WO2025112290A1 - 一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂的制备及其应用 - Google Patents

一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂的制备及其应用 Download PDF

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WO2025112290A1
WO2025112290A1 PCT/CN2024/091914 CN2024091914W WO2025112290A1 WO 2025112290 A1 WO2025112290 A1 WO 2025112290A1 CN 2024091914 W CN2024091914 W CN 2024091914W WO 2025112290 A1 WO2025112290 A1 WO 2025112290A1
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calcium
silicon
aluminum
alooh
doped
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孙冰冰
李敏
王慧洋
薛长颖
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Dalian University of Technology
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61P31/22—Antivirals for DNA viruses for herpes viruses
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    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a preparation method and application of an AlOOH nano-adjuvant doped with calcium and silicon elements, and specifically to a method of doping calcium and silicon elements into the AlOOH nano-adjuvant, which can be used for the development of vaccines and for the prevention and treatment of various infectious diseases.
  • Infectious diseases have always been one of the major threats to human life and health, and a large number of cases die from infectious diseases every year. For example, according to the World Health Organization (WHO), 296 million people were infected with chronic hepatitis B in 2019, resulting in an estimated 820,000 deaths, most of which were caused by cirrhosis and hepatocellular carcinoma (primary liver cancer), with 1.5 million new cases of infection each year.
  • WHO World Health Organization
  • adjuvants there are six types of adjuvants approved for use by the US FDA, including aluminum salt adjuvants, MF59, AS03, AS04, CpG ODN and AS01B. Among them, the use of aluminum salts in adjuvant vaccines is extremely important. Aluminum salt adjuvants are widely used in tetanus, diphtheria, pertussis, polio, hepatitis A, hepatitis B vaccines, etc.
  • the present invention aims to provide a preparation and application of a calcium- and silicon-doped aluminum oxyhydroxide (AlOOH) nanoadjuvant capable of inducing a balanced immune response, which is based on the AlOOH adjuvant synthesis method and doped with calcium and silicon during the preparation process.
  • AlOOH aluminum oxyhydroxide
  • the prepared AlOOH adjuvant has good A good and balanced immune response can induce a more balanced humoral and cellular immune response, so as to better design preventive or therapeutic vaccines, and can provide a new platform for the design of aluminum adjuvant vaccines.
  • a calcium- and silicon-doped AlOOH nano-adjuvant includes calcium- and silicon-doped AlOOH adjuvants, wherein calcium or silicon atoms exist in the AlOOH lattice, and the adjuvant body is AlOOH nanoparticles.
  • AlOOH is synthesized using an Al source, and a calcium source or a silicon source is simultaneously used in a reaction solution to dope Ca or Si elements, and an alkaline solution is added to precipitate Al(OH) 3 and dopants, and a Ca or Si-doped AlOOH adjuvant is prepared using a hydrothermal synthesis method.
  • the Al source is various inorganic or organic aluminum salts such as aluminum nitrate, aluminum chloride, and aluminum ethoxide.
  • the aluminum element exists in the reaction solution in the form of aluminum ions, and the concentration of aluminum ions in the reaction system is 0.01-10 mol/L, preferably 0.01-2 mol/L.
  • the calcium source is an inorganic or organic soluble calcium salt such as calcium chloride, calcium nitrate, tricalcium citrate, etc., and the molar ratio of calcium element to aluminum element is: 0.01-100:1, preferably 0.01-40:1; the calcium element exists in the reaction solution in the form of calcium ions, with a concentration of 0.01-10 mol/L, and the preferred concentration of calcium element is 0.01-2 mol/L.
  • the silicon source is an organic silane such as tetraethoxysilane, a soluble silicate such as silicic acid or sodium silicate, potassium silicate, etc.
  • the molar ratio of silicon element to aluminum element is: 0.01-100:1, preferably 0.01-40:1; the silicon element exists in the reaction solution in the form of ions or organic matter containing silicon element, and the concentration of silicon element in the reaction solution is 0.01-10 mol/L, preferably 0.01-2 mol/L.
  • the alkaline solution is an aqueous solution such as NaOH, KOH, ammonia water or various alkaline buffer solutions, and the concentration of the alkaline solution is 0.01-10 mol/L, preferably 0.01-2 mol/L.
  • the calcium or silicon doped AlOOH nano-adjuvant is a nanoparticle, and the nanoparticle has a morphology of 20nm-1000nm (preferably 50-800nm).
  • the preparation method of the above-mentioned AlOOH nano-adjuvant doped with calcium or silicon comprises the following steps:
  • reaction solution For the synthesis of calcium-doped aluminum hydroxide: at a temperature of 20-60° C., an alkaline solution is added dropwise to an aluminum source solution containing a calcium source until the pH is 4-13, and the mixture is stirred throughout the process to obtain a dispersed system (reaction solution);
  • reaction solution For the synthesis of silicon-doped aluminum hydroxide: at a temperature of 20-60° C., an alkaline solution containing a silicon source is added dropwise to an aluminum source solution, and then the alkaline solution is added dropwise until the pH value is 4-13, and the mixture is stirred throughout the process to obtain a dispersed system (reaction solution);
  • step 2 Add the dispersed system of step 1 into the reactor, and prepare the reaction product by hydrothermal synthesis, the temperature is 50-300° C., preferably 90-250° C., and the hydrothermal time is 2-48 h, preferably 4-36 h;
  • step 3 Centrifuge the reaction product of step 2, remove the supernatant, wash it with ultrapure water, and then store it or dry it.
  • the concentration of the silicon source in the alkaline solution containing the silicon source is 0.01-10 mol/L, preferably 0.02-5 mol/L;
  • the preparation method of the alkaline solution containing the silicon source is: adding the silicon source to the alkaline solution to obtain the alkaline solution containing the silicon source.
  • the stirring rate is 300-1500 rpm, preferably 300-900 rpm, and the stirring time is 3 min-3 h.
  • step 3 the speed of the centrifuge during washing is 8000-15000 rpm, preferably 11000 rpm; the time is 10-30 min, preferably 25 min.
  • the present invention also provides the use of the above-mentioned AlOOH nano adjuvant doped with calcium or silicon in various preventive and therapeutic adjuvant vaccines.
  • the vaccine adjuvant is an AlOOH nanoadjuvant doped with calcium or silicon
  • the corresponding vaccine antigens include but are not limited to hepatitis B surface antigen, hepatitis B core antigen, human papillomavirus-like particles, novel coronavirus spike protein receptor binding domain, human papillomavirus antigen, varicella zoster virus recombinant glycoprotein, etc.
  • corresponding hepatitis B virus vaccines, human papillomavirus vaccines, novel coronavirus vaccines, herpes zoster virus vaccines, etc. are constructed.
  • the AlOOH nanoadjuvant doped with calcium or silicon in the present invention has been verified by in vivo experiments in mice to be able to induce efficient humoral immunity and cellular immunity at the same time.
  • the preparation method of AlOOH capable of simultaneously inducing efficient humoral immunity and cellular immunity of the present invention is simple, easy to operate, has good repeatability, and mild reaction conditions, and finally obtains vaccine nanoparticles with uniform dispersion and uniform particle size, which has good application prospects in the prevention and treatment of infectious virus.
  • the present invention includes 9 drawings,
  • Figure 1 is a transmission electron micrograph of AlOOH adjuvants (Si/Ca-AlOOH-1/2/3/4) doped with different concentrations of calcium and silicon elements, where the scale bar is 200 nm.
  • FIG2 is an X-ray diffraction (XRD) pattern of AlOOH adjuvants (Si/Ca-AlOOH-1/2/3/4) doped with different concentrations of calcium and silicon elements.
  • XRD X-ray diffraction
  • Figure 3 shows the X-ray photoelectric characteristics of AlOOH adjuvant (Si/Ca-AlOOH-1/4) doped with different concentrations of calcium and silicon. XPS.
  • Figure 4 is a comparison of the element content results in different parts of AlOOH adjuvants (Si/Ca-AlOOH-1/4) doped with different concentrations of calcium and silicon.
  • the bulk doping element content (orange) was determined using an inductively coupled plasma atomic emission spectrometer (ICP), and the surface element content (green) was determined using XPS. The results show that silicon and calcium are uniformly doped in AlOOH.
  • FIG5 shows the in vitro immune evaluation of AlOOH adjuvants (Si/Ca-AlOOH-1/2/3/4) doped with different concentrations of calcium (A) and silicon (B).
  • Figure 6 shows the results of different concentrations of calcium and silicon-doped AlOOH adjuvants (Si/Ca-AlOOH-1/4) inducing the expression of CD86 (A) and MHC II (B) on the surface of BMDC cells and the release of IL-1 ⁇ (C), IL-6 (D), IL-12 (E) and TNF- ⁇ (F) cytokines.
  • Si/Ca-AlOOH-1/4 calcium and silicon-doped AlOOH adjuvants
  • Figure 7 shows the level of hepatitis B antibody induced by Si/Ca-AlOOH-1/4 vaccine using 6-8 week old C57BL/6 mice as a model; the specific hepatitis B antigen immunization strategy is to intramuscularly inject Si/Ca-AlOOH-1/4 containing 2 ⁇ g of hepatitis B surface antigen on day 0, inject an equal amount of Si/Ca-AlOOH-1/4 again on day 21, and collect serum and spleen on day 42 to detect humoral immunity and cellular immunity levels.
  • Figures 7A and 7B show the levels of specific antibodies in serum, which are total IgG and IgG 1 levels, respectively.
  • Figures 7C to 7I respectively show the expression of CD69 on the surface of CD4 + T cells (C), the expression of CD69 on the surface of CD8 + T cells (D), the expression of FasL on the surface of CD8 + T cells (E), the expression of CD44 high CD62 low on the surface of CD4 + T cells (F), the expression of CD44 high CD62 low on the surface of CD8 + T cells (G), the expression of CD69 on the surface of B cells (H), and the expression of CD27 on the surface of B cells (I).
  • FIG8 is the immunopathological analysis of the main organs of mice.
  • Figure 9D shows a radar chart of functional T cell and B cell activation.
  • Figures 9E-9F are cell activation spectra of CD69 + /CD4 + , CD69 + /CD8 + , FasL + /CD8 + , CD44 high CD62 low /CD4 + , CD44 high CD62L low / CD8 + , CD69 + /CD19 + and CD27 + /CD19 + analyzed by flow cytometry after spleen cells of immunized mice were restimulated with gE ( 2 ⁇ g /mL) for 120 h, wherein Figure 9E is a radar chart showing the production of Th1 (TNF- ⁇ , IFN- ⁇ , IL-2) and Th2 (IL-4) cytokines in CD4 + T cells, and Figure 9F is a radar chart showing the production of Th1 (TNF- ⁇ , IFN- ⁇ , IL-2) and Th2 (IL-4) cytokines in CD8 + T cells.
  • gE 2 ⁇ g /mL
  • Figure 9G and Figure 9H show the number of gE-specific IFN- ⁇ (G) and (H) IL-2-producing spot-forming cells detected by ELISPOT after restimulation of spleen cells of immunized mice with gE (2 ⁇ g/mL) for 24 hours.
  • Figure 9I and Figure 9J show the levels of IL-4 (I) and IFN- ⁇ (J) secreted by spleen cells of immunized mice 5 days after restimulation of spleen cells with gE (2 ⁇ g/mL).
  • a method for preparing a calcium-doped AlOOH nanoadjuvant comprising the following steps:
  • the prepared calcium-doped AlOOH nanoadjuvants (Ca-AlOOH-1/2/3/4) products were respectively recorded as AlOOH, Ca-AlOOH-1 (calcium to aluminum element molar ratio of 0.05), Ca-AlOOH-2 (calcium to aluminum element molar ratio of 0.1), Ca-AlOOH-3 (calcium to aluminum element molar ratio of 0.15), and Ca-AlOOH-4 (calcium to aluminum element molar ratio of 0.2).
  • step 3 Add NaOH solution to the mixed solution in step 2 until the pH is 7, stir at 600 rpm throughout the process, and the reaction temperature is 25°C to obtain a dispersed system;
  • step 4 Add the dispersed system of step 3 into the reactor, and prepare it by hydrothermal method at a temperature of 120° C. for 4 h to obtain a reaction product;
  • step 5 Centrifuge the reaction product of step 4, remove the supernatant, wash it with ultrapure water and then store it or dry it.
  • the centrifuge speed during washing is 11000 rpm and the time is 25 minutes.
  • a method for preparing a silicon-doped AlOOH nanoadjuvant comprising the following steps:
  • Dilute aluminum solution dilute AlCl 3 ⁇ 6H 2 O solution to 25mL, with a concentration of 0.5mol/L.
  • the morphology of the products in Examples 1 and 2 was detected by transmission electron microscopy (TEM) (as shown in FIG1 ). The results showed that as the silicon-aluminum ratio increased, the morphology of the material changed from rod-like to leaf-like. As the calcium-aluminum ratio increased, the morphology changed from rod-like to granular.
  • TEM transmission electron microscopy
  • XRD analysis shows that with the increase of Si and Ca elements, the XRD spectrum of doped AlOOH does not show any impurity peaks other than AlOOH, indicating that Si and Ca are uniformly distributed in the AlOOH structure.
  • XPS analysis (as shown in Figure 3) further confirmed the successful doping of silicon and calcium.
  • the silicon and calcium elements were quantified by XPS and ICP (as shown in Figure 4), and the results showed that the doping amount of elements on the surface and in the bulk of the material was similar, indicating that the AlOOH particles were uniformly doped with silicon and aluminum.
  • the MTS method was used to determine the cell viability of bone marrow-derived dendritic cells (BMDCs) after treatment with nanomaterials at different concentrations (0, 100, 200, 300, 400 and 500 ⁇ g/mL, respectively).
  • the experimental results (as shown in Figure 5) showed that the adjuvant had good biocompatibility.
  • BMDC cell activation and cytokine production were analyzed (as shown in Figure 6).
  • the results showed that with the increase of silicon content, the doped adjuvant induced higher IL-12 and IL-6 production, indicating that doped silicon induced the release of Th1 and Th2 type cytokines.
  • With the increase of calcium content the doped adjuvant induced an increase in IL-12, while the IL-6 cytokine remained unchanged, suggesting that calcium doping induced enhanced release of Th1 type cytokines, but not Th2 type cytokines.
  • mice 6-8 week old C57BL/6 mice were used as animal models to detect the humoral immunity and cellular immunity induced by Si-AlOOH-1/2/3/4 and Ca-AlOOH-1/2/3/4 prepared in Examples 1 and 2.
  • the method comprises the following steps: On day 0, mice were intramuscularly injected with 50 ⁇ L of vaccine, which contained 2 ⁇ g of hepatitis B surface antigen (HBV), 50 ⁇ g of aluminum, silicon and calcium in total, and the vaccine was dispersed in a physiological saline system. On day 21, the same amount of the above vaccine was injected again, and on day 42, serum and spleen were collected to detect the levels of total IgG and IgG 1 in serum and the maturation and differentiation of spleen cells.
  • HBV hepatitis B surface antigen
  • control groups were added: a physiological saline group (each mouse was injected with 50 ⁇ L of physiological saline), an HBV group (each mouse was injected with only 2 ⁇ g of hepatitis B surface antigen), and an HBV+Alum group (each mouse was injected with a commercial aluminum hydroxide adjuvant containing 2 ⁇ g HBV and 50 ⁇ g of aluminum element ( adjuvant 2%, InvivoGen) mixture, abbreviated as Alum group), each group had 6 experimental mice.
  • Example 7 the characterization results of Example 5 show that the antibody titer experimental results show that the doped aluminum adjuvant can produce higher IgG and IgG 1 antibody titers, proving that the doped AlOOH can produce higher humoral immunity levels.
  • the results of spleen cell maturation and differentiation show that the doped aluminum adjuvant can promote CD4 + , CD8 + cell activation and high expression of CTL cell killing mediators.
  • Each dose contained 5 ⁇ g gE and 50 ⁇ g equivalent Al (Si-AlOOH-4 prepared in Example 1 or Ca-AlOOH-4 prepared in Example 2) or 100 ⁇ g equivalent Al (Si-AlOOH-4 prepared in Example 1, denoted as Si-AlOOH-4-H, or Ca-AlOOH-4 prepared in Example 2, denoted as Ca-AlOOH-4-H).
  • gE (5 ⁇ g) was used as a control.
  • gE-specific serum was detected on day 42.
  • Splenocytes of immunized mice were restimulated with gE (2 ⁇ g/mL) for 120 h, and the cell activation spectra of CD69 + /CD4 + , CD69 + /CD8 + , FasL + /CD8 + , CD44 high CD62 low /CD4 + , CD44 high CD62L low /CD8 + , CD69 + /CD19 + and CD27 + /CD19 + were analyzed by flow cytometry.
  • Th1-type (TNF- ⁇ , IFN- ⁇ , IL-2) and Th2-type (IL-4) cytokines in CD4 + T cells is shown in Figure 9E.
  • Intracellular cytokine analysis showed that Si-AlOOH-4 and Si-AlOOH-4-H induced higher proportions of IL-4, IFN- ⁇ , IL-2 and TNF- ⁇ in CD4 + T cells compared with the gE group, indicating that Si-doped AlOOH nanoadjuvants induced CD4 + T cells to produce Th2 and Th1-type cytokines, which was independent of the adjuvant dose.
  • Ca-AlOOH-4 induced higher proportions of IFN- ⁇ and TNF- ⁇ in CD4 + T cells; Ca-AlOOH-4-H induced a higher ratio of IL-4 and IL-2 in CD4 + T cells.
  • the results showed that Ca-AlOOH-4 has the potential to induce the production of Th2 and Th1 type cytokines in CD4 + T cells, and its level is determined by the adjuvant dose.
  • Th1-type (TNF- ⁇ , IFN- ⁇ , IL-2) and Th2-type (IL-4) cytokines in CD8 + T cells is shown in Figure 9F.
  • Si-AlOOH-4 enhanced the production of intracellular IFN- ⁇ , IL-2, and TNF- ⁇ in CD8 + T cells.
  • Si-AlOOH-4-H induced an increase in IFN- ⁇ in CD8 + T cells.
  • Ca-AlOOH-4 nanoadjuvant induced an increase in the proportion of CD8 + T cells containing IFN- ⁇ and TNF- ⁇ in cells
  • Ca-AlOOH-4-H induced an increase in the proportion of CD8 + T cells containing IL-4 in cells.
  • Ca-AlOOH-4 and Ca-AlOOH-4-H induced an increase in IL-4 secretion to 11.6 times and 7.1 times, and the increase decreased with the increase in Ca-AlOOH-4 dose.
  • Ca-AlOOH-4 and Ca-AlOOH-4-H induced an increase in IFN- ⁇ secretion to 3.3 times and 7.6 times, indicating that the increase in IFN- ⁇ release induced by Ca-AlOOH-4 was adjuvant dose-dependent.
  • the calcium or silicon-doped AlOOH nanoadjuvant of the present invention was verified by in vivo experiments in mice to be able to induce efficient humoral immunity and cellular immunity at the same time.

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Abstract

一种基于钙、硅元素掺杂的羟基氧化铝纳米佐剂的制备方法及应用。该AlOOH佐剂颗粒形貌为50-800nm的纳米颗粒，其中，硅、钙元素掺杂在AlOOH晶格中，且钙元素或硅元素与铝元素摩尔比为0.01-100：1。利用Al源合成AlOOH，在反应液中同时使用钙源或硅源进行Ca或Si元素的掺杂，通过加入碱性溶液使Al(OH)3及掺杂物进行沉淀，利用水热合成法制备Ca或Si掺杂的AlOOH佐剂。在乙肝表面抗原和水痘-带状疱疹病毒糖蛋白E抗原模型中，钙或硅元素掺杂的AlOOH纳米佐剂经小鼠体内实验验证可以同时诱导高效的体液免疫和细胞免疫。

Description

一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂的制备及其应用 技术领域
本发明涉及一种基于钙、硅元素掺杂的AlOOH纳米佐剂的制备及其应用，具体而言是将钙、硅元素分别掺杂到AlOOH纳米佐剂中的方法，其可以用于疫苗的开发，用于各种传染病的预防和治疗。
背景技术
传染性疾病一直是人类生命健康安全的重大威胁之一，每年有大量病例因传染性疾病死亡。例如，据世界卫生组织(WHO)报道，2019年有2.96亿人感染慢性乙型肝炎，估计导致82万人死亡，其中大多数死于肝硬化和肝细胞癌(原发性肝癌)，每年有150万新感染病例。
疫苗作为预防传染性疾病最重要的武器，它的发展经历了几个阶段。1798年英国医生琴纳研发全球最早的牛痘疫苗，开启了疫苗的应用历史。直到20世纪中后期，疫苗的发展进入了黄金时代。作为疫苗中的一种重要组成成分，佐剂在增强抗原的免疫应答方面发挥着极其重要的作用。佐剂的发展经历了由天然成分到人工合成的工程疫苗佐剂两个阶段，其中1926年铝盐的佐剂效应的发现具有划时代的意义。至今为止，美国FDA批准使用的疫苗当中，有六类佐剂，包括铝盐佐剂，MF59，AS03，AS04，CpG ODN和AS01B。其中铝盐在佐剂疫苗的使用极其重要。铝盐佐剂被广泛应用于破伤风、白喉、百日咳、脊髓灰质炎、甲肝、乙肝疫苗等。
然而，单纯的铝盐佐剂一般只能增强体液免疫水平，而不能提高宿主的细胞免疫水平，不能够提供平衡的免疫应答。此外，为改善抗原与佐剂的相互作用通常会在疫苗配方中加入赋性剂，而这一过程又会增加疫苗生产的复杂性。因此，从疫苗佐剂本身出发，设计一种能够工艺简单、配方简单、能够引发强大且平衡的免疫应答的传染性疫苗在传染性疾病预防和治疗方面具有重大意义。
发明内容
为解决现有技术的问题，本发明的目的在于提供了一种能够引发平衡免疫应答的基于钙、硅元素掺杂的羟基氧化铝(AlOOH)纳米佐剂的制备及应用，其以AlOOH佐剂合成方法为基础，在制备过程中掺杂钙、硅元素。所制备的AlOOH佐剂具有良 好的、平衡的免疫应答，可以诱导更平衡的体液、细胞免疫反应，以更好的设计预防或治疗性疫苗，能够为铝佐剂疫苗设计提供新的平台。
一种基于钙、硅元素掺杂的AlOOH纳米佐剂，包括钙、硅元素掺杂和AlOOH佐剂，钙或硅原子存在于AlOOH晶格中，佐剂主体为AlOOH纳米颗粒。利用Al源合成AlOOH，在反应液中同时使用钙源或硅源进行Ca或Si元素的掺杂，通过加入碱性溶液使Al(OH)3及掺杂物进行沉淀，利用水热合成法制备Ca或Si掺杂的AlOOH佐剂。
进一步地，所述Al源为硝酸铝、氯化铝、乙氧基铝等各种无机或有机铝盐，铝元素在反应液中的存在形式为铝离子，铝离子在反应体系中的浓度为0.01-10mol/L，优选浓度为0.01-2mol/L。
进一步地，所述钙源为氯化钙，硝酸钙，柠檬酸三钙等无机或有机可溶性钙盐，钙元素与铝元素摩尔比为：0.01-100：1，优选为0.01-40：1；钙元素在反应液中的存在形式为钙离子，浓度为0.01-10mol/L，钙元素优选浓度为0.01-2mol/L。
进一步地，所述硅源为四乙氧基硅烷等有机硅烷，硅酸或硅酸钠、硅酸钾等可溶性硅酸盐，硅元素与铝元素摩尔比为：0.01-100：1，优选为0.01-40：1；硅元素在反应液中的存在形式为含硅元素的离子或有机物，硅元素在反应液中的浓度为0.01-10mol/L，优选浓度为0.01-2mol/L。
进一步地，所述碱性溶液为NaOH，KOH，氨水等水溶液或各种碱性缓冲液，碱性溶液浓度为0.01-10mol/L，优选为0.01-2mol/L。
进一步地，所述钙或硅元素掺杂的AlOOH纳米佐剂为纳米颗粒，纳米颗粒的形貌20nm-1000nm(优选为50-800nm)的纳米颗粒。
上述基于钙或硅元素掺杂的AlOOH纳米佐剂的制备方法，所述制备方法包括以下步骤：
①对于钙元素掺杂的羟基氧化铝的合成：在温度为20-60℃下，向含有钙源的铝源溶液滴加碱性溶液至pH为4-13，全程搅拌，得到分散体系(反应液)；
对于硅元素掺杂的羟基氧化铝的合成：在温度为20-60℃下，向铝源溶液滴加含有硅源的碱性溶液后，滴加碱性溶液至pH为4-13，全程搅拌，得到分散体系(反应液)；
②将步骤①的分散体系加入至反应釜中，用水热合成法制备得到反应产物，温度为50-300℃，优选为90-250℃，水热时间2-48h，优选为4-36h；
③离心步骤②的反应产物，除去上清液，用超纯水离心洗涤后保存或烘干保存。
进一步地，步骤①中，含有硅源的碱性溶液中硅源的浓度为0.01-10mol/L，优选为0.02-5mol/L；所述的含有硅源的碱性溶液的制备方法为：将硅源加入到碱性溶液中，得到含有硅源的碱性溶液。
进一步地，步骤①中，搅拌的速率为300-1500rpm，优选为300-900rpm，搅拌时间为3min-3h。
进一步地，步骤③中，所述洗涤时离心机转速为8000-15000rpm，优选为11000rpm；时间为10-30min，优选为25min。
本发明还提供上述基于钙或硅元素掺杂的AlOOH纳米佐剂在各种预防性和治疗性佐剂疫苗中的应用。
进一步地，所述疫苗佐剂为钙或硅元素掺杂的AlOOH纳米佐剂，相应配苗抗原包括但不限于乙肝表面抗原、乙肝核心抗原、人乳头瘤病毒样颗粒、新型冠状病毒刺突蛋白受体结合域、人乳头瘤病毒抗原、水痘带状疱疹病毒重组糖蛋白等。基于以上抗原构建相应的乙肝病毒疫苗、人乳头瘤病毒疫苗、新冠病毒疫苗、带状疱疹病毒疫苗等等。
本发明有益效果为：
本发明中的基于钙或硅元素掺杂的AlOOH纳米佐剂经小鼠体内实验验证可以同时诱导高效的体液免疫和细胞免疫。
本发明所述的可以同时诱导高效的体液免疫和细胞免疫的AlOOH的制备方法简单，易操作，重复性好，反应条件温和，最终得到分散均匀、粒径均一的疫苗纳米颗粒，在传染病病毒预防和治疗上具有良好的应用前景。
附图说明
本发明附图9幅，
图1为不同浓度钙、硅元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/2/3/4)的透射电镜图。其中：标尺为200nm。
图2为不同浓度钙、硅元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/2/3/4)的X射线衍射(XRD)图。
图3为不同浓度钙、硅元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/4)X射线光电 子能谱(XPS)。
图4为不同浓度钙、硅元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/4)的不同部位元素含量结果对比。使用电感耦合等离子体原子发射光谱仪(ICP)测定体相掺杂元素含量(橙色)，使用XPS测定表面元素含量(绿色)，结果表明硅、钙在AlOOH中均匀掺杂。
图5为不同浓度钙(A)、硅(B)元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/2/3/4)的体外免疫评估。
图6为不同浓度钙、硅元素掺杂的AlOOH佐剂(Si/Ca-AlOOH-1/4)诱导BMDC细胞表面CD86(A)和MHC II(B)表达及IL-1β(C)、IL-6(D)、IL-12(E)和TNF-α(F)细胞因子的释放结果。
图7为以6-8周C57BL/6小鼠为模型检测Si/Ca-AlOOH-1/4疫苗诱导的乙肝抗体水平；乙肝抗原免疫策略具体为第0天肌内注射含2μg乙肝表面抗原的Si/Ca-AlOOH-1/4，第21天再次注射等量Si/Ca-AlOOH-1/4，第42天取血清和脾脏检测体液免疫和细胞免疫水平。图7A、图7B为血清中特异性抗体水平，分别为总IgG和IgG1水平。其中，HBsAg为单纯的乙肝表面抗原，Alum为HBsAg与商业化的羟基氧化铝佐剂的混合物，所有佐剂的注射量为：铝、钙、硅元素总质量为50μg。图7C-图7I分别为CD4+T细胞表面CD69的表达(C)、CD8+T细胞表面CD69的表达(D)、CD8+T细胞表面FasL的表达(E)、CD4+T细胞表面CD44highCD62low的表达(F)、CD8+T细胞表面CD44highCD62low的表达(G)、B细胞表面CD69的表达(H)和B细胞表面CD27的表达(I)情况。
图8为小鼠主要器官的免疫病理分析。
图9A-图9C为以雌性C57BL/6小鼠(8周，n＝6)在第0、21天通过肌肉注射接种掺杂AlOOH与水痘-带状疱疹病毒糖蛋白E抗原(VZV gE)混合物后，第42天检测gE特异性血清总IgG(A)，(B)IgG1(B)和IgG2c滴度(C)。图9D为功能T细胞和B细胞激活的雷达图表示。图9E-图9F为用gE(2μg/mL)重新刺激免疫小鼠脾细胞120h，用流式细胞术分析CD69+/CD4+、CD69+/CD8+、FasL+/CD8+、CD44highCD62low/CD4+、CD44highCD62Llow/CD8+、CD69+/CD19+和CD27+/CD19+的细胞活化谱，其中图9E为CD4+T细胞中Th1型(TNF-α、IFN-γ、IL-2)和Th2型(IL-4)细胞因子产生的雷达图表示，图9F为CD8+T细胞中Th1型(TNF-α、IFN-γ、IL-2)和Th2 型(IL-4)细胞因子产生的雷达图表示。图9G、图9H为用gE(2μg/mL)重新刺激免疫小鼠的脾细胞24小时，采用酶联免疫斑点法(ELISPOT)检测gE特异性的IFN-γ(G)和(H)IL-2产生斑点形成细胞的数量(H)。图9I、图9J为用gE(2μg/mL)重新刺激免疫小鼠脾细胞5天后脾细胞分泌IL-4(I)和IFN-γ(J)的水平。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1
一种钙元素掺杂的AlOOH纳米佐剂的制备方法，所述方法包括如下步骤：
①配制1.6mol/L AlCl3·6H2O溶液，0.8mol/L无水CaCl2溶液，1mol/L NaOH溶液。
②将上述溶液混合稀释得到不同比例、每种25mL的钙掺杂铝溶液：将1.6mol/LAlCl3·6H2O溶液和0.8mol/L无水CaCl2溶液以不同的体积进行混合、稀释，制备成钙、铝元素摩尔比(Ca/Al)为：0、0.05、0.1、0.15和0.2的混合溶液，其中铝离子浓度为0.5mol/L，制备的钙元素掺杂的AlOOH纳米佐剂(Ca-AlOOH-1/2/3/4)产物分别记作AlOOH，Ca-AlOOH-1(钙、铝元素摩尔比为0.05)、Ca-AlOOH-2(钙、铝元素摩尔比为0.1)、Ca-AlOOH-3(钙、铝元素摩尔比为0.15)、Ca-AlOOH-4(钙、铝元素摩尔比为0.2)。
③向步骤②中混合溶液滴加NaOH溶液至pH＝7，全程600rpm搅拌，反应温度25℃，得到分散体系；
④将步骤③的分散体系加入至反应釜中，用水热法水热制备，温度为120℃，水热时间为4h，得到反应产物；
⑤离心步骤④的反应产物，除去上清液，用超纯水离心洗涤后保存或烘干保存，洗涤时离心机转速为11000rpm，时间为25min。
实施例2
一种硅元素掺杂的AlOOH纳米佐剂的制备方法，所述方法包括如下步骤：
①配制1.6mol/L AlCl3·6H2O溶液，1mol/L NaOH溶液；取正硅酸乙酯TEOS，加入到1mol/LNaOH溶液中，得到含有0.5mol/L TEOS的NaOH溶液。
②稀释铝溶液：将AlCl3·6H2O溶液稀释至25mL，浓度为0.5mol/L。
③向步骤②中稀释的铝溶液滴加不同体积的含有TEOS的NaOH溶液(原硅酸钠溶液)，再滴加NaOH溶液至pH＝7，全程600rpm搅拌，反应温度为25℃；根据含有TEOS的NaOH溶液的加入量不同，制备成硅、铝元素摩尔比(Si/Al)为：0、0.05、0.1、0.15和0.2的溶液，制备的硅元素掺杂的AlOOH纳米佐剂(Si-AlOOH-1/2/3/4)产物分别记作AlOOH，Si-AlOOH-1(硅、铝元素摩尔比为0.05)、、Si-AlOOH-2(硅、铝元素摩尔比为0.1)、Si-AlOOH-3(硅、铝元素摩尔比为0.15)、Si-AlOOH-4(硅、铝元素摩尔比为0.2)。
④后续水热、洗涤等后续过程与实施例1中钙掺杂相同。
实施例3
一种实施例1和2所制备的钙、硅元素掺杂的AlOOH纳米佐剂物化特性的检测：
AlOOH、Si-AlOOH-1/2/3/4和Ca-AlOOH-1/2/3/4分别在水和pH＝7.4，10mMKNO3中的水合粒径(Hydrodynamic size)、Zeta电位(Zeta potential)见表1。
表1
通过透射电镜(TEM)检测实施例1、2中的产物的形貌(如图1所示)，结果表明，随着硅铝比的增加，材料的形态由棒状向叶状转变。随着钙铝比的增加，其形貌由棒状变为颗粒状。
XRD分析(如图2所示)表明，随着Si和Ca元素的增加，掺杂的AlOOH的XRD谱没有出现除AlOOH外的杂峰，说明Si和Ca均匀分布在AlOOH结构中。
XPS分析(如图3所示)进一步证明了硅和钙的成功掺杂。用XPS和ICP(如图4所示)对硅和钙元素进行了定量，结果表明材料表面和体相中元素的掺杂量相似，表明AlOOH颗粒中均匀掺杂硅、铝。
实施例4
采用MTS法测定不同浓度(分别为0、100、200、300、400和500μg/mL)的纳米材料处理后骨髓来源树突状细胞(BMDCs)的细胞活力。实验结果(如图5所示)表明佐剂具有良好的生物相容性。分析了BMDC细胞激活和细胞因子的产生(如图6所示)。结果表明随着硅含量的增加，掺杂的佐剂诱导了更高的IL-12和IL-6的产生，这表明掺杂硅诱导了Th1和Th2型细胞因子的释放。随着钙含量的增加，掺杂佐剂诱导IL-12升高，IL-6细胞因子不变，提示掺杂钙诱导Th1型细胞因子释放增强，而非Th2型细胞因子释放增强。
实施例5
以6-8周C57BL/6小鼠为动物模型，检测实施例1和2中制备的Si-AlOOH-1/2/3/4和Ca-AlOOH-1/2/3/4诱导体液免疫和细胞免疫水平，所述方法包括如下步骤：第0天小鼠肌内注射50μL疫苗，其中有乙肝表面抗原(HBV)2μg，铝、硅和钙总和为50μg，疫苗分散在生理盐水体系中。第21天再次注射等量上述疫苗，第42天取血清和脾脏，检测血清中的总IgG、IgG1水平以及脾细胞成熟分化情况。其中，加入对照组：生理盐水组(每只小鼠注射50μL生理盐水)，HBV组(每只小鼠只注射2μg的乙肝表面抗原)，HBV+Alum组(每只小鼠注射含2μg HBV与含50μg铝元素的商业化的羟基氧化铝佐剂(adjuvant 2％，InvivoGen)的混合物，简写为Alum组)，每组实验小鼠数量为6只。
血清中的总IgG和IgG1如图7A-图7B所示。
脾细胞成熟分化情况如图7C-图7I所示。
如图7所示，实施例5的表征结果表明，抗体滴度实验结果表明掺杂的铝佐剂能够产生更高的IgG、IgG1抗体滴度，证明掺杂的AlOOH能产生更高的体液免疫水平。脾细胞成熟分化结果表明，掺杂的铝佐剂能够促进CD4+、CD8+细胞活化及CTL细胞杀伤介质高表达。
此外，主要器官的免疫病理分析(如图8所示)表明掺杂的铝佐剂具有良好的生物 安全性，具有很强的应用潜力。
实施例6
雌性C57BL/6小鼠(8周，n＝6)在第0、21天通过肌肉注射接种掺杂AlOOH与水痘-带状疱疹病毒糖蛋白E抗原(VZV gE)混合物。每剂含有5μggE和50μg当量Al(实施例1制备的Si-AlOOH-4或实施例2制备的Ca-AlOOH-4)或100μg当量Al(实施例1制备的Si-AlOOH-4，记作Si-AlOOH-4-H，或实施例2制备的Ca-AlOOH-4，记作Ca-AlOOH-4-H)。以gE(5μg)为对照。第42天检测gE特异性血清。
血清中gE特异性总IgG,IgG1和IgG2c滴度如图9A-图9C所示。结果表明，掺杂硅和钙的AlOOH纳米佐剂均可诱导佐剂剂量依赖性的gE特异性抗体反应的增加。
脾细胞成熟分化情况如图9D所示。与gE组相比，Si掺杂可诱导更高的CD69+/CD4+、CD69+/CD8+、FasL+/CD8+、CD62LlowCD44high/CD4+、CD69+/CD19+和CD27+/CD19+细胞百分比，并且随着佐剂含量的增加，FasL+/CD8+和CD27+/CD19+细胞进一步增加。结果表明，Si-AlOOH-4诱导了CTL细胞和记忆B细胞激活的剂量依赖性增强，以及其他功能性T和B细胞的非剂量依赖性的激活。与gE组相比，Ca掺杂诱导的CD69+/CD4+、CD69+/CD8+、FasL+/CD8+、CD62LlowCD44high/CD4+、CD62LlowCD44high/CD8+、CD69+/CD19+、CD27+/CD19+细胞比例较高，且随着佐剂含量的增加，CD69+/CD4+、CD69+/CD8+、FasL+/CD8+、CD69+/CD19+细胞比例进一步升高。这提示了Ca-AlOOH-4诱导CD4+T细胞、CD8+T细胞、CTL细胞和B细胞的激活增强呈剂量依赖性，而记忆T和B细胞的激活呈非剂量依赖性。
用gE(2μg/mL)重新刺激免疫小鼠脾细胞120h，用流式细胞术分析CD69+/CD4+、CD69+/CD8+、FasL+/CD8+、CD44highCD62low/CD4+、CD44highCD62Llow/CD8+、CD69+/CD19+和CD27+/CD19+的细胞活化谱。
CD4+T细胞内Th1型(TNF-α，IFN-γ，IL-2)和Th2型(IL-4)细胞因子产生如图9E所示。细胞内细胞因子分析显示，与gE组相比，Si-AlOOH-4和Si-AlOOH-4-H诱导CD4+T细胞内IL-4、IFN-γ、IL-2和TNF-α的比例更高，表明Si掺杂AlOOH纳米佐剂诱导CD4+T细胞产生Th2和Th1型细胞因子，其与佐剂剂量无关。与gE组相比，Ca-AlOOH-4诱导CD4+T细胞内IFN-γ和TNF-α的比例更高；与Ca-AlOOH-4组相比， Ca-AlOOH-4-H诱导CD4+T细胞内IL-4和IL-2的比例更高。结果表明，Ca-AlOOH-4具有诱导CD4+T细胞内产生Th2和Th1型细胞因子的潜力，其水平由佐剂剂量决定。
CD8+T细胞内Th1型(TNF-α、IFN-γ、IL-2)和Th2型(IL-4)细胞因子产生如图9F所示。Si-AlOOH-4增强CD8+T细胞中细胞内IFN-γ、IL-2和TNF-α的产生。与Si-AlOOH-4相比，Si-AlOOH-4-H诱导CD8+T细胞中IFN-γ增加。Ca-AlOOH-4纳米佐剂诱导细胞内含IFN-γ和TNF-α的CD8+T细胞的比例增加，Ca-AlOOH-4-H诱导胞内含IL-4的CD8+T细胞的比例增加。这些结果表明，CD8+T细胞内Th2和Th1型细胞因子的产生是由Ca-AlOOH-4纳米佐剂的剂量决定的。
在VZV gE抗原(2μg/mL)体外重新刺激激免疫小鼠的脾细胞24小时后，通过IFN-γ和IL-2酶联免疫斑点(ELISPOT)实验检测gE特异性T细胞反应如图9G-图9H所示。结果表明，与gE组相比，高剂量的Ca-AlOOH-4组具有更多的IFN-γ和IL-2斑点形成细胞，说明Ca元素的掺杂可促进IFN-γ和IL-2产生细胞的剂量依赖性增加。然而，无论佐剂的剂量高低，Si-AlOOH-4并没有引起细胞因子释放量的显著增加，说明Si-AlOOH-4纳米佐剂诱导的T细胞反应并不显著。
用gE(2μg/mL)重新刺激免疫小鼠脾细胞5天后从脾细胞中分泌到细胞外的IL-4和IFN-γ的水平如图9I-图9J所示。结果表明，与gE组相比，Si-AlOOH-4诱导IL-4和IFN-γ分泌量增加至6.0倍和1.7倍，而随着Si-AlOOH-4剂量的增加，IL-4和IFN-γ分泌减少至2.1倍和1.0倍。与gE组比较，Ca-AlOOH-4和Ca-AlOOH-4-H诱导的IL-4分泌增加至11.6倍和7.1倍，其增加幅度随Ca-AlOOH-4剂量的增加而降低。此外，Ca-AlOOH-4和Ca-AlOOH-4-H诱导IFN-γ分泌量增加至3.3倍和7.6倍，说明Ca-AlOOH-4诱导的IFN-γ释放量增加具有佐剂剂量依赖性。
在乙肝表面抗原和水痘-带状疱疹病毒糖蛋白E抗原模型中，本发明钙或硅元素掺杂的AlOOH纳米佐剂经小鼠体内实验验证可以同时诱导高效的体液免疫和细胞免疫。

Claims (10)

  1. 一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：利用Al源合成羟基氧化铝，在反应液中使用钙源或硅源进行Ca或Si元素的掺杂，通过加入碱性溶液使Al(OH)3及掺杂物进行沉淀，利用水热合成法制备Ca或Si掺杂的羟基氧化铝佐剂。
  2. 根据权利要求1所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：所述钙源为无机或有机可溶性钙盐，所述无机或有机可溶性钙盐包括但不限于氯化钙，硝酸钙或柠檬酸三钙，钙元素与铝元素的摩尔比为0.01-100：1，钙元素在反应液中的浓度为0.01-100mol/L。
  3. 根据权利要求1所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：所述硅源为有机硅烷、硅酸或可溶性硅酸盐，所述有机硅烷包括四乙氧基硅烷，所述可溶性硅酸盐包括硅酸钠或硅酸钾，硅元素与铝元素的摩尔比为0.01-100：1。
  4. 根据权利要求1所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：所述碱性溶液包括NaOH，KOH或氨水的水溶液或缓冲液，浓度为0.01-10mol/L。
  5. 根据权利要求1所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：所述Al源为无机或有机铝盐，所述无机或有机铝盐包括硝酸铝、氯化铝或乙氧基铝，铝元素在反应液中的浓度为0.01-10mol/L。
  6. 根据权利要求1所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂，其特征在于：所述钙或硅元素掺杂的AlOOH纳米佐剂为20nm-1000nm的纳米颗粒。
  7. 权利要求1-6中任意一项所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂的制备方法，其特征在于：包括如下步骤：
    ①在温度为20-60℃下，向含有钙源的铝源溶液滴加碱性溶液至pH为4-13，或向铝源溶液滴加含有硅源的碱性溶液后，滴加碱性溶液至pH为4-13，全程搅拌，得到分散体系；
    ②将步骤①的分散体系水热合成制备羟基氧化铝佐剂；水热温度为50-300℃，水热时间为2-48h，得到反应产物；
    ③离心步骤②的反应产物，除去上清液，用超纯水离心洗涤后保存或烘干后保存。
  8. 根据权利要求7所述的制备方法，其特征在于，步骤①中，含有硅源的碱性溶液中硅源的浓度为0.01-10mol/L；搅拌的速率为300-1500rpm，搅拌时间为3min-3h； 步骤③中，所述洗涤时离心机转速为8000-15000rpm，时间为10-30min。
  9. 权利要求1-8中任意一项所述的一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂在预防性和治疗性佐剂疫苗中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述疫苗包括乙肝病毒疫苗、人乳头瘤病毒疫苗、新冠病毒疫苗、带状疱疹病毒疫苗。
PCT/CN2024/091914 2023-12-01 2024-05-09 一种基于钙或硅元素掺杂的羟基氧化铝纳米佐剂的制备及其应用 Pending WO2025112290A1 (zh)

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