ALUMINUM NANOCRYSTAL DELIVERY SYSTEM, AND SELF-ASSEMBLED PARTICLE ADJUVANT VACCINE BASED ON BINDING OF ALUMINUM NANOCRYSTAL DELIVERY SYSTEM AND VACCINE ANTIGEN MOLECULE

Description

TECHNICAL FIELD

The present invention relates to the technical field of biomedicine technology and vaccines, and particularly relates to an aluminum nanocrystal vaccine delivery system and a preparation method thereof, and a self-assembled particle adjuvant vaccine based on specific binding of an Fc affinity protein covering a surface of an aluminum nanocrystal in the aluminum nanocrystal vaccine delivery system and a vaccine antigen molecule.

BACKGROUND

Adjuvants are crucial to the efficacy of subunit vaccines. Since Glenny and his colleagues first discovered in 1926 that combining aluminum salts (KAl(SO₄)₂·12H₂O) with diphtheria toxoid could increase vaccine potency, aluminum salt adjuvants had become the only vaccine adjuvants for human and were widely used in vaccine preparations. Existing aluminum salt adjuvants include potassium aluminum sulfate, aluminum phosphate, and aluminum hydroxide. The aluminum hydroxide adjuvant is the most widely used. It can greatly improve the immunogenicity of vaccines.

The specific surface area and uniformity of the aluminum hydroxide adjuvant determine the adsorption efficiency of the aluminum adjuvant to antigens. The smaller the aluminum hydroxide adjuvant particles, the larger the specific surface area, the more antigens can be adsorbed, and the more conducive to improving the immunogenicity of the vaccine. The existing aluminum hydroxide adjuvant generally activates humoral immunity but rarely T cell-mediated immunity, which is related to the presentation mode of adsorbed antigen of the aluminum hydroxide adjuvant. In addition to adjuvants, the antigen density, intracellular presentation level, and spatiotemporal characteristics also play an extremely important role in the production of antibodies (Nature Biomedical Engineering 2020, 4 (6), 636-648. Advanced Drug Delivery Reviews 2020, 158, 91-115.). After the micron-sized aluminum hydroxide adjuvant adsorbs the antigens, the aluminum adjuvant generally binds to the cell membranes of antigen-presenting cells such as dendritic cells (DC cells) to induce the formation of lipid rafts, causing the activation of downstream pathways, which cannot interfere with the fate of the presented antigens in the cell. The antigens are presented by the major histocompatibility complex II (MHC-II) after being processed by a lysosomal pathway, rather than by the cross-presentation of the major histocompatibility complex I (MHC-I). Nanocrystallization of the aluminum hydroxide adjuvant may change the uptake by antigen-presenting cells, thereby affecting the presentation mode of its adsorbed antigens. Although administration by adsorbing the antigens by the aluminum adjuvant is the most common vaccine combination mode, in the presence of serum or interstitial fluid, the surface adsorbed antigens will be rapidly desorbed from the aluminum adjuvant surface due to competitive adsorption, affecting the immune response ability of the vaccine (Nature Medicine. 2020, 26 (3), 430-440.). Moreover, some antigens are too strongly adsorbed by aluminum adjuvants, resulting in changes in antigen conformation, thereby slowing down and affecting the production of antigen-specific antibodies. Although there are some limitations in the application process of aluminum adjuvants, due to the strict requirements for vaccine safety, the successful development of novel adjuvants requires a long process and huge financial investment. Therefore, it will be of great practical value to develop a universal delivery platform for subunit vaccines based on aluminum adjuvant particles to reduce the impact of nonspecific adsorption of antigen adjuvants on antigens and increase the dose of antigens taken up by antigen-presenting cells. The inventors aim to disclose a structurally stable aluminum nanocrystal delivery system and a preparation method for a self-assembled particle adjuvant vaccine based on specific binding of an Fc affinity protein covering a surface of an aluminum nanocrystal and an antigen molecule. The method is a universal antigen self-assembled aluminum adjuvant delivery platform, which is non-selective for the charge of the antigen and can conveniently construct a self-assembled particle vaccine by binding the Fc affinity tag commonly used in antigen purification as an assembly element.

SUMMARY

The present invention provides an aluminum nanocrystal vaccine delivery system and a preparation method thereof, and a self-assembled particle adjuvant vaccine based on specific binding of an Fc affinity protein covering a surface of an aluminum nanocrystal in the aluminum nanocrystal vaccine delivery system and a vaccine antigen molecule. The aluminum nanocrystal according to the present invention can be effectively bound to the Fc affinity protein through a modified molecule so as to construct the aluminum nanocrystal delivery system, and can effectively carry immune antigens to further construct the self-assembled particle adjuvant vaccine, so that the stability of carrying the antigens can be remarkably improved, the density of the antigens carried on the surface of the aluminum nanocrystal is increased, and the immune antigens can be effectively delivered to lymph node tissues; and moreover, the cellular internalization of the immune antigens is greatly enhanced, immune cells are activated extremely efficiently, and thus the antibody generation level is enhanced.

In order to achieve above objective of the present invention, the present invention provides the following technical solution:

- the present invention provides an aluminum nanocrystal delivery system constructed by effectively binding an aluminum nanocrystal to an Fc affinity protein through a modified molecule.
- the average particle size of the aluminum nanocrystal is 5-500 nm; and the surface of the aluminum nanocrystal carries the modified molecule that can be linked to a group of the Fc affinity protein through host-guest coordination and chemoselectivity covalent modification.

Further, the aluminum nanocrystal is prepared by the following steps:

- adding a modified molecule solution to an aluminum salt solution, and stirring and fully mixing to obtain a mixed solution; and
- adding the mixed solution to an alkaline solution to obtain a reaction solution, adjusting the pH of the reaction solution to be 5.5-8.0, then reacting, standing, centrifuging and taking precipitate to obtain the aluminum nanocrystal.

The aluminum salt is more than one of aluminum chloride, aluminum nitrate, aluminum sulfate, and aluminum acetate; the solvent used in the aluminum salt solution is pure water, or a sodium acetate solution with the concentration of 0.01 mol/L; the concentration of the aluminum salt is 0.01-0.5 mol/L; and the stirring speed is 100-1,400 rpm, and the stirring time is 10 min to 5 h.

The modified molecule is more than one of L-O-phosphoserine, citric acid, oxalic acid, salicylic acid, and ATP; the concentration of the modified molecule solution is 0.01-0.5 mol/L; and in the mixed solution, the molar ratio of the aluminum salt to the modified molecules is (10:1)-(1:10).

The alkaline solution is more than one of NaOH, KOH and ammonia solution; the concentration of the alkaline solution is 0.01-0.5 mol/L; and the rate of adding the alkaline solution to the mixed solution is 1-20 mL/min.

Further, the reaction temperature is 25-90° C.; the reaction time is 10 min to 10 h; and the standing time is 1-12 h.

Further, the modified molecule includes one that can perform amino, carboxyl and sulfydryl functional group functionalization and chemoselectivity covalent modification group functionalization on a surface functional group of the aluminum nanocrystal.

Furthermore, the modified molecule is specifically more than one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, maleimide, succinimide, azide and alkyne.

Further, the corresponding modified molecule includes a corresponding reactant that can perform covalent binding with the Fc affinity protein, such as N-hydroxysuccinimide (NHS) obtained from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/(N-hydroxysuccinimide) NHS reaction.

The present invention also provides a preparation method for an aluminum nanocrystal delivery system. The preparation method includes the following steps:

- (1) adding an aluminum nanocrystal and a modified molecule to a solvent, and stirring the mixture to allow reaction, thereby obtaining a mixed solution 1;
- (2) adding an Fc affinity protein and a modified molecule to a solvent, and stirring the mixture to allow reaction, thereby obtaining a mixed solution 2; and
- (3) uniformly mixing the mixed solution 1 in step (1) and the mixed solution 2 in step (2), then reacting, centrifugally washing, taking the precipitate, and resuspending to obtain the aluminum nanocrystal delivery system.

Specifically, the solvent is specifically injection water, ultrapure water, normal saline, a Tris buffer solution, or a phosphate buffer solution; the mass-to-volume ratio of the aluminum nanocrystal to the solvent is 0.5-60 mg/mL; the molar ratio of aluminum ions to the modified molecule in the aluminum nanocrystal is 1:(0.001-1000);

- in step (1), the stirring reaction temperature is 4-25° C., the stirring reaction time is 0.5-24 h, and the stirring rate of the stirring reaction is 100-600 rpm;
- in step (2), the stirring reaction temperature is 4-25° C., and the stirring reaction time is 0.5-24 h; and
- in step (3), the reaction time is 0.5-24 h, the centrifugal washing is performed twice, and the product is resuspended into the buffer solution with the pH of 7.4-9.

Based on that, the present invention also provides a self-assembled particle adjuvant vaccine based on binding of an aluminum nanocrystal delivery system and an antigen molecule, wherein the aluminum nanocrystal delivery system is the abovementioned aluminum nanocrystal delivery system, and/or the aluminum nanocrystal delivery system is obtained by the abovementioned preparation method, and specifically, the surface of the aluminum nanocrystal prepared by the abovementioned method is covered with an Fc affinity protein and a specifically bound self-assembled vaccine antigen molecule.

The Fc affinity protein is a G protein and/or an A protein with high affinity to an Fc region; the G protein and/or A protein is a bacterial protein or a recombinant protein;

- the bacterial protein is derived from Group G Streptococcus and Staphylococcus aureus;
- the recombinant protein is arbitrarily modified by gene recombination, but still maintains the Fc affinity, and specifically is a recombinant A protein/G protein, an A protein or G protein with C-terminal recombinant cysteine or phosphoserine, and a polypeptide fragment derived from the A protein and/or G protein; and
- the polypeptide fragment from the A protein and/or G protein is recombinant peptide formed by different amino acid sequences and chemically synthesized polypeptide sequences.

Specifically, the Fc affinity protein is derived from the species of human, murine, rabbits, pigs and sheep.

The binding mode of the Fc affinity protein and the aluminum nanocrystal may be that the aluminum nanocrystal is bound with the Fc affinity protein in a non-covalent mode, such as electrostatic or host-guest coordination adsorption, or covalent modification, chemoselectivity specific binding and other forms.

The vaccine antigen molecule includes one or more of an Fc fragment containing pathogenic subunit antigen, recombinant subunit antigen, antigen epitope peptide and nucleic acid antigen.

The pathogen includes viruses, bacteria and/or parasites; and

Preferably, the viruses are selected from DNA virus and/or RNA virus.

More preferably, the viruses are selected from coronaviridae, herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, picornaviridae, hepadnaviridae, flaviviridae, papillomaviridae, poxviridae and retroviridae.

Further preferably, the viruses are selected from novel coronavirus, influenza virus, herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV.

Preferably, the bacteria are selected from gram-positive bacteria and/or gram-negative bacteria.

More preferably, the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter frazier, Pesudomonas pyocyaneum, Acinetobacter baumannii, Mycobacterium tuberculosis and Helicobacter pylori.

Further preferably, the bacteria are selected from Streptococcus pneumoniae, salmonella, Neisseria meningitidis, Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Helicobacter pylori and the like.

The parasites are selected from one or more of plasmodium, Toxoplasma gondii, trypanosome, schistosome, filariasis and leishmania.

The present invention also provides a preparation method of a self-assembled particle adjuvant vaccine based on binding of the aluminum nanocrystal delivery system and the vaccine antigen molecule, specifically, the method includes: uniformly mixing an Fc fragment containing vaccine antigen molecule with an aluminum nanocrystal delivery system solution, so as to obtain a self-assembled particle vaccine based on an aluminum nanocrystal with a surface covered with an Fc affinity protein and an antigen.

Specifically, materials are mixed by a rotary shaker at 4-25° C. for 10-60 min so as to be uniformly mixed; and the concentration of the aluminum nanocrystal is 0.5-60 mg/mL, the concentration of antigen is 5-200 mg/mL, and the mass ratio of the vaccine antigen molecules to aluminum ions in the aluminum nanocrystal is 1:(0.1-100).

An administration method of the self-assembled particle adjuvant vaccine includes oral perfusion, intravenous injection, intramuscular injection, intraperitoneal injection and any combination thereof.

More preferably, the administration method of the adjuvant vaccine bound reagent is intramuscular injection.

The present invention has the technical effects that:

The aluminum nanocrystal provided by the present invention can be effectively bound with the Fc affinity protein and effectively carry the immune antigen, thus obtaining more excellent immunotherapy effect with lower antigen dosage and lower injection dosage; and the immune cells are efficiently activated to implement balanced body fluid and cell immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

FIG. 1 shows TEM images of aluminum nanocrystals obtained according to Examples 1, 2 and 3 of the present invention;

FIG. 2 shows an XRD image of an aluminum nanocrystal obtained according to an Example 1 of the present invention;

FIG. 3 shows BMDCs activation level and MHC expression level by an adjuvant according to an Example 17 of the present invention;

FIG. 4 shows RBD specific IgG antibody titer in mouse serum 19, 35 and 56 days after vaccination according to an Example 19 of the present invention;

FIG. 5 shows pseudovirus neutralizing antibody titer in mouse serum 56 days after vaccination according to an Example 19 of the present invention; and

FIG. 6 is a schematic diagram of aluminum nanocrystal surface Fc binding protein modification and antigen self-assembly process according to the present invention.

DETAILED DESCRIPTION

The present invention discloses an aluminum nanocrystal vaccine delivery system and a preparation method thereof, and a self-assembled particle adjuvant vaccine based on specific binding of an Fc affinity protein covering a surface of an aluminum nanocrystal in the aluminum nanocrystal vaccine delivery system and a vaccine antigen molecule. Those skilled in the art may refer to the content herein with appropriate modification of the process parameters. It is particularly to be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and the skill in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the disclosure, spirit and scope of the present invention to implement and apply the technology of the present invention.

The aluminum nanocrystal vaccine delivery system and the preparation method thereof, and the self-assembled particle adjuvant vaccine based on specific binding of the Fc affinity protein covering the surface of the aluminum nanocrystal in the aluminum nanocrystal vaccine delivery system and the vaccine antigen molecule, and raw materials or reagents used in the application of vaccine immune synergy can be commercially purchased.

The aluminum nanocrystal adjuvant used in the examples of the present invention is homemade, and the experimental solution is shown in the Examples.

The present invention is further described below in conjunction with the Examples:

Example 1

AIC13.6H₂O was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.055 mol/L of aluminum ion; the obtained aluminum salt solution and 0.05 mol/L of L-O-phosphoserine (purchased from Sigma-Aldrich, CAS No. 407-41-0) were stirred for 2 h at a stirring speed of 300 rpm; after fully mixing, 0.1 mol/L of NaOH solution was added at a speed of 7 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 7. The molar ratio of Al³⁺ to the L-O-phosphoserine was 1:2; and the reaction temperature was 35° C., and the stirring reaction was carried out for 2 h; then, standing was allowed for 7 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 2

AIC13.6H₂O was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.45 mol/L of aluminum ion; the obtained aluminum salt solution and 0.35 mol/L of sodium citrate were stirred for 5 h at a stirring speed of 100 rpm; after fully mixing, 0.01 mol/L of NaOH solution was added at a speed of 1 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 5.5. The molar ratio of Al³⁺ to the sodium citrate was 1:6; and the reaction temperature was 45° C., and the stirring reaction was carried out for 5 h; then, standing was allowed for 1 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 3

AlCl₃·6H₂O was dissolved in pure water to prepare an aluminum salt solution containing 0.01 mol/L of aluminum ion; the obtained aluminum salt solution and 0.02 mol/L of salicylic acid were stirred for 5 h at a stirring speed of 200 rpm; after fully mixing, 0.5 mol/L of ammonia solution was added at a speed of 9 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 7.5. The molar ratio of Al³⁺ to the salicylic acid was 7:1; and the reaction temperature was 25° C., and the stirring reaction was carried out for 10 h; then, standing was allowed for 5 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Comparative Example 1

AlCl₃·6H₂O was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.5 mol/L of aluminum ion; then, 0.15 mol/L of NaOH solution was added at a speed of 7 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 7. The reaction temperature was 90° C., the stirring reaction was carried out for 10 min, and the stirring speed was 900 rpm; then, standing was allowed at room temperature for 5 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare aluminum nanocrystal without modification molecules.

Example 4

Aluminum sulfate was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.45 mol/L of aluminum ion; the obtained aluminum salt solution and 0.35 mol/L of sodium citrate were stirred for 3 h at a stirring speed of 800 rpm; after fully mixing, 0.01 mol/L of KOH solution was added at a speed of 1 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 5.5. The molar ratio of Al³⁺ to the sodium citrate was 1:6; and the reaction temperature was 45° C., and the stirring reaction was carried out for 5 h; then, standing was allowed for 1 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 5

Aluminum nitrate was dissolved in pure water to prepare an aluminum salt solution containing 0.01 mol/L of aluminum ion; the obtained aluminum salt solution and 0.02 mol/L of aqueous ATP were stirred for 5 h at a stirring speed of 1,000 rpm; after fully mixing, 0.5 mol/L of ammonia solution was added at a speed of 9 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 7.5. The molar ratio of Al³⁺ to the salicylic acid was 7:1; and the reaction temperature was 25° C., and the stirring reaction was carried out for 10 h; then, standing was allowed for 5 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 6

AlCl₃·6H₂O was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.5 mol/L of aluminum ion; the obtained aluminum salt solution and 0.01 mol/L of oxalic acid were stirred for 10 min at a stirring speed of 1,400 rpm; after fully mixing, 0.15 mol/L of ammonia solution was added at a speed of 10 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 5. The reaction temperature was 90° C., and the stirring reaction was carried out for 10 h; then, standing was allowed at room temperature for 1 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 7

Aluminum acetate was dissolved in 0.01 mol/L of NaAc to prepare an aluminum salt solution containing 0.25 mol/L of aluminum ion; the obtained aluminum salt solution and 0.5 mol/L of ATP were stirred for 40 min at a stirring speed of 1,200 rpm; after fully mixing, 0.05 mol/L of KOH solution was added at a speed of 20 ml/min through a peristaltic pump; and the pH of the mixed reaction solution was adjusted to be about 8. The reaction temperature was 55° C., and the stirring reaction was carried out for 2 h; then, standing was allowed at room temperature for 12 h after the reaction was finished; centrifuging followed by washing was performed; and finally sterilizing by high-pressure steam or by a filter membrane was performed to prepare an aluminum nanocrystal.

Example 8
(1) Physicochemical Property Characterization of Aluminum Nanocrystal

An aluminum nanocrystal was diluted to be 10 μg/ml at 25° C., and then dripped on a common carbon support membrane, so as to observe the structure of the aluminum nanocrystal through an electron microscope, and the aluminum nanocrystal was displayed as particles under a transmission electron microscope (FEI company, model: Tecnai G2 20S-TWIN). As shown in FIG. 1, FIG. 1 shows TEM images of the nanoparticles obtained when the aluminum element and the modified molecule were in different molar ratios in Example 1. FIG. 1A shows an electron microscope image of the product in the Example 1, FIG. 1B shows an electron microscope image of the product without a modified molecule in the Comparative Example 1, FIG. 1C shows an electron microscope image of the product in the Example 2, and FIG. 1D shows an electron microscope image of the product in the Example 3.

As shown in FIG. 1, when the modified molecule exists, the obtained aluminum nanocrystal adjuvant has smaller average size. The average size of the aluminum nanocrystals obtained in the Example 1 is 20 nanometers, the average size of the aluminum nanocrystals obtained in the Example 2 is 57.1 nanometers, and the average size of the aluminum nanocrystals obtained in the Example 3 is 25.3 nanometers. When the modified molecule does not exist, the obtained products are clustered aggregated particles with the average size of about 350 nanorods. Therefore, in the presence of the modified molecule, the obtained aluminum nanocrystal has more uniform size, better dispersity and better solution stability.

(2) XRD Characterization of Aluminum Nanocrystal

10 mL of the aluminum nanocrystal prepared in the Example 1 was subjected to freeze-drying to obtain a powder sample, XRD characterization was performed on the powder sample, and then the powder sample was analyzed with Jade, as shown in FIG. 2. FIG. 2 shows an XRD image of product nanoparticles obtained in the Example 1.

As shown in FIG. 2, the obtained aluminum nanocrystal can be correspondingly of an aluminum hydroxide structure.

(3) Particle Size and Surface Charge Characterization for Aluminum Nanocrystal

The aluminum nanocrystals prepared in the Examples 1-7 and the aluminum nanocrystal without the modified molecule prepared in the Comparative Example 1 were respectively diluted to be 10 μg/ml at 25° C., and the particle size and Zeta potential off the aluminum nanocrystals were tested by a nano particle analyzer (purchased from Malvern company, model: Zetasizer Nano ZS). The result is shown in Table 1.

The physicochemical properties of the aluminum nanocrystal particles prepared in the Examples 1-7 and the aluminum nanocrystal without modified molecules prepared in the Comparative Example 1 are shown in Table 1.

TABLE 1

Physicochemical properties of two aluminum nanocrystals obtained with and without modified molecule

Comparative

Example 1
Example 2
Example 3
Example 1
Example 4
Example 5
Example 6
Example 7

Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum
Aluminum

nanocrystal
nanocrystal
nanocrystal
nanocrystal
nanocrystal
nanocrystal
nanocrystal
nanocrystal

Test items
particles
particles
particles
particles
particles
particles
particles
particles

Average
33.7 nm
71.4 nm
31.6 nm
451.6 nm
76.2 nm
5.6 nm
158.2 nm
482.1 nm

hydrated

particle

size

Potential
−23
−16
−14
26
−18
−21
−17
−13

(mV)

As shown in Table 1, the aluminum nanocrystals that were nano-sized and uniformly dispersed were prepared in the Examples 1 to 7 and Comparative Example 1.

Example 9