Nanometer aluminum adjuvant, lipid nanoparticle, preparation method and use thereof

Abstract

Disclosed is a nano-aluminum adjuvant comprising flaky aluminum hydroxide, the flaky aluminum hydroxide having a length L, a width W and a thickness T, wherein the length L is in the range of 20-100 nm, the width W is in the range of 20-100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller one of the length L and the width W. Also disclosed are a preparation method of the nano-aluminum adjuvant, a lipid nanoparticle comprising the nano-aluminum adjuvant, a method for preparing the lipid nanoparticle comprising the nano-aluminum adjuvant, and a use of the nano-aluminum adjuvant for preparing an mRNA-liposome drug.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nano-aluminum adjuvant, a method for preparing an aluminum adjuvant, lipid nanoparticles containing the nano-aluminum adjuvant, a method for preparing lipid nanoparticles, and the use of the nano-aluminum adjuvant in the preparation of lipid nanoparticles or liposome drugs. Background Technology

[0002] Nucleic acid drugs are biological macromolecules developed based on nucleic acid molecules such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). They treat diseases by regulating gene expression or protein synthesis. RNA drugs are currently one of the most widely used classes of nucleic acid drugs, mainly including antisense oligonucleotides (RNA, DNA, DNA, DNA, DNA, DNA). A nti S ense O ligonucleotide (ASO), small interfering RNA ( s mall i nterfering RNA siRNA), messenger RNA ( m essenger RNA These drugs are classified into types such as mRNA. Their core advantages include targeting traditionally undrugable targets, long-lasting effects (some drugs require dosing only once every six months), and short production cycles.

[0003] Among various nucleic acid drugs, mRNA-based therapies, by delivering exogenous mRNA to cells to express therapeutic proteins, have shown great potential in combating a variety of diseases (Pardi, et al., Nat. Rev. Drug Discov. 2018, 17, 261-279. Chaudhary, et al., Nat. Rev. Drug Discov. 2021, 20, 817.). However, mRNA is readily degraded by nucleases present in the extracellular fluid under physiological conditions, and its negatively charged nature limits cellular uptake efficiency. Furthermore, after entering the cell, mRNA needs to escape from the endosome to the cytoplasm to initiate protein translation. Therefore, developing efficient and biosafe delivery vectors capable of targeted delivery of mRNA to the cytoplasm of target cells has become one of the key challenges in the field of nucleic acid drugs. An ideal delivery system should be able to protect mRNA from nuclease degradation in vivo, achieve efficient cellular uptake and endosome escape, and promote target protein expression (Hajj, et. al., Nat. Rev. Mater. 2017, 2, 17056. Wang, et. al., Mol. Cancer 2021, 20, 33.).

[0004] Currently, lipid nanoparticles composed of ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol (PEG) lipids are being developed. L ipid N ano P LNP (Liver-Nutrient NP) is the most advanced delivery platform for RNA therapy in clinical practice (Han, et al., Nat. Chem. 2024, 16, 1687-1697.). Two COVID-19 mRNA vaccines based on the LNP delivery system have been approved by the U.S. Food and Drug Administration (FDA) for use in combating the COVID-19 pandemic, fully validating the safety and efficacy of the LNP platform in mRNA vaccines (Mullard, et al., Nat. Rev. Drug Discov. 2021, 20, 500-501. Baden, et al., N. Engl. J. Med. 2021, 384, 403-416.).

[0005] However, this field still faces a key challenge: the release of mRNA cargo loaded in LNP systems into the cytoplasm is extremely limited. Specifically, after drug-loaded LNPs enter the cell via endocytosis, they form early endosomes, gradually mature into late endosomes, and eventually fuse with lysosomes. For effective delivery, the mRNA cargo must be released into the cytoplasm before the late endosomes fuse with the lysosomes; otherwise, most of the mRNA cannot escape from the endocytosis / lysosome pathway in time, will be degraded by enzymes in the lysosomes, and thus cannot be translated into the target protein, directly affecting the efficacy and therapeutic effect of the vaccine. Endosome escape is a key bottleneck in mRNA-LNP drug delivery, and releasing mRNA before lysosome fusion is a crucial step in achieving efficient delivery. In existing technologies, although most (95%) of LNPs are taken up by the cell, less than 5% of the mRNA delivered via LNPs can escape the endosomes and reach the cytoplasm (Zhao, et al., Nat. Nanotech. 2024, 19, 1702-1711.). Since mRNA must reach the cytoplasm to achieve its mechanism of action, improving endosome escape efficiency is of great significance for enhancing the therapeutic effect of mRNA.

[0006] Generally, in LNP delivery systems, endosome escape is primarily attributed to the ionizable lipid components of the LNP (Huang, et al., Adv. Mater. 2022, 34, e2107946.). In an acidic endosome environment, ionizable lipids protonate and become positively charged, interacting electrostatically with the negatively charged endosome membrane, disrupting the membrane structure and thus promoting the release of mRNA into the cytoplasm. Therefore, most studies focus on optimizing LNP components to improve endosome escape efficiency.

[0007] Recent studies have found that manganese ions encapsulated in LNPs can be released into endosomes, further promoting endosome escape and improving transfection efficiency (Fan, et al., Sci. Adv. 2022, 8, 51.). Similar findings have been observed in other nucleic acid delivery systems. Nanocarriers containing inorganic ions such as calcium and zinc ions can induce an increase in inorganic ion concentration within lysosomes after entering the lysosome, causing a sharp rise in osmotic pressure, disrupting osmotic balance, and thus promoting the release of nucleic acids into the cytoplasm (Semple et al., Nat. Biotechnol. 2010, 28, 172-176. Choi et al., ACS Nano, 2014, 8, 4559-4570.).

[0008] Aluminum adjuvants are among the most widely used pharmaceutical adjuvants, and their safety and efficacy have been well-established (Reed, et al., Nat. Med. 2013, 19, 1597-1608.). Since the immunomodulatory effect of alum was first discovered in 1926, aluminum adjuvants have become the most widely used human vaccine adjuvants globally, with a cumulative usage exceeding 3 billion doses. Aluminum hydroxide was the first human immunomodulator approved by the US FDA. Aluminum adjuvants carry a positively charged surface, allowing them to effectively load negatively charged mRNA (Li, et al., Chem. Soc. Rev. 2018, 47, 4954-4980.). However, traditional aluminum adjuvants are difficult to encapsulate in liquid nitrogen nanoparticles (LNPs) due to their large particle size and tendency to aggregate. Currently, aluminum adjuvants have not been integrated as functional components into the internal structure of LNPs.

[0009] Therefore, there is an urgent need in the pharmaceutical field to develop an aluminum adjuvant that can be used in LNP. Summary of the Invention

[0010] To address the above and other issues, the inventors, through in-depth research, developed a nano-aluminum adjuvant applicable to LNPs. This nano-aluminum adjuvant can load mRNA and, as an essential component, embed or encapsulate it within the LNP lipid bilayer, forming nano-aluminum adjuvant-embedded lipid particles for mRNA delivery. Under acidic endosomal conditions, the nano-aluminum adjuvant in the LNP rapidly dissolves, releasing Al. 3+ This leads to a sharp increase in ion concentration within the endosome lumen, disrupting endosome membrane stability and causing osmotic imbalance, thereby promoting endosome escape of mRNA and ultimately increasing the transfection efficiency of mRNA in target cells.

[0011] At least one embodiment of this disclosure provides a nano-aluminum adjuvant comprising flake aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W.

[0012] In some examples, the nano-aluminum adjuvant satisfies one or more of the following:

[0013] (1) It has a pore size in the range of 1 nm to 10 nm;

[0014] (2) Having a total pore volume in the range of 0.09 cm³ / g to 0.41 cm³ / g; and

[0015] (3) It has a specific surface area BET in the range of 18.29 m² / g to 227.58 m² / g.

[0016] At least one embodiment of this disclosure also provides a method for preparing a nano-aluminum adjuvant comprising flake-shaped aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W, the method comprising: dissolving aluminum chloride in an alkaline solution, heating the resulting solution, and then centrifuging to obtain the nano-aluminum adjuvant.

[0017] In some examples, the method satisfies one or more of the following:

[0018] (1) The alkaline solution is selected from sodium hydroxide, potassium hydroxide, C 1~4 Sodium alkanol, C 1~4 An aqueous solution of potassium alkoxide or any combination thereof;

[0019] (2) The molar ratio of the aluminum chloride to the alkali is in the range of 1:2 to 1:5;

[0020] (3) The heating temperature is 80°C to 200°C;

[0021] (4) The heating time is from 6 hours to 80 hours; and

[0022] (5) The centrifugation speed is from 3,000 rpm to 25,000 rpm.

[0023] At least one embodiment of this disclosure also provides a lipid nanoparticle comprising a nano-aluminum adjuvant, wherein the nano-aluminum adjuvant comprises flake-shaped aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W, the lipid nanoparticle having a lipid bilayer, and the nano-aluminum adjuvant being encapsulated in the lipid bilayer and / or at least partially embedded in the lipid bilayer.

[0024] In some examples, the lipid nanoparticles also contain ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol lipids.

[0025] In some examples, the lipid nanoparticles also contain a drug. For example, the drug is an mRNA drug. For example, the mRNA drug includes one or more of Herceptin-mRNA, insulin-mRNA, and interleukin-mRNA.

[0026] In some examples, the nano-aluminum adjuvant is stable at pH > 7.4 and degrades to release Al at pH 5.0 ~ 6.8. 3+ .

[0027] At least one embodiment of this disclosure also provides a method for preparing lipid nanoparticles containing a nano-aluminum adjuvant, wherein the nano-aluminum adjuvant comprises flake aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W; the method comprises: (1) dissolving aluminum chloride in an alkaline solution, heating the resulting solution, and then centrifuging to obtain the nano-aluminum adjuvant; (2) mixing the nano-aluminum adjuvant prepared in (1) with ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol lipids in an aqueous medium to obtain the lipid nanoparticles containing the nano-aluminum adjuvant.

[0028] In some examples, the method further includes mixing the nano-aluminum adjuvant with a drug before mixing the nano-aluminum adjuvant with the ionizable lipid, cholesterol, neutral lipid, and polyethylene glycol lipid to load the drug onto the nano-aluminum adjuvant. For example, the drug includes an mRNA drug.

[0029] In some examples, the aqueous medium is a phosphate-buffered saline solution.

[0030] At least one embodiment of this disclosure also provides the use of a nano-aluminum adjuvant in the preparation of a liposomal drug, wherein the nano-aluminum adjuvant comprises sheet-like aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W, and the liposomal drug has improved endosome escape efficiency and / or target cell transfection efficiency. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0032] Figure 1 A flowchart illustrating the preparation of lipid nanoparticles (LNPs) comprising nano-aluminum adjuvants loaded with messenger ribonucleic acid (mRNA) according to one embodiment of the present invention is shown.

[0033] Figure 2 A transmission electron microscope image of the non-porous nano-aluminum adjuvant prepared according to Example 1 of this disclosure is shown;

[0034] Figure 3 Transmission electron microscopy (TEM) image of a small-pore (pore size of about 2 nm) nano-aluminum adjuvant prepared according to Example 2 of this disclosure is shown.

[0035] Figure 4 Transmission electron microscopy (TEM) image of macroporous (pore size about 5 nm) nano-aluminum adjuvant prepared according to Example 3 of this disclosure is shown.

[0036] Figure 5 Transmission electron microscopy (TEM) image of lipid nanoparticles (Alum-mRNA-LNP) containing an aluminum nanoparticle adjuvant loaded with mRNA, prepared according to Example 5 of this disclosure;

[0037] Figure 6The results of a hemolysis experiment performed according to Example 6 of this disclosure are shown, wherein: a - a schematic diagram of the hemolysis experiment; b - photographs of hemolyzed samples from 96-well plates treated with PBS, LNP, and Alum-LNP under different pH conditions, with Triton used as a positive control; c - absorbance of the hemolyzed samples from 96-well plates presented in bar chart form, with data expressed as mean ± standard deviation (n = 4);

[0038] Figure 7 The changes in serum markers in C57BL / 6 mice after three intramuscular injections of PBS, mOVA (model antigen ovalbumin), Alum-mOVA, mOVA-LNP, and Alum-mOVA-LNP (10 µg mOVA per injection) are shown. Among them, α-CD 3+ CD 4+ IFN-γ+, β-CD 3+ CD 4+ IL 4+ ;

[0039] Figure 8 The tumor growth inhibition curves of B16 tumor-bearing mice treated with PBS, mOVA, Alum-mOVA, mOVA-LNP, and Alum-mOVA-LNP (10 µg mOVA per injection) are shown.

[0040] Figure 9 The levels of corresponding antibodies or proteins in mouse serum treated with Herceptin-mRNA, insulin-mRNA, and interleukin 2-mRNA encapsulated with LNP and Alum-LNP are shown, where *P < 0.05, **P < 0.01, and ***P < 0.001. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this invention.

[0042] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such combinations to obtain further embodiments.

[0043] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety.

[0044] In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this disclosure, the use and terminology of this disclosure shall prevail.

[0045] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0046] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0047] Unless otherwise stated, when disclosing or claiming protection for any type of range (e.g., the number of carbon atoms), it is intended to separately disclose or claim protection for each possible numerical value that the range may reasonably cover, including any subranges included therein. For example, in this document, if the numerical range is 1 to 100, it indicates any numerical value or subrange or endpoint contained within that range.

[0048] As used herein, the words “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms “containing” or “comprising (including)” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently composed of” or “composed of”.

[0049] It should be understood that, unless otherwise specified, the singular form used in this disclosure (such as "a / an / a kind") may include plural references.

[0050] The reagents and raw materials used in this disclosure are commercially available or can be prepared by conventional chemical synthesis methods.

[0051] This article uses the term "optional" to describe a situation that may or may not occur.

[0052] mRNA drugs are a class of biotechnology drugs that use messenger ribonucleic acid (mRNA) to guide cells in synthesizing specific proteins. The core principle is the delivery of artificially synthesized or modified exogenous mRNA molecules into human cells, where the target protein is generated through the cell's own protein synthesis mechanisms, thereby triggering an immune response or replenishing deficient proteins. To protect mRNA from degradation and achieve precise delivery, mRNA drugs are typically encapsulated in delivery systems such as LNPs, entering the target cell via endocytosis and then being released from endosomes (i.e., endosome escape) into the cytoplasm to exert their biological effects.

[0053] Endosomal escape is one of the key bottlenecks currently hindering the delivery and intracellular therapeutic efficacy of mRNA nanomedicines. If mRNA drugs loaded in LNP delivery systems cannot achieve endosome escape, they will be trapped in the endosome-lysosome degradation pathway, leading to inactivation or degradation. One of the current problems with mRNA drugs is that the amount of mRNA cargo loaded on drug-loaded LNPs released into the cytoplasm is extremely limited. Drug-loaded LNPs enter the cell through endocytosis, forming early endosomes, which gradually mature into late endosomes and eventually fuse with lysosomes. For effective delivery, the mRNA cargo must be released into the cytoplasm before the late endosomes fuse with the lysosomes; otherwise, most of the mRNA will be degraded by enzymes in the lysosomes (Zhang, et al., Chem. Soc. Rev. 2024, 53, 317-360.). Therefore, releasing as much mRNA as possible before lysosomal maturation is a crucial stage for achieving efficient delivery. Of the existing mRNA-LNP drugs, although most (95%) of LNPs can be taken up by cells, less than 5% of the mRNA delivered via LNPs successfully escapes endosomes and reaches the cytoplasm (Zhao, et al., Nat. Nanotech. 2024, 19, 1702-1711.). Since mRNA must reach the cytoplasm to exert its function / effect, improving endosome escape efficiency is crucial for enhancing the therapeutic effect of mRNA.

[0054] Using adjuvants in mRNA drug delivery systems to induce endosome escape is currently a hot research topic. Aluminum adjuvants are the most widely used adjuvants for protein subunit vaccines, and their safety and efficacy have been well-established (Reed, et al., Nat. Med. 2013, 19, 1597-1608.). Aluminum adjuvants have a positively charged surface, enabling them to effectively load negatively charged mRNA (Li, et al., Chem. Soc. Rev. 2018, 47, 4954-4980.). However, traditional aluminum adjuvants are difficult to encapsulate in LNPs due to their large particle size and tendency to aggregate.

[0055] Through in-depth research, the inventors discovered that nano-sized aluminum adjuvants are characterized by their small size and good dispersibility, and can dissolve and release aluminum ions (Al) under low endobolic pH conditions. 3+ Therefore, it is suitable as a component of LNP, by releasing Al 3+ It promotes the endosome escape of mRNA. In addition, the stability of nanoscale aluminum adjuvants is affected by factors such as size and morphology, which determines the rate at which they rapidly dissolve and release aluminum ions under low endosome pH conditions (Lindblad, et al., Vaccine 2004, 22, 3658-3668.).

[0056] To address the above issues, the inventors designed a sheet-like aluminum nanoparticle adjuvant capable of loading mRNA and embedding or encapsulating it within the lipid bilayer of LNPs. After the drug-loaded LNP is internalized by target cells, the mRNA-loaded aluminum nanoparticle adjuvant rapidly dissolves and releases Al in response to the low pH environment of the endosomal environment. 3+ By disrupting endosome membrane stability and inducing osmotic imbalance, this promotes endosome escape of mRNA, ultimately improving mRNA transfection efficiency in target cells. The inventors' research shows that encapsulating nano-aluminum adjuvants within LNPs enhances LNP delivery performance, providing new insights for the development of next-generation LNP platforms and the optimization of mRNA therapeutic strategies.

[0057] One embodiment of this disclosure provides a nano-aluminum adjuvant comprising flake aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W.

[0058] As used herein, the term "nano-aluminum adjuvant" refers to a pharmaceutically acceptable nanoscale aluminum-containing adjuvant and is used interchangeably with "Alum". Aluminum adjuvants are among the most widely used pharmaceutical adjuvants, and their safety and efficacy have been well-established. In some examples, the nano-aluminum adjuvants described in this disclosure may contain or be aluminum hydroxide (or hydrated aluminum oxide).

[0059] As described above, the nano-aluminum adjuvants of this disclosure have a sheet-like shape, with their length L and thickness W dimensions being much larger than their thickness T dimension, i.e., they have a "two-dimensional (2D)" structure. In some examples, the length L and width W can independently be in the range of about 1 to 100 nm, for example, in any combination of the numerical ranges of 1 to 90 nm, 1 to 80 nm, 1 to 70 nm, 1 to 60 nm, 1 to 50 nm, 5 to 50 nm, 10 to 50 nm, 15 to 100 nm, 20 to 50 nm, or above. In some examples, the thickness T can be much smaller than the smaller of the length L and the thickness W, for example, the thickness T can be less than or equal to 1 / 10 of the smaller of the length L and the width W, such as less than or equal to 1 / 15, 1 / 20, 1 / 25, 1 / 40, 1 / 50, 1 / 75, 1 / 100, etc. For example, the thickness of the nano-aluminum adjuvant can be in the range of 0.01~1 nm, 0.01~0.8 nm, 0.01~0.7 nm, 0.01~0.6 nm, 0.01~0.5 nm, 0.01~0.4 nm, 0.01~0.3 nm, 0.01~0.2 nm, 0.01~0.1 nm, and 0.05~0.1 nm. The nano-aluminum adjuvant having the length T, width W, and thickness T specified in this disclosure can enter cells via endocytosis and effectively dissolve and release Al. 3+ This leads to a surge in ion concentration within the endosome lumen, disrupting endosome membrane stability and causing osmotic imbalance, thereby promoting endosome escape of mRNA and ultimately increasing the transfection efficiency of mRNA in target cells. Furthermore, the disclosed nano-aluminum adjuvant also possesses the adjuvant effect inherent in aluminum adjuvants themselves; that is, aluminum adjuvants activate the TH2 pathway, and LNPs activate the TH1 pathway, effectively achieving synergistic effects between different pathways.

[0060] In some embodiments, the nano-aluminum adjuvants described herein may be non-porous (non-porous) or porous. For example, the nano-aluminum adjuvant may have a pore size in the range of about 0 nm to 10 nm, wherein when the pore size of the nano-aluminum adjuvant is about 0 nm, it means that the nano-aluminum adjuvant does not have noticeably visible pores, i.e., it is non-porous or non-porous. In some examples, the pore size of the nano-aluminum adjuvant may be in the range of about 0 to 10 nm, for example, in the ranges of about 0 to 8 nm, about 0 to 7 nm, about 0 to 6 nm, about 0 to 5 nm, about 1 to 10 nm, about 1 to 8 nm, about 1 to 7 nm, about 1 to 6 nm, about 1 to 5 nm, about 2 to 5 nm, etc., for example, about 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 nm, etc. Alternatively, the nano-aluminum adjuvant may have a pore size greater than or equal to 0.09 cm⁻¹. 3 / g to 0.11 cm 3 / g, 0.11 cm3 / g to 0.14 cm 3 / g, 0.32 cm 3 / g to 0.36 cm 3 / g, 0.36 cm 3 / g to 0.38 cm 3 / g, 0.38 cm 3 / g to 0.41 cm 3 The total pore volume is [value missing]. Alternatively, nano-aluminum adjuvants may have a specific surface area (BET) ranging from 18.29 m² / g to 227.58 m² / g. Generally, one of the mechanisms of action of aluminum adjuvants is the "warehouse effect," which promotes the action of drug molecules by adsorbing and releasing them. Porosity and specific surface area are important physicochemical parameters of aluminum adjuvants, which have a certain influence on the performance and final effect of drug formulations. Careful control of microscopic dimensions such as specific surface area and porosity can further improve the performance of aluminum adjuvants during design and optimization. Aluminum adjuvants with the porosity and specific surface area defined in this disclosure exhibit good drug loading and release capabilities.

[0061] Another aspect of this disclosure provides a method for preparing any of the above-mentioned nano-aluminum adjuvants, the method comprising: dissolving aluminum chloride in an alkaline solution, heating the resulting solution, and then centrifuging to obtain the nano-aluminum adjuvant.

[0062] In some embodiments, the potassium chloride used in the methods described herein may be anhydrous potassium chloride or hydrated potassium chloride (e.g., potassium chloride hexahydrate KCl·6H2O, etc.).

[0063] In some embodiments, the alkaline solution used in the methods described herein can be an aqueous solution of any suitable inorganic or organic base. For example, the alkaline solution may include an aqueous solution of an inorganic base such as sodium hydroxide or potassium hydroxide; or, for example, C... 1~4 Sodium alkanol, C 1~4 Aqueous solutions of organic bases such as potassium alkoxide, for example, aqueous solutions of sodium methoxide, potassium methoxide, sodium tert-butoxide, potassium tert-butoxide, etc.

[0064] In some implementations, the molar ratio of aluminum chloride to alkali can be in the range of 1:2 to 1:5, such as 1:2~1:4.5, 1:2~1:4, 1:2~1:3, 1:2.5~1:3.5, 1:2.75~1:3.75, etc. By adjusting the molar ratio of aluminum chloride to alkali, the pore size of the prepared nano-aluminum adjuvant can be adjusted, thereby adjusting the total pore volume and / or specific surface area of ​​the nano-aluminum adjuvant, thus improving the drug loading and release capacity of the nano-aluminum adjuvant.

[0065] In some implementations, the mixture of potassium chloride and alkaline solution can be heated at a temperature of 80-200°C, for example, at a temperature of 100-150°C; the heating time can be 6-80 hours, for example, 12-72 hours, 18-60 hours, 24-48 hours, etc.

[0066] In some embodiments, the reaction mixture can be centrifuged at a speed of 3,000 to 25,000 rpm, for example, at 5,000 to 20,000 rpm; the centrifugation time can be 5 to 60 min, for example, 10 to 45 min or 10 to 30 min, etc.

[0067] Another aspect of this disclosure provides a lipid nanoparticle comprising any of the above-described nano-aluminum adjuvants; wherein the lipid nanoparticle has a lipid bilayer, and the nano-aluminum adjuvant is encapsulated in and / or at least partially embedded in the lipid bilayer.

[0068] As used herein, the term "lipid nanoparticle" is used interchangeably with "lipid nanoparticle" or "LNP" and refers to a nanoscale delivery carrier composed of lipids, characterized by a bilayer structure formed by the directional arrangement of lipid molecules in an aqueous phase, with hydrophobic fatty acid chains aggregated inward and hydrophilic polar heads facing outward, i.e., a lipid bilayer. LNPs can encapsulate and protect bioactive molecules such as mRNA for intracellular delivery. In some embodiments, the LNPs described herein may contain any of the aluminum nanoparticle adjuvants described herein, and also contain ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol lipids. The ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol (PEG) lipids that can be used in the LNPs described herein can be any ionizable lipid, cholesterol, neutral lipid, and polyethylene glycol lipid commonly used in the art to form LNPs. In some examples, ionizable lipids may include, but are not limited to, ALC-0315, SM-102, DLin-MC3-DMA, C12-200, TT3, and 5A2-SC8; cholesterol may be cholesterol and cholesterol derivatives (such as β-sitosterol); neutral lipids (auxiliary lipids) may include, but are not limited to, distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, and sphingomyelin; PEG lipids may include, but are not limited to, PEG-DMG (such as DMG-PEG2000, which is a standard component of many marketed LNP formulations), PEG-DMA, PEG-DPPE (such as DPPE-PEG2000), and PEG-modified ceramides. However, those skilled in the art will understand that any ionizable lipid, cholesterol, neutral lipid, and PEG lipid suitable for forming LNPs may be used to form the LNPs of this disclosure, and this disclosure does not limit such use.

[0069] In some embodiments, the LNP described herein may also include a drug, such as an mRNA drug. The mRNA drug that may be included in the LNP described herein may be any mRNA drug suitable for delivery via an LNP, such as, but not limited to, one or more of Herceptin-mRNA, insulin-mRNA, and interleukin-mRNA. However, those skilled in the art will understand that other drugs suitable for delivery via an LNP may also be included in the LNP described herein, and this disclosure does not limit such inclusion.

[0070] In some embodiments, the nano-aluminum adjuvants described in this disclosure are stable at pH > 7.4 and dissolve to release Al at pH 5.0–6.8. 3+ Therefore, the nano-aluminum adjuvant contained in LNP is stable before entering the cell via endocytosis and, after endocytosis, is able to rapidly dissolve and release Al in response to the low pH of the endosome. 3+ This leads to an increase in ion concentration within the endosome lumen, disrupting endometrial stability and causing osmotic imbalance, resulting in endosome rupture and promoting endosome escape of mRNA, ultimately improving mRNA transfection efficiency in target cells. Furthermore, the nano-aluminum adjuvant also exhibits an adjuvant effect; the aluminum adjuvant activates the TH2 pathway, while the LNP activates the TH1 pathway. This allows the LNP containing the nano-aluminum adjuvant disclosed in this invention to simultaneously achieve nucleic acid delivery (mRNA transfection) and the aforementioned adjuvant effect through the same vector, thereby achieving a synergistic enhancement of immunogenicity.

[0071] Another aspect of this disclosure provides a method for preparing any of the above-mentioned LNPs, the method comprising: (1) dissolving aluminum chloride in an alkaline solution, heating the resulting solution, and then centrifuging to obtain a nano-aluminum adjuvant; (2) mixing the nano-aluminum adjuvant prepared in (1) with ionizable lipids, cholesterol, neutral lipids and PEG lipids in an aqueous medium to obtain an LNP containing the nano-aluminum adjuvant.

[0072] In some embodiments, the method further includes mixing the nano-aluminum adjuvant with mRNA before mixing it with ionizable lipids, cholesterol, neutral lipids, and PEG lipids to load the mRNA onto the nano-aluminum adjuvant.

[0073] In some implementations, the aqueous medium may be a phosphate-buffered saline solution.

[0074] Another aspect of this disclosure also discloses the use of any of the above-mentioned nano-aluminum adjuvants in the preparation of mRNA-liposome drugs, wherein the mRNA-liposome drugs have improved endosome escape efficiency and / or target cell transfection efficiency.

[0075] This disclosure has at least the following advantages over the prior art:

[0076] 1. The LNP containing nano-aluminum adjuvant disclosed herein has a unique encapsulation / embedded unit structure, wherein the nanoscale (e.g., 20-50 nm in size) aluminum adjuvant serves as the core, which is encapsulated and / or partially embedded in the lipid bilayer, forming a composite structure rather than simple surface adsorption or physical mixing;

[0077] 2. The LNP containing nano-aluminum adjuvant disclosed herein has an acid-responsive release structure, wherein the nano-aluminum adjuvant is stable at pH > 7.4, and directionally dissolves and releases Al³⁺ in endosomes at pH 5.0~6.8; by utilizing the endosome disruption mechanism of Al³⁺ (i.e., osmotic imbalance → endosome rupture, not the traditional lipid protonation pathway), it promotes endosome escape of mRNA by increasing the ion concentration in the endosome lumen, disrupting the stability of the endosome membrane, and inducing osmotic imbalance, thereby ultimately improving the transfection efficiency of mRNA in target cells.

[0078] 3. The LNP containing nano-aluminum adjuvant disclosed herein can be formed by one-pot mixing via a single-step co-self-assembly process, simplifying the preparation process;

[0079] 4. The LNP containing nano-aluminum adjuvant disclosed herein achieves simultaneous nucleic acid delivery (mRNA transfection) and adjuvant effect (Al³⁺ activates the TH2 pathway, and LNP activates the TH1 pathway) through the same vector, thereby achieving synergistic enhancement of immunogenicity;

[0080] 5. Compared to conventional aluminum adjuvants (micron-sized) that cannot be embedded in the lipid layer due to particle size mismatch, the nano-aluminum adjuvants disclosed herein have a suitable size that can be encapsulated and / or embedded in the lipid bilayer, thereby forming a robust composite structure with other components of the LNP.

[0081] Example

[0082] The following embodiments further illustrate the invention, but should not be construed as limiting the invention to the details described in the embodiments. Unless otherwise stated, the instruments and reagents used in the following embodiments are commercially available products.

[0083] Example 1: Preparation of non-porous nano-aluminum adjuvant

[0084] 241 mg of aluminum chloride hydrate (AlCl3·6H2O) was ultrasonically dispersed in 10 mL of deionized water, and 110 mg of sodium hydroxide was ultrasonically dispersed in 5 mL of deionized water. The two solutions were mixed and added to a 50 mL hydrothermal reactor, which was then placed in a 120°C oven for 48 h. The reaction mixture was cooled to room temperature and centrifuged at 5,000 rpm for 10 minutes, discarding the supernatant. The precipitate was washed three times with 10 mL of deionized water to obtain the final non-porous (pore size approximately 0 nm) nano-aluminum adjuvant. Figure 2 Its transmission electron microscope image is shown.

[0085] Example 2: Preparation of microporous nano-aluminum adjuvant

[0086] 241 mg of aluminum chloride hydrate (AlCl3·6H2O) was ultrasonically dispersed in 10 mL of deionized water, and 130 mg of sodium hydroxide was ultrasonically dispersed in 5 mL of deionized water. The two solutions were mixed and added to a 50 mL hydrothermal reactor, which was then placed in a 120°C oven for 48 h. The reaction mixture was cooled to room temperature and centrifuged at 15,000 rpm for 10 minutes, discarding the supernatant. The precipitate was washed three times with 10 mL of deionized water to obtain nano-aluminum adjuvants with final micropores (approximately 2 nm in diameter). Figure 3 Its transmission electron microscope image is shown.

[0087] Example 3: Preparation of macroporous nano-aluminum adjuvant

[0088] 241 mg of aluminum chloride hydrate (AlCl3·6H2O) was ultrasonically dispersed in 10 mL of deionized water, and 150 mg of sodium hydroxide was ultrasonically dispersed in 5 mL of deionized water. The two solutions were mixed and added to a 50 mL hydrothermal reactor, which was then placed in a 120°C oven for 48 h. The reaction mixture was cooled to room temperature and centrifuged at 20,000 rpm for 10 minutes, discarding the supernatant. The precipitate was washed three times with 10 mL of deionized water to obtain the final macroporous (approximately 5 nm) aluminum adjuvant nanoparticles. Figure 4 Its transmission electron microscope image is shown.

[0089] Example 4: Preparation of aluminum nanoparticle adjuvant loaded with mRNA (Alum-mRNA)

[0090] The non-porous, small-pore, and macroporous aluminum adjuvants prepared in Examples 1-3 were dissolved in PBS to prepare aluminum adjuvant dispersions. mRNA was dissolved in the PBS solution.

[0091] The dispersion containing 20 μg of nano-aluminum adjuvant was mixed with a diluent containing 4 μg of mRNA and vortexed at high speed for 2 min to ensure homogeneity. The reaction mixture was allowed to stand at 4°C for 2 hours, and then centrifuged at 5,000 rpm for 2 min to obtain nano-aluminum adjuvant loaded with mRNA (Alum-mRNA).

[0092] Add 300 μL of acetic acid solution (10 mM) to the prepared Alum-mRNA to obtain an Alum-mRNA mixture.

[0093] Example 5: Lipid nanoparticles containing unloaded aluminum nanoparticle adjuvants (Alum-LNP) or loaded with... Preparation of lipid nanoparticles with aluminum nanoadjuvant for mRNA (Alum-mRNA-LNP)

[0094] The LNP precursor was prepared using a ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5 and an aqueous phase:ethanol phase = 3:1. The precursor was then subjected to two ultrafiltrations using a 100 kDa ultrafiltration tube to remove acidic substances and other impurities from the system, resulting in an LNP precursor mixture.

[0095] Under vortex conditions, 100 μL of the LNP precursor mixture prepared above was added dropwise to the Alum-mRNA mixture prepared in Example 4 or the dispersion of nano-aluminum adjuvant (Alum) without mRNA.

[0096] The reaction mixture was ultrafiltered using a 100 kDa dialysis tube at 4000 rpm for 60 min. PBS was added to the concentrate, and ultrafiltration and concentration were continued. These steps were repeated three times to obtain the title compounds Alum-mRNA-LNP and Alum-LNP. A transmission electron microscopy image of Alum-mRNA-LNP is shown below. Figure 5 As shown. Because the electron density of nano-aluminum adjuvants is relatively high, the contrast is darker; the darker areas in the figure represent nano-aluminum adjuvants.

[0097] Example 6: Performance Testing

[0098] 6.1 Hemolysis Test

[0099] Hemolysis experiments were conducted by co-incubating mouse erythrocytes (RBCs) with lipid nanoparticles (LNPs) under specific pH conditions.

[0100] The 4% mouse erythrocytes used in the experiment (purchased from Hongquan Biotechnology Co., Ltd.) were stored in Agger's solution. The experimental procedure was as follows: First, the mouse erythrocytes were washed with cold PBS (pH 7.4) and centrifuged at 1000 g for 5 minutes, repeated five times to ensure that only intact and dispersed erythrocytes were retained. Then, the erythrocytes were resuspended in PBS (pH 7.4) and 20 mM citrate buffer solution (pH 6.2) to adjust the concentration to 4% (blood cell volume / buffer volume). Erythrocyte solutions with different pH values ​​were added to two different 96-well plates (100 μL per well) and mixed with equal volumes of PBS, LNP, and Alum-LNP, respectively. 1 wt% Triton X-100 was used as a positive control. The 96-well plates were incubated at 37°C for 1 hour, then centrifuged at 1000 g for 5 minutes at 4°C, and the supernatant was collected. The obtained supernatant was transferred to new 96-well plates, and the absorbance was measured at 450 nm.

[0101] like Figure 6As shown, at pH 7.4, there was no significant difference in hemolytic activity between lipid nanoparticles based on aluminum nanoparticle adjuvant (Alum-LNP) and lipid nanoparticles (LNP), indicating that both are safe; however, at pH 6.2-6.8, the hemolytic activity of Alum-LNP was higher than that of LNP, indicating that Alum-LNP has a better ability to escape from the body than LNP.

[0102] 6.2 Immune Response Test

[0103] The nanoparticle formulation (mOVA, mOVA-Alum, mOVA-LNP, and Alum-mRNA-LNP (10 µg mOVA per injection)) was injected intravenously into C57BL / 6 mice (30 mice purchased from Vital River) every 7 days for a total of three times. The immune activation of the mice was then analyzed by flow cytometry. On day 21, the mice were euthanized, and the spleens were removed, ground with the end of a syringe, and repeatedly washed with PBS until no obvious red color remained. The solution was centrifuged at 500 g for 5 minutes, and the supernatant was discarded. Red blood cell lysis buffer was added to lyse the red blood cells, obtaining purified spleen cells. After counting the cell suspension using a hemocytometer or other instruments, the cell concentration was adjusted to approximately 1 * 102 7 / mL. For mouse samples, purified CD16 / CD32 monoclonal antibody binds to FcγRⅢ / Ⅱ receptors, thereby blocking non-specific staining and reducing background fluorescence of negative cells to the level of unlabeled cells. Add 1 μg of purified anti-mouse CD16 / 32 monoclonal antibody to the sample and incubate at room temperature for 30 minutes. For IFN-γ and IL-4 cytokines, cell fixation and permeabilization should be performed according to the manufacturer's instructions before staining. Finally, add fluorescently labeled antibody and incubate at 4°C in the dark for 30 minutes. Staining results can be immediately analyzed by flow cytometry.

[0104] The fluorescent antibodies used in this embodiment include: Elab Fluor Violet-labeled anti-mouse CD45 antibody, PerCP / Cyanine 5.5-labeled anti-mouse CD3 antibody, FITC-labeled anti-mouse CD4 antibody, APC-labeled anti-mouse IFN-γ antibody, and PE-labeled anti-mouse IL-4 antibody.

[0105] The production of IFN-γ and IL-4 are key indicators of the immune response of type 1 helper T cells (Th1) and type 2 helper T cells (Th2) in vivo (Tuzlak, S. et al., Nat. Immunol. 2021, 22, 1210-1217.).

[0106] like Figure 7As shown, compared with the PBS control group, mice treated with LNP and Alum-LNP showed increased average levels of the Th1 cytokine IFN-γ at the injection site, indicating that both LNP (SM-102 LNP) and Alum-LNP (SM-102 LNP) can activate Th1 immune responses. Furthermore, compared with the PBS control group, mice treated with LNP showed no significant change in the average level of the Th2 cytokine IL-4 at the injection site, while mice treated with Alum-LNP showed an increased average level of the Th2 cytokine IL-4 at the injection site. This indicates that LNP can elicit a potent type 1 helper T cell (Th1) response, and the Alum-LNP vaccine, due to the addition of aluminum adjuvant, can elicit both potent Th1 and Th2 immune responses, which is beneficial for improving the efficacy of tumor vaccines.

[0107] 6.3 Tumor Suppression Test

[0108] Six- to eight-week-old C57BL / 6 mice were intravenously injected with a nanoparticle containing 10 μg of mOVA-mRNA, once every other week for a total of three times, to activate the mouse's immune system to resist tumor cell invasion. Subsequently, in a tumor prevention experiment, B16-F10-OVA cells (1 * 10^6 cells) were subcutaneously injected into the hind limbs of the mice. 6 (Each cell). Tumor growth was observed every three days, including taking photos, measuring animal weight, and monitoring tumor size.

[0109] like Figure 8 As shown, compared with the PBS control group, the mOVA-LNP and Alum-mOVA-LNP injection groups exhibited tumor suppression effects. In the Alum-LNP group, tumors were completely suppressed in 5 out of 6 mice, and overall survival time was prolonged. There were no significant differences in tumor size among the PBS, mOVA, and mOVA-Alum groups. Therefore, the results indicate that mice injected with Alum-LNP showed stronger tumor growth inhibition compared to the LNP group, suggesting that Alum-LNP has a stronger preventive effect than LNP.

[0110] 6.4 Transfection efficiency test

[0111] To detect the transfection level of the corresponding mRBA in serum, BALB / c mice were inoculated with different nano-formulations on days 1, 2, and 3, and blood samples were collected 24 hours after inoculation. The content of the target protein in serum was determined by ELISA.

[0112] The specific steps of the ELISA experiment are as follows: Using a flat-bottomed 96-well plate, add 2 μg of specific protein (dissolved in 100 mM carbonate buffer, pH 9.6) to each well and coat overnight at 4°C. Then block with PBS-Tween solution containing 10% fetal bovine serum. Take serum samples from immunized animals, dilute 100-fold with PBS-T solution, and then perform four-fold serial dilutions. Add the serum samples before and after dilution to the wells and incubate at 37°C for 2 hours. Use horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, diluted 1:5000 in PBS-T solution containing 10% fetal bovine serum for the labeling reaction. Finally, add horseradish peroxidase substrate and measure the absorbance at 450 nm using a microplate reader.

[0113] The results above show that Alum-LNP can significantly improve the transfection efficiency of various mRNAs, demonstrating a broad-spectrum effect in enhancing mRNA transfection efficiency. For example... Figure 9 As shown, the serum IL2 level in the Alum-LNP group mice exceeded 254.3 ± 45.6 ng / L, which was 2.3 times higher than that in the LNP group (112.0 ± 40.6 ng / L). The serum insulin level in the Alum-LNP group mice exceeded 25.6 ± 3.9 ng / L, which was 2.1 times higher than that in the LNP group (12.4 ± 4.9 ng / L). The serum trastuzumab level in the Alum-LNP group mice exceeded 186.5 ± 18.0 ng / L, which was 3.2 times higher than that in the LNP group. In terms of safety, there was almost no difference between the Alum-LNP and LNP groups.

[0114] Although specific aspects of the invention have been explained and described, it will be apparent to those skilled in the art that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, the appended claims are intended to cover all such changes and modifications that fall within the scope of the invention.

Claims

1. A nano-aluminum adjuvant, characterized in that... It comprises flake aluminum hydroxide, said flake aluminum hydroxide having a length L, a width W and a thickness T, wherein, The length L is in the range of 20~100 nm. The width W is in the range of 20~100 nm, and The thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W.

2. The nano-aluminum adjuvant as described in claim 1, characterized in that, The nano-aluminum adjuvant satisfies one or more of the following: (1) It has a pore size in the range of 1 nm to 10 nm; (2) Having a total pore volume in the range of 0.09 cm³ / g to 0.41 cm³ / g; and (3) It has a specific surface area BET in the range of 18.29 m² / g to 227.58 m² / g.

3. A method for preparing nano-aluminum adjuvants, characterized in that, The nano-aluminum adjuvant comprises flake-shaped aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20–100 nm, the width W is in the range of 20–100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W. The method includes: dissolving aluminum chloride in an alkaline solution, heating the resulting solution, and then centrifuging to obtain the nano-aluminum adjuvant.

4. The method as described in claim 3, characterized in that, The method satisfies one or more of the following: (1) The alkaline solution is selected from sodium hydroxide, potassium hydroxide, C 1~4 Sodium alkanol, C 1~4 An aqueous solution of potassium alkoxide or any combination thereof; (2) The molar ratio of the aluminum chloride to the alkali is in the range of 1:2 to 1:5; (3) The heating temperature is 80°C to 200°C; (4) The heating time is from 6 hours to 80 hours; and (5) The centrifugation speed is from 3,000 rpm to 25,000 rpm.

5. Lipid nanoparticles containing nano-aluminum adjuvant, characterized in that, The nano-aluminum adjuvant comprises flake-shaped aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20–100 nm, the width W is in the range of 20–100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W. The lipid nanoparticles have a lipid bilayer, and the nano-aluminum adjuvant is encapsulated in the lipid bilayer and / or at least partially embedded in the lipid bilayer.

6. The lipid nanoparticles as described in claim 5, characterized in that, The lipid nanoparticles also contain ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol lipids.

7. The lipid nanoparticles as described in claim 6, characterized in that, The lipid nanoparticles also contain a drug.

8. The lipid nanoparticles as described in claim 7, characterized in that, The drug in question is an mRNA drug.

9. The lipid nanoparticles as described in claim 8, characterized in that, The mRNA drug includes one or more of Herceptin-mRNA, insulin-mRNA, and interleukin-mRNA.

10. The lipid nanoparticles according to any one of claims 5 to 9, characterized in that, The nano-aluminum adjuvant is stable at pH > 7.4 and dissolves to release Al at pH 5.0 ~ 6.

8. 3+ .

11. A method for preparing lipid nanoparticles containing nano-aluminum adjuvant, characterized in that, The nano-aluminum adjuvant comprises flake aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W. The method includes: (1) Dissolve aluminum chloride in an alkaline solution, heat the resulting solution, and then centrifuge to obtain the nano-aluminum adjuvant; (2) The nano-aluminum adjuvant prepared in (1) is mixed with ionizable lipids, cholesterol, neutral lipids and polyethylene glycol lipids in an aqueous medium. To obtain the lipid nanoparticles containing the nano-aluminum adjuvant.

12. The method as described in claim 11, characterized in that, The method further includes: Before mixing the nano-aluminum adjuvant with the ionizable lipids, cholesterol, neutral lipids, and polyethylene glycol lipids, the nano-aluminum adjuvant is mixed with the drug to load the drug onto the nano-aluminum adjuvant.

13. The method as described in claim 12, characterized in that, The drugs include mRNA drugs.

14. The method according to any one of claims 11 to 13, characterized in that, The aqueous medium is a phosphate buffered saline solution.

15. The use of nano-aluminum adjuvant in the preparation of mRNA-liposome drugs, characterized in that, The nano-aluminum adjuvant comprises flake-shaped aluminum hydroxide having a length L, a width W, and a thickness T, wherein the length L is in the range of 20 to 100 nm, the width W is in the range of 20 to 100 nm, and the thickness T is less than or equal to 1 / 10 of the smaller of the length L and the width W, and the mRNA-liposome drug has improved endosome escape efficiency and / or target cell transfection efficiency.

Images