Antibody-targeted modified dual-strain prebiotic delivery system, preparation method and application

Abstract

This invention relates to the field of biopharmaceutical technology, and in particular to an antibody-targeted modified dual-strain prebiotic delivery system, its preparation method, and its applications. This invention modifies VCAM-1 antibody fragments onto *AKK* bacteria and *Bifidobacterium lactis* Probio-M8, then utilizes fructooligosaccharides and inulin as prebiotics to enhance probiotic metabolism and promote intestinal colonization. Finally, it coats the system with hydroxypropyl methylcellulose phthalate (HPMCP) enteric coating to construct the antibody-targeted modified dual-strain prebiotic delivery system—V-PM8-Am-FI-HPMC. The V-PM8-Am-FI-HPMC provided by this invention can simultaneously achieve intestinal flora regulation, lipid metabolism balance, inflammation suppression, ischemia improvement, and vascular anti-calcification, ultimately achieving the purpose of treating and / or improving atherosclerosis, while ensuring targeting and safety.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to an antibody-targeted modified dual-strain prebiotic delivery system, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is a chronic vascular disease characterized by lipid deposition, fibrosis, and calcification in the arterial wall, forming atherosclerotic plaques. This leads to thickening and hardening of the vessel wall and narrowing of the lumen, ultimately causing blood flow obstruction and organ ischemia. The development of AS is closely related to multidimensional lipid metabolism abnormalities. Its core mechanism involves low-density lipoprotein cholesterol (LDL-C) inducing plaque formation through deposition in the vessel wall. While high-density lipoprotein cholesterol (HDL-C) can play a protective role by reverse cholesterol transport, its elevation alone has limited effect on improving cardiovascular risk. Elevated triglycerides (TG) are often accompanied by decreased HDL-C; both increase risk synergistically through residual particle deposition and inflammatory responses. Severely elevated TG (≥5.6 mmol / L) may also induce pancreatitis. Total cholesterol (TC), as the sum of all lipoprotein cholesterols, is mainly contributed by LDL-C and requires comprehensive evaluation in conjunction with non-HDL-C (TC-HDL-C). Furthermore, lipoprotein (a) [Lp(a)] has prothrombotic and proinflammatory properties independent of the risk of elevated LDL-C, and the risk of cardiovascular events is significantly increased when both coexist. Therefore, prevention and treatment strategies should prioritize LDL-C while controlling TG, non-HDL-C, and Lp(a), with particular attention to the synergistic management of multiple lipid indicators in individuals with metabolic disorders.

[0003] Low-density lipoprotein (LDL) deposited in the endothelial space is easily oxidized to oxidized low-density lipoprotein (ox-LDL). This process is a key step in the early stages of atherosclerosis, and ox-LDL is more pathogenic. ox-LDL can activate the Toll-like receptor 4-nuclear transcription factor κB (TLR4-NF-κB) pathway in endothelial cells, inducing the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). These adhesion molecules can recruit peripheral monocytes to migrate to the subendothelial space and differentiate into macrophages. Macrophages engulf ox-LDL through scavenger receptors, forming foam cells. Simultaneously, macrophages release inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), further exacerbating endothelial damage and LDL deposition, forming a vicious cycle of "lipid enrichment-inflammatory amplification." Cholesterol crystals released after foam cell necrosis can activate nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes, leading to increased intraplaque inflammation, plaque instability, and increased risk of cardiovascular events such as acute myocardial infarction. While traditional lipid-regulating drugs such as statins and proprotein convertase subtilisin 9 (PCSK9) inhibitors can lower plasma LDL levels, they cannot remove cholesterol already deposited on the vessel wall, nor can they inhibit inflammasome activation or foam cell formation. Anti-inflammatory drugs such as colchicine and IL-1β antibodies can alleviate local inflammation, but they have no regulatory effect on lipid metabolism disorders and are unable to prevent new lipid deposition. Although peptide drugs with both lipid-regulating and anti-inflammatory potential have been developed, they only inhibit inflammation and calcification through glutathione, without addressing the upstream interaction mechanism of "lipid deposition-inflammatory activation," thus failing to fundamentally solve the problem. Meanwhile, gut microbiota imbalance is also a significant upstream cause of cardiovascular disease. Pathogenic bacteria in the gut can metabolize choline and carnitine to produce trimethylamine (TMA), which is then oxidized in the liver to trimethylamine N-oxide (TMAO). TMAO promotes platelet activation, enhances macrophage uptake of ox-LDL, and inhibits cholesterol reverse transport, accelerating the formation of atherosclerotic plaques. Gut microbiota imbalance leading to impaired intestinal barrier function results in lipopolysaccharide (LPS) entering the bloodstream, activating a systemic inflammatory response and further exacerbating vascular endothelial damage. Furthermore, coronary artery obstruction leads to myocardial cell hypoxia, interrupting ATP production and triggering cell necrosis and apoptosis. Inflammatory factors such as calcium salts and bone morphogenetic protein 2 (BMP-2) released from necrotic cells can induce vascular smooth muscle cells (VSMCs) to differentiate into osteoblast-like cells, activating osteogenic transcription factors such as runt-related transcription factor 2 (RUNX2) and promoting vascular calcification. Calcification reduces vascular elasticity, further aggravating myocardial ischemia and creating a vicious cycle of ischemia-calcification.

[0004] Existing single-target or dual-target drugs cannot cover the multiple pathological processes involved in AS, and various intervention programs have limitations: lipid-lowering drugs only target some blood lipid indicators and cannot intervene in inflammation and gut microbiota imbalance; anti-inflammatory drugs cannot improve blood lipids and may also cause side effects. Overall, existing single-target or dual-target drugs cannot achieve multi-target synergy of "blood lipid regulation-inflammation suppression-microbiota optimization," resulting in poor AS lesion reversal effects. Probiotic preparations lack stability and are easily affected by the gastrointestinal environment, leading to reduced activity; they lack targeting and cannot accumulate in AS-related lesion sites or intestinal colonization areas. Therefore, current technologies are unable to improve existing plaques and vascular calcification, cannot effectively regulate the imbalance of the "gut microbiota-metabolism-inflammation" axis, and cannot meet the needs of high-risk AS patients for deep blood lipid regulation, thus failing to fundamentally stop disease progression. Moreover, long-term use of existing single-target or dual-target drugs can easily lead to safety risks, including potential damage to liver and kidney function and gut microbiota dysbiosis; at the same time, the multi-drug combination administration method leads to low patient medication adherence, affecting long-term treatment efficacy.

[0005] In summary, while current treatment strategies can be effective at single points, they struggle to overcome core bottlenecks. To effectively halt disease progression and precisely repair damaged myocardium and vascular function, the key lies in simultaneously regulating multiple pathological processes. This requires addressing systemic inflammation caused by gut microbiota imbalance, reversing lipid-driven plaque formation and vascular calcification, and simultaneously addressing the oxygen supply and metabolic needs of ischemic myocardium. Current technologies have not yet achieved the synergistic intervention capabilities required for these multiple dimensions. Furthermore, existing technologies suffer from poor targeting and insufficient safety.

[0006] Therefore, the core challenge facing the field is how to construct a multi-axis synergistic system that can simultaneously achieve "gut microbiota regulation, lipid metabolism balance, inflammation suppression, ischemia improvement, and vascular anti-calcification," while ensuring targeting and safety, and forming a treatment closed loop of "source intervention, lesion repair, and long-term prevention." Summary of the Invention

[0007] The purpose of this invention is to provide an antibody-targeted modified dual-strain prebiotic delivery system, its preparation method, and its applications, in order to solve the problems existing in the prior art. The V-PM8-Am-FI-HPMC provided by this invention can simultaneously achieve intestinal flora regulation, lipid metabolism balance, inflammation suppression, ischemia improvement, and vascular anti-calcification, ultimately achieving the purpose of treating and / or improving atherosclerosis, while ensuring targeting and safety.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a method for preparing an antibody-targeted modified dual-strain prebiotic delivery system, comprising the following steps:

[0010] VCAM-1 single-chain antibody solution was mixed with N-hydroxysuccinimide-biotin, incubated and purified to obtain biotinylated VCAM-1scFv;

[0011] Akkermansia muciniphila (AKK) and Bifidobacterium lactis were modified with avidin to obtain avidin-modified Akkermansia muciniphila and avidin-modified Bifidobacterium lactis bacterial cultures, respectively.

[0012] The avidin-modified Ackermania obliterans bacterial solution and the avidin-modified Bifidobacterium lactis bacterial solution were respectively mixed with biotinylated VCAM-1scFv to obtain Ackermania obliterans targeted bacterial solution and Bifidobacterium lactis targeted bacterial solution.

[0013] The targeted Akermansia bacterium suspension and the targeted Bifidobacterium lactis bacterium suspension were mixed to obtain a targeted dual-bacterium complex;

[0014] Fructooligosaccharides and inulin are mixed to obtain a prebiotic solution;

[0015] The targeted dual-bacterial complex and the prebiotic solution are mixed to obtain an antibody-targeted modified dual-bacterial-prebiotic delivery system.

[0016] More preferably, the Bifidobacterium lactis includes Bifidobacterium lactis Probio-M8.

[0017] In this invention, the *AKK* bacteria regulate the intestinal immune system by secreting specific peptide molecules such as p9 protein, thereby inhibiting NF-κB pathway activation, reducing the release of pro-inflammatory factors such as IL-1β and TNF-α, and improving inflammatory responses in metabolic diseases. *AKK* bacteria use mucin in intestinal mucus as a nutrient source. During the degradation of mucin, it stimulates intestinal goblet cells to secrete more mucin, thereby promoting the renewal and thickening of the mucus layer. The mucus layer is an important component of the intestinal physical barrier; its increased thickness helps enhance intestinal barrier function and prevent the invasion of pathogens and harmful substances. *AKK* bacteria can upregulate the expression of tight junction proteins such as closing small cyclic protein 1 (ZO-1), blocking protein, and tight junction protein-1. Tight junction proteins are important components of the connections between intestinal epithelial cells; increased expression of these proteins can enhance intercellular connections and reduce intestinal permeability. Reduced intestinal permeability can reduce the entry of toxins, inflammatory factors, and other substances from the intestine into the bloodstream, thereby alleviating systemic inflammatory responses, reducing chronic damage to the blood vessel walls caused by systemic inflammation, and thus delaying the calcification process. Furthermore, *AKK* bacteria can produce acetic acid and propionic acid by degrading intestinal mucin. Other gut bacteria utilize the acetic acid and propionic acid produced by AKK bacteria to further metabolize butyrate. Therefore, AKK bacteria can also indirectly promote butyrate production. Acetic acid can activate the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway by activating the G protein-coupled receptor 43 (GPR43) receptor. After AMPK activation, it can promote fatty acid β-oxidation, accelerate lipid breakdown, and inhibit the activity of enzymes related to de novo lipid synthesis, thus reducing triglyceride production. This process helps improve hepatic lipid metabolism disorders and has an indirect ameliorative effect on hypertriglyceridemia and abnormal cholesterol metabolism. Propionic acid can inhibit the expression of "cholesterol transporter" (Niemann-Pick C1-like protein 1 (NPC1L1)) in intestinal epithelial cells, thereby reducing the absorption of dietary cholesterol and bile acid-bound cholesterol, and thus preventing high-fat diet-induced hypercholesterolemia and atherosclerosis. After being absorbed through the intestines, butyrate reaches the liver via the bloodstream, activating the AMPK signaling pathway and subsequently inhibiting the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol synthesis, thus reducing hepatic cholesterol synthesis. Butyrate can also reduce calcium deposition by inhibiting the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Short-chain fatty acids such as acetic acid and propionic acid can also promote the differentiation and proliferation of Treg cells, enhancing their immunosuppressive function. Treg cells can secrete anti-inflammatory cytokines such as IL-10, and Treg cell activation can indirectly inhibit the activity of pro-inflammatory cells, further reducing the levels of pro-inflammatory factors. Bacterial cell wall capsule polysaccharides can activate macrophage burial, clearing away already formed foam cells.

[0018] In this invention, the *Bifidobacterium lactis* Probio-M8 is a probiotic strain that can improve gastrointestinal function, promote bone development and height growth in children, and enhance physical function in elderly patients with sarcopenia by regulating the structure and metabolites of the intestinal flora. Clinical trials have confirmed that *Bifidobacterium lactis* Probio-M8 can reduce serum TMA and TMAO levels, and not only can it metabolize butyrate, but it can also indirectly increase butyrate levels by regulating the abundance of butyrate-producing bacteria in the intestine. Furthermore, as a member of the *Bifidobacterium* genus, it is naturally resistant to gastric acid and bile.

[0019] In this invention, vascular cell adhesion molecule-1 (VCAM-1) is a key adhesion molecule on the surface of endothelial cells, mediating the adhesion and migration of leukocytes such as monocytes and neutrophils by binding to integrin α4β1. During atherosclerotic plaque formation, VCAM-1 expression is upregulated, promoting the recruitment of inflammatory cells to the vessel wall and accelerating plaque progression. VCAM-1 antibodies, by blocking this pathway, can significantly reduce inflammatory cell adhesion, decrease local inflammatory responses, and inhibit atherosclerotic plaque growth. Pathological conditions such as cerebral ischemia activate endothelial cell Notch1 signaling through exosomes, leading to high VCAM-1 expression and endothelial cell senescence. Anti-VCAM-1 antibodies can block the Notch1 signaling-induced inflammatory cascade response, and even under persistent activation of endothelial Notch1 signaling, they can still alleviate vascular damage by inhibiting VCAM-1 expression. By inhibiting VCAM-1 expression, the expression of endothelial cell senescence markers such as p16 and p53 is reduced, delaying vascular endothelial dysfunction. Furthermore, the mechanism of action of VCAM-1 antibody is independent of lipid regulation, directly targeting inflammatory pathways without interfering with lipid metabolism.

[0020] This invention modifies VCAM-1 antibody fragments on AKK bacteria and Bifidobacterium lactis Probio-M8, then utilizes fructooligosaccharides and inulin as prebiotics to enhance probiotic metabolism and promote probiotic colonization in the intestine. Finally, it coats the probiotics with hydroxypropyl methylcellulose phthalate (HPMCP) enteric coating to construct an antibody-targeted modified dual-bacterial-prebiotic delivery system—V-PM8-Am-FI-HPMC. The V-PM8-Am-FI-HPMC provided by this invention can simultaneously achieve intestinal flora regulation, lipid metabolism balance, inflammation suppression, ischemia improvement, and vascular anti-calcification, ultimately achieving the purpose of treating and / or improving atherosclerosis, while ensuring targeting and safety.

[0021] Preferably, the molar ratio of the VCAM-1 single-chain antibody to the N-hydroxysuccinimide-biotin is 1:2 to 1:4.

[0022] More preferably, the molar ratio of the VCAM-1 single-chain antibody to the N-hydroxysuccinimide-biotin is 1:3.

[0023] Preferably, the volume ratio of the avidin-modified Akkermansia bacterium culture to the biotinylated VCAM-1scFv is 1:1.5-1:2.5;

[0024] The volume ratio of the avidin-modified Bifidobacterium lactis culture to the biotinylated VCAM-1scFv is 1:1.5 to 1:2.5.

[0025] More preferably, the volume ratio of the avidin-modified Akkermansia bacterium culture to the biotinylated VCAM-1scFv is 1:2;

[0026] The volume ratio of the avidin-modified Bifidobacterium lactis culture to the biotinylated VCAM-1scFv is 1:2.

[0027] Preferably, the volume ratio of the targeted Ackermaniasis bacterial solution to the targeted Bifidobacterium lactis bacterial solution is 1:1.

[0028] Preferably, the effective viable bacteria count in the targeted dual-strain complex is 8 × 10⁻⁶. 8 CFU / mL.

[0029] Preferably, the mass ratio of the fructooligosaccharide to inulin is 1:1; and the concentration of prebiotics in the prebiotic solution is 50 mg / mL.

[0030] Preferably, the volume ratio of the targeted dual-bacterial complex to the prebiotic solution is 4:1.

[0031] This invention provides an antibody-targeted modified bimicrobial-prebiotic delivery system prepared by the above-described method.

[0032] This invention provides the application of the above-described antibody-targeted modified bimicrobial-prebiotic delivery system in the preparation of drugs for treating and / or improving atherosclerosis.

[0033] The present invention provides a medicament for treating and / or improving atherosclerosis, the medicament comprising the above-described antibody-targeted modified bimicrobial-prebiotic delivery system.

[0034] The present invention discloses the following technical effects:

[0035] 1. VCAM-1 targeted modification can precisely anchor intestinal and vascular lesion sites, block the recruitment of inflammatory cells and endothelial senescence, and is adapted to dual probiotic carriers, with outstanding targeting and no interference with lipid metabolism.

[0036] 2. The synergistic effect of the two probiotics (AKK bacteria + Bifidobacterium lactis Probio-M8) can enhance the intestinal barrier, regulate lipid metabolism, reduce the levels of pro-inflammatory factors and TMAO, clear foam cells, and delay vascular calcification. They have strong biocompatibility and complementary functions.

[0037] 3. The two prebiotics, fructooligosaccharides and inulin, can promote the colonization and metabolic enhancement of bacteria. The enteric coating of HPMCP can protect the system activity from the destruction of gastric acid and bile, making it suitable for oral delivery and highly stable.

[0038] 4. The preparation process of this invention is simple and controllable, and the oral administration is convenient and has high patient compliance. Compared with traditional drugs and invasive treatments, it significantly reduces systemic side effects and treatment costs, and achieves precise, safe and comprehensive intervention for cardiovascular diseases, meeting the needs of clinical applications.

[0039] 5. The targeted modified dual-strain-prebiotic delivery system (VCAM-1 targeted modified dual-strain-prebiotic enteric-coated synergistic delivery system) – V-PM8-Am-FI-HPMC – provided by this invention can simultaneously achieve intestinal flora regulation, lipid metabolism balance, inflammation suppression, ischemia improvement, and vascular anti-calcification, ultimately achieving the purpose of treating and / or improving atherosclerosis, while ensuring targeting and safety. The V-PM8-Am-FI-HPMC provided by this invention relies on HPMCP enteric coating and possesses the core enteric-coating characteristic of "stable in gastric acid and released in the intestine." After incubation in a gastric acid environment for 2 hours, the bacterial survival rate is ≥85%, far exceeding that of the uncoated group. In lipid metabolism regulation experiments, it was confirmed that AKK bacteria and Bifidobacterium lactis Probio-M8 can synergistically achieve multiple regulation including "inhibiting foam cell formation, reducing intracellular total cholesterol accumulation, and regulating dyslipidemia," while simultaneously promoting short-chain fatty acid production with the help of prebiotics, further enhancing lipid metabolism balance. In in vitro and in vivo efficacy evaluations, it effectively inhibited the release of inflammatory factors (TNF-α, IL-6, IL-1β) in vitro (≥50%) and significantly reduced the number of foam cells (≥65%); in vivo, it significantly reduced the release of ApoE... - / -This treatment demonstrated excellent intervention effects in a mouse model of atherosclerosis, sustainably reducing LDL-C, improving lipid indicators such as HDL-C and TG, increasing the abundance of beneficial gut bacteria, and reducing the area of ​​coronary artery calcification and the proportion of plaque in the aortic root. Its effects were superior to those of the traditional drug combination group. Furthermore, it induced the activation of immune-regulating Treg cells, enhancing the anti-inflammatory effect. In biosafety evaluation, healthy ICR mice showed stable physiological status and normal weight gain after oral administration. A single high-dose administration (20 times the effective dose) showed no acute toxicity, and continuous administration for 12 weeks did not damage liver or kidney function or induce persistent inflammatory responses. Enteric coating further improved safety, and biocompatibility was good. Ultimately, this V-PM8-Am-FI-HPMC, relying on its multi-dimensional advantages of precise targeting, multi-axis synergy, and stable safety, achieved good therapeutic effects in an atherosclerosis model through synergistic intervention of "gut microbiota regulation, lipid metabolism balance, inflammation suppression, and vascular anti-calcification," providing an efficient, precise, and safe comprehensive solution for the treatment of cardiovascular diseases. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 A statistical chart showing the binding status of the target;

[0042] Figure 2 A statistical chart showing the dissolution of simulated gastric acid and simulated intestinal solutions over 0-4 hours;

[0043] Figure 3 A statistical chart simulating bacterial survival in gastric acid over 0-2 hours;

[0044] Figure 4 This is a statistical graph of in vitro inflammatory factor levels;

[0045] Figure 5 A statistical graph of foam cell clearance;

[0046] Figure 6 A statistical graph showing the regulation of total cholesterol within cells;

[0047] Figure 7 The graph shows the changes in LDL-C in each group from week 0 to week 8.

[0048] Figure 8 The results are statistical results of blood lipid indicators; where A is HDL-C; B is TG; C is TC; and D is Lp(a).

[0049] Figure 9 The values ​​represent the serum levels of various inflammatory factors; where A represents TNF-α, B represents IL-6, and C represents TMAO.

[0050] Figure 10 A statistical chart showing the relative abundance of bacteria in feces;

[0051] Figure 11 This is a statistical chart of the area of ​​coronary artery calcification.

[0052] Figure 12 This is a map showing the percentage of patch area.

[0053] Figure 13 This is a graph showing the changes in mouse body weight from 0 to 14 days.

[0054] Figure 14 This is a graph showing the changes in body weight of mice from 0 to 12 weeks.

[0055] Figure 15 This is a graph showing changes in biomarkers of liver and kidney injury; where A represents alanine aminotransferase and aspartate aminotransferase; B represents creatinine; and C represents blood urea nitrogen.

[0056] Figure 16 This is a graph showing changes in the inflammatory factor IL-10.

[0057] Figure 17 This is a graph showing the changes in the inflammatory factor IFN-γ. Detailed Implementation

[0058] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0059] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0060] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0061] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0062] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0063] Example 1: Preparation method of antibody-targeted modified dual-strain prebiotic delivery system

[0064] 1. Materials:

[0065] Akkermansia muciniphila (AKK strain) was purchased from CASMA Mall, product number BNCC323275;

[0066] Bifidobacterium lactis Probio-M8 was provided by Hebei University. This strain is published in the literature "Bifidobacterium lactis Probio-M8 prevents nonalcoholic fatty liver disease in high-fat diet-fed rats: The potential role in modulating gut microbiota" (Fan, W., Tang, K., Deng, Y., Zheng, C., Pan, M., Pi, D., Liang, Z., Zhen, J., Yang, Q., & Zhang, Y. (2024). Bifidobacterium lactis Probio-M8 prevents nonalcoholic fatty liver disease in high-fat diet-fed rats: The potential role in modulating gut microbiota. Food Bioengineering, 3, 29–40.).

[0067] VCAM-1 single-chain antibody (scFv) was purchased from CASMA Mall, product number VCAM-1, brand PEPROTECH.

[0068] N-hydroxysuccinimide (NHS)-Biotin was purchased from CASMA Mall, product number S832894.

[0069] 2. The preparation method of the antibody-targeted modified dual-strain-prebiotic delivery system is as follows:

[0070] Step 1: Cultivation and Collection of Probiotics

[0071] Akkermansia myxophilus was inoculated into modified brain and heart extract broth (mBHI) medium (Brain and heart extract powder (BHI) (manufacturer: Solarbio, catalog number: LA0360) 20-30 g / L; heme chloride: 0.5-1.0 mg / L; L-cysteine ​​hydrochloride: 1-2 mg / L; N-acetylglucosamine: 1-2 g / L; yeast extract: 2-5 g / L; peptone: 2-5 g / L) and cultured under strict anaerobic conditions at 37°C for 5-7 days until the logarithmic growth phase to obtain Akkermansia myxophilus bacterial suspension—Am.

[0072] Bifidobacterium lactis Probio-M8 was inoculated into De Man, Rogosa and Sharpe (MRS) medium and cultured with shaking at 37°C and 5% CO2 for 18-24 hours until the logarithmic growth phase, yielding Bifidobacterium lactis Probio-M8 bacterial culture—PM8.

[0073] Take 10 mL of *Akermansia myxophilus* and *Bifidobacterium lactis* Probio-M8 bacterial suspensions respectively, centrifuge at 7000 rpm and 4℃ for 10 minutes, collect the bacterial cells, and gently wash three times with pH 7.4 PBS buffer to remove culture medium residue; adjust the concentration of both bacterial suspensions to 1×10⁻⁶. 9 CFU / mL, refrigerate at 4℃ for later use.

[0074] Step 2: Biotin labeling of VCAM-1scFv

[0075] Take 1 mg / mL of VCAM-1 single-chain antibody (scFv) solution and mix it with N-hydroxysuccinimide (NHS)-biotin at a molar ratio of 1:3. Incubate at room temperature in the dark for 2 hours to biotinylate the lysine residues of the antibody, obtaining the biotinylated reaction solution. Purify the labeled antibody using a PD-10 desalting column. Equilibrate the PD-10 desalting column with 10 mL of PBS at a flow rate of 1 mL / min and discard the initial eluent. Slowly load the biotinylated reaction solution (1 mL) and collect the eluent (containing the labeled antibody). Continue washing the PD-10 desalting column with 10 mL of PBS and combine all eluents. Determine the total protein concentration using the BCA method, ensuring no column leakage, and verify free biotin residue by HPLC. Remove unbound biotin using an affinity chromatography column to further purify the antibody. Load the eluent from the PD-10 desalting column at a flow rate of 0.5 mL / min. Wash with PBS until the UV absorbance (A280) baseline stabilizes to remove unbound NHS-biotin. Elute with a low-pH glycine buffer using a protein A / G column, immediately neutralizing to pH 7.0. Then perform streptavidin column purification to remove non-specific binding. Equilibrate the streptavidin agarose beads in the streptavidin column with PBS at a flow rate of 1 mL / min. Load the purified antibody solution from the protein A / G column slowly through the column (1 mL / min) to capture unbound free biotin. Wash the column with 10 mL of PBS to remove residual contaminants. Elute the target antibody to improve antibody purity and activity. HPLC verification showed a labeling rate >95%. Adjust the purified antibody concentration to 0.5 mg / mL to obtain biotinylated VCAM-1scFv, stored at -20°C protected from light.

[0076] Step 3: Targeted Modification of Two Probiotics

[0077] Glutaraldehyde solution was added to both bacterial cultures to a final concentration of 0.01% (v / v), and the cultures were incubated at 25°C with shaking for 15 minutes to activate the surface amino groups. The cultures were then washed twice, twice by centrifugation at 3000 rpm and 4°C for 5 minutes with PBS buffer to remove free glutaraldehyde, yielding two activated bacterial cultures. Both activated bacterial cultures (with an effective viable count of 4 × 10⁻⁶) were then further processed. 8CFU / mL) was mixed with 1 mg / mL avidin solution at a volume ratio of 1:5 and incubated at 37°C for 30 minutes. Avidin was then covalently coupled to amino-aldehyde groups, and the mixture was washed twice with PBS buffer at 3000 rpm and 4°C for 5 minutes. After washing, the avidin modification level was determined using the quinoline carboxylic acid (BCA) method, which showed a modification level of 500-800 molecules / bacterium. The two avidin-modified bacterial cultures were then mixed with biotinylated VCAM-1scFv at a volume ratio of 1:2 and incubated at 25°C for 20 minutes to form a targeting complex. The two targeting complexes were then mixed at a volume ratio of 1:1, and PEG-4000 was added to a final concentration of 1 wt% to optimize stability. After standing for 30 minutes, the mixture was centrifuged at 3000 rpm and 4°C for 5 minutes, and the precipitate was collected and resuspended in PBS to a total bacterial concentration of 8 × 10⁻⁶. 8 CFU / mL was used to obtain the targeted dual-bacterial complex—V-PM8-Am, in which the ratio of viable bacteria of Akkermansia myxophilus and Bifidobacterium lactis Probio-M8 in the targeted dual-bacterial complex was 1:1.

[0078] Step 4: Prebiotic Complex Formulation

[0079] Fructooligosaccharides and inulin were weighed and mixed at a mass ratio of 1:1. Sterile deionized water was added, and the mixture was stirred and dissolved in a 60°C water bath to prepare a 50 mg / mL prebiotic stock solution (i.e., the concentration of fructooligosaccharides in the prebiotic stock solution was 25 mg / mL, and the concentration of inulin was 25 mg / mL). After cooling to room temperature, the solution was autoclaved at 121°C for 20 minutes and then allowed to cool naturally to room temperature. The targeted dual-strain complex was mixed with the prebiotic stock solution at a volume ratio of 4:1, and gently stirred at 20°C for 30 minutes to ensure uniform dispersion of the prebiotics. The final prebiotic concentration in the system was 10 mg / mL, yielding the prebiotic complex system, namely the antibody-targeted modified dual-strain-prebiotic delivery system—V-PM8-Am-FI.

[0080] Step 5: Preparation of enteric coating of hydroxypropyl methylcellulose phthalate (HPMCP)

[0081] Weigh HPMC and add it to an ethanol-water mixed solvent (ethanol:water = 6:4, v / v). Dissolve the solution in a 50°C water bath by stirring to prepare a 10% (w / v) coating solution. Cool the solution to 30°C for later use. Spray dry the prebiotic complex system to prepare 100-200μm microparticles as the core material. Coat the particles using a fluidized bed coating machine with the following settings: inlet air temperature 40°C, outlet air temperature 30°C, atomization pressure 0.2MPa, and coating solution spray rate 5mL / min. The coating weight gain is 15%-20%. After coating, dry the film at 35°C and 40% relative humidity for 2 hours to solidify the coating film, thus obtaining V-PM8-Am-FI-HPMC. Seal and store at 4°C.

[0082] Example 2: Targeted Binding Verification

[0083] Human umbilical vein endothelial cells (HUVECs) were purchased from CASMA Mall, catalog number CP-H082;

[0084] IgG-FITC was purchased from CASMA Mall, product number D16866.

[0085] Human umbilical vein endothelial cells (HUVECs) were selected, and the cells were first cultured at a concentration of 1×10⁻⁶. 5 VCAM-1 was seeded at a density of 1 cell / well in 6-well cell culture plates and cultured to 80%-90% confluence. The cells were then incubated with 50 μg / mL oxidized low-density lipoprotein (ox-LDL) for 24 h to induce high expression of VCAM-1. Four parallel experiments were set up: a targeted group (V-PM8-Am-FI-HPMC prepared in Example 1), a non-targeted group (PM8-Am-FI-HPMC, i.e., V-PM8-Am-FI-HPMC prepared in Example 1 without "Step 2: Biotinylation of VCAM-1scFv" and "Step 3: Targeted Modification by Two Probiotics"), an isotype antibody control group (IgG-FITC), and a blank control group (i.e., no treatment). V-PM8-Am-FI-HPMC and PM8-Am-FI-HPMC were labeled with fluorescein isothiocyanate (FITC) at a ratio of 1×10⁻⁶. 8 HUVEC cells were co-incubated with CFU / mL at a concentration of 10 μg / mL for 1 h (37℃, 5% CO2), and IgG-FITC at a concentration of 10 μg / mL was also co-incubated with HUVEC cells for 1 h (37℃, 5% CO2). After incubation, the cells were gently washed three times with pre-cooled PBS buffer to remove unbound carriers or antibodies and eliminate interference from non-specific binding. The cell-binding fluorescence intensity was detected by flow cytometry, and the carrier-cell binding rate was calculated. The results are shown below. Figure 1 As shown. By Figure 1 It can be seen that the cell binding rate of the targeted group is ≥35%, which is significantly higher than that of the non-targeted group, the isotype antibody control group and the blank control group, indicating that the V-PM8-Am-FI-HPMC prepared in Example 1 can specifically bind to the target cells and the targeting binding effect is significant.

[0086] Example 3: Determination of enteric coating properties

[0087] 100 mg of V-PM8-Am-FI-HPMC prepared in Example 1 was added to 10 mL of simulated gastric acid (pH 1.2 hydrochloric acid solution containing 0.32% (v / v) pepsin) and simulated intestinal solution (pH 6.8 phosphate buffer containing 1% (v / v) pancreatin), respectively, and the mixture was shaken at 37°C and 100 rpm. Samples were then taken at 0.5 h, 1 h, 2 h, and 4 h, and the dissolution rate was assessed by determining the prebiotic content in the samples using high-performance liquid chromatography (HPLC). The results are as follows: Figure 2 As shown.

[0088] Take 100 mg each of the experimental group (V-PM8-Am-FI-HPMC prepared in Example 1) and the uncoated control group (V-PM8-Am-FI prepared in Example 1), add 10 mL of simulated gastric acid to each, and measure bacterial survival for 0-2 hours. The results are as follows. Figure 3 As shown.

[0089] Depend on Figure 2 and Figure 3 It was found that after incubation in a gastric acid environment for 2 hours, the bacterial survival rate was ≥85%, while the survival rate of the uncoated control group was ≤30%. After incubation in the intestinal environment for 4 hours, the coating membrane completely dissolved, and the prebiotic release was ≥90%, proving that HPMCP enteric coating can resist gastric acid damage and accurately release prebiotics in the intestine.

[0090] Example 4: Validation of In Vitro Inflammation Suppression

[0091] RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS, 1 μg / mL), and then grouped and treated with PBS, PM8 (prepared in Example 1), Am (prepared in Example 1), V-PM8-Am (prepared in Example 1), V-PM8-Am-FI (prepared in Example 1), and V-PM8-Am-FI-HPMC (prepared in Example 1), respectively. The amount of PBS added was 200 μL / well, and the amount of the other reagents added was 1 × 10⁻⁶. 8 CFU / well, after incubation for 24 h, the levels of TNF-α, IL-6, and IL-1β in the supernatant were detected by ELISA. The results are as follows: Figure 4 As shown. By Figure 4 It was found that, compared with PBS treatment, V-PM8-Am-FI-HPMC treatment reduced the levels of TNF-α, IL-6, and IL-1β by ≥50%.

[0092] Example 5: Validation of in vitro lipid metabolism regulation

[0093] Macrophages were induced to form foam cells using ox-LDL (50 μg / mL), and then grouped into groups. PBS, PM8 (prepared in Example 1), Am (prepared in Example 1), V-PM8-Am (prepared in Example 1), V-PM8-Am-FI (prepared in Example 1), and V-PM8-Am-FI-HPMC (prepared in Example 1) were added to each well. The amount of PBS added was 200 μL / well, and the amount of the other formulations added was 1 × 10⁻⁶. 8 CFU / well, after incubation for 48 h, the number of foam cells was counted using Oil Red O staining, and the intracellular total cholesterol (TC) content was detected using a cholesterol quantification kit. Results are as follows: Figure 5 and Figure 6 As shown. By Figure 5 and Figure 6 It can be seen that, compared with PBS treatment, V-PM8-Am-FI-HPMC treatment reduced the number of foam cells by ≥65% and the intracellular total cholesterol (TC) content by ≥45%, significantly inhibiting foam cell formation and lipid accumulation.

[0094] Example 6: In vivo effect evaluation

[0095] Select ApoE for 6-8 week old infants - / - Mice weighing 18-22g were induced to develop an atherosclerosis model for 10 weeks using a high-fat diet (the high-fat diet consisted of a basal diet supplemented with 21wt% lard (fat source), 0.15wt% cholesterol, and 0.2wt% sodium cholate, with fat providing 45% of the energy and cholesterol content of 0.15g / 100g of feed; the basal diet was purchased from CASMA, catalog number D10012M). The model mice were randomly divided into 7 groups (n=5) and orally administered equal volumes of physiological saline, PM8 (prepared in Example 1), Am (prepared in Example 1), V-PM8-Am (prepared in Example 1), V-PM8-Am-FI (prepared in Example 1), V-PM8-Am-FI-HPMC (prepared in Example 1), and atorvastatin + colchicine, respectively. The dosage of physiological saline, PM8, Am, V-PM8-Am, V-PM8-Am-FI, and V-PM8-Am-FI-HPMC was 200 μL / mouse (containing 1.6 × 10⁻⁶ effective viable bacteria). 8 CFU / mouse, converted to 8 × 10⁸ CFU / mouse based on mouse weight 7(CFU / kg), In the atorvastatin + colchicine treatment, the dosage of atorvastatin was 10 mg / kg and the dosage of colchicine was 0.2 mg / kg. Atorvastatin and colchicine were dissolved in 200 μL of normal saline and administered by gavage, once daily orally. Treatment continued for 8 weeks with free access to food and water. Low-density lipoprotein cholesterol (LDL-C) was measured periodically; after 8 weeks of treatment, high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), total cholesterol (TC), and lipoprotein (a) [Lp(a)] were measured; serum levels of TNF-α, IL-6, and TMAO were also measured. Results are as follows. Figures 7-9 As shown.

[0096] Eight weeks after drug administration, fecal samples were collected for 16S rRNA sequencing to detect the relative abundance of AKK bacteria, Bifidobacterium lactis Probio-M8 (Probio-M8), and butyric acid-producing bacteria (Prevotella faecalis). Coronary arteries were collected for Alcian blue-Alizarin Red staining to quantify the calcification area. Aortic root samples were collected for Oil Red O staining, and ImageJ software was used to calculate the proportion of plaque area to the vascular lumen area. Results are as follows: Figures 10-12 As shown.

[0097] Depend on Figure 7 and Figure 8 It can be seen that V-PM8-Am-FI-HPMC treatment significantly regulates lipid metabolism in mice, breaking the limitations of traditional single-target regulation. LDL-C decreased continuously over time, and HDL-C, TG, TC, Lp(a) and other indicators were effectively improved. However, V-PM8-Am-FI-HPMC treatment was significantly more effective in regulating lipid disorders than single / double groups of PM8 and Am, as well as atorvastatin + colchicine treatment.

[0098] Depend on Figure 9 It can be seen that V-PM8-Am-FI-HPMC treatment of mice inhibited serum TNF-α levels by >50%, and IL-6 and TMAO were significantly reduced simultaneously, effectively alleviating the inflammatory state in the process of atherosclerosis.

[0099] Depend on Figure 10It was found that the abundance of AKK bacteria in the feces of mice treated with V-PM8-Am-FI-HPMC was significantly increased, by more than 150% compared with the saline group, eventually reaching more than 8%, far exceeding the PM8, Am and other treatments as well as the atorvastatin + colchicine treatment, verifying that the enteric coating of HPMC specifically enhances the colonization of AKK bacteria; the abundance of butyric acid-producing bacteria (Prevotella faecalis) was >12%, forming a synergistic effect with Bifidobacterium lactis Probio-M8 (>6.5%); compared with other drug groups (such as uncoated V-PM8-Am-FI treatment, traditional drug combination treatment (atorvastatin + colchicine treatment)), the enteric coating of HPMC significantly enhanced the ability to regulate beneficial bacteria, laying a microecological foundation for subsequent metabolic and inflammation improvement.

[0100] Depend on Figure 11 and Figure 12 It was found that the area of ​​coronary artery calcification and aortic root plaque in mice treated with V-PM8-Am-FI-HPMC was significantly reduced compared to the saline group, and the lesion severity was milder than that treated with atorvastatin + colchicine. In summary, the V-PM8-Am-FI-HPMC provided by this invention can exert a multi-target synergistic anti-atherosclerotic effect by regulating lipid metabolism, inhibiting inflammatory responses, and improving gut microbiota structure, and its efficacy is superior to that of traditional drug combination groups, demonstrating potential clinical application value.

[0101] Example 7 Biosafety Evaluation

[0102] ICR mice (n=20, half male and half female) were administered a single oral gavage dose of 20 times the effective dose of V-PM8-Am-FI-HPMC (prepared in Example 1), and their behavior and weight changes were observed over 14 days. Another ICR mouse (n=20, half male and half female) was administered a continuous oral gavage dose of the effective dose of V-PM8-Am-FI-HPMC (200 μL V-PM8-Am-FI-HPMC containing 1.6 × 10⁻⁶ effective live bacteria). 8 CFU / mouse) for 12 weeks, with untreated ICR mice as controls. Weight was measured weekly, and liver and kidney function and levels of inflammatory factors (IL-10, IFN-γ) were regularly assessed. Results are as follows: Figures 13-17 As shown.

[0103] Depend on Figure 13 It was found that during the 14-day observation period, the body weight of mice in the male and female treatment groups showed a consistent trend with the control group, with no significant abnormal fluctuations, and the mice exhibited normal behavior and no symptoms of poisoning. This indicates that after a single oral administration of 20 times the effective dose of V-PM8-Am-FI-HPMC, mice did not experience acute toxicity, and the preparation has good acute safety.

[0104] Depend on Figure 14It can be seen that the weight gain trend of the male and female drug administration groups and the control group is consistent, with no significant difference, indicating that long-term drug administration did not have an adverse effect on the growth and development of mice.

[0105] Depend on Figure 15 It was found that after 12 weeks of continuous administration, the fluctuation range of liver injury markers (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and kidney injury markers (creatinine (Cr) and blood urea nitrogen (BUN)) in both the female and male treatment groups was within ±15% of the normal value, and there were no significant differences between the groups. This further confirms from the perspective of liver and kidney function that long-term continuous gavage administration of effective doses of V-PM8-Am-FI-HPMC does not cause damage to liver and kidney function in ICR mice.

[0106] Depend on Figure 16 and Figure 17 It can be seen that in the long-term toxicity experiment, although there were individual fluctuations in serum IL-10 and IFN-γ levels between male and female groups, they were all within the normal range and did not show abnormal increases or decreases over time. The IL-10 / IFN-γ ratio remained stable between 0.8 and 1.2 (the baseline range for healthy mice), indicating that long-term administration did not induce a persistent inflammatory response.

[0107] Based on the combined results of changes in body weight, levels of inflammatory factors (IL-10, IFN-γ), and liver and kidney function markers, V-PM8-Am-FI-HPMC did not have significant adverse effects on the growth and development, inflammatory status, or liver and kidney function of ICR mice, whether it was a single high-dose acute exposure or long-term continuous administration, indicating good biosafety.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an antibody-targeted modified dual-strain prebiotic delivery system, characterized in that, Includes the following steps: VCAM-1 single-chain antibody solution was mixed with N-hydroxysuccinimide-biotin, incubated and purified to obtain biotinylated VCAM-1scFv; Akkermansia myxophilus ( Akkermansia muciniphila ) and Bifidobacterium lactis ( Bifidobacterium lactis The samples were modified with avidin to obtain avidin-modified Ackermania viride and avidin-modified Bifidobacterium lactis. The Bifidobacterium lactis was Bifidobacterium lactis Probio-M8. The catalog number of the Ackermania viride was BNCC323275. The avidin-modified Ackermania obliterans bacterial solution and the avidin-modified Bifidobacterium lactis bacterial solution were respectively mixed with biotinylated VCAM-1scFv to obtain Ackermania obliterans targeted bacterial solution and Bifidobacterium lactis targeted bacterial solution. The targeted Akermansia bacterium suspension and the targeted Bifidobacterium lactis bacterium suspension were mixed to obtain a targeted dual-bacterium complex; Fructooligosaccharides and inulin are mixed to obtain a prebiotic solution; The targeted dual-bacterial complex and the prebiotic solution are mixed to obtain an antibody-targeted modified dual-bacterial-prebiotic delivery system.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the VCAM-1 single-chain antibody to the N-hydroxysuccinimide-biotin is 1:2 to 1:

4.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the avidin-modified Akkermansia bacterium culture to the biotinylated VCAM-1scFv is 1:1.5-1:2.

5. The volume ratio of the avidin-modified Bifidobacterium lactis culture to the biotinylated VCAM-1scFv is 1:1.5 to 1:2.

5.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the targeted Ackermania ulmoides bacterial solution to the targeted Bifidobacterium lactis bacterial solution is 1:

1.

5. The preparation method according to claim 1, characterized in that, The effective viable bacteria number in the targeted double bacterial complex is 8 x 10 8 CFU / mL.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the oligofructose to inulin is 1:1; the concentration of prebiotics in the prebiotic solution is 50 mg / mL.

7. The preparation method according to claim 1, characterized in that, The volume ratio of the targeted dual-strain complex to the prebiotic solution is 4:

1.

8. The antibody-targeted modified bimicrobial-prebiotic delivery system prepared by the preparation method according to any one of claims 1-7.

9. The use of the antibody-targeted modified bimicrobial-prebiotic delivery system according to claim 8 in the preparation of a drug for treating atherosclerosis.

10. A drug for treating atherosclerosis, characterized in that, The drug comprises the antibody-targeted modified bimicrobial-prebiotic delivery system of claim 8.

Images