3 Np at the highest dose used in this research was not significantly different compared to those of control, Alhydrogel® and Al(OH)3 Mp-treated group. No permanent behavioral alterations were induced during 14-day observation period. To complete the histological data, further organ-related biochemical analysis and measurement of Al(OH)3 accumulation in major organs should be carried out in the future for probing any abnormality in the organ.


The findings of this study describes the intraperitoneal acute toxicity profile of Al(OH)3 Np. Although further toxicological assays are needed to complete this study, the current data indicated the low intraperitoneal toxicity of Al(OH)3 Np as its obtained data was not significantly different compared to those of control counterparts. Thus, it will help the researcher to uncover the high potency of Al(OH)3 Np and can be beneficial for further development of a new generation vaccine adjuvant.


This study is supported by Research Grant from Ministry of Science, Technology and Higher Education of Republic Indonesia (INSINAS 2019).

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