A biological hybrid nano-preparation based on cholesterol regulation and a preparation method and application thereof

Abstract

This invention discloses a cholesterol-regulated bio-hybrid nanoparticle formulation, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method includes: preparing a gold nanoparticle solution; mixing the gold nanoparticle solution with a photosynthetic bacteria culture to obtain a PSB@GNP complex; mixing the PSB@GNP complex with a Proteus mirabilis culture to obtain a PM-PSB@GNP core solution; and modifying the PM-PSB@GNP core solution with a coating solution followed by spray drying to obtain the bio-hybrid nanoparticle formulation EC-PM-PSB@GNP. The preparation process of this invention is simple and controllable, requiring no complex or precise equipment. Oral administration is convenient, resulting in high patient compliance. Compared to traditional chemical drugs and injectable formulations, it significantly reduces systemic toxicity and environmental pollution risks, while also reducing treatment procedures and medication costs, achieving efficient, safe, and precise colorectal cancer treatment and meeting clinical application needs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a cholesterol-regulated bio-hybrid nano-formulation, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is a common and frequently occurring malignant tumor in clinical practice, and its treatment efficacy is significantly limited by the complex characteristics of the tumor microenvironment. Among these, the synergistic effect of local cholesterol homeostasis imbalance and the immunosuppressive microenvironment poses a major challenge to current treatment strategies. To restore tumor sensitivity to treatment and stimulate a potent and specific anti-tumor immune response, the key lies in effectively regulating the tumor microenvironment to achieve precise targeted delivery and efficient functioning of the treatment system. This process requires simultaneously addressing multiple dimensions of issues, including cholesterol-mediated treatment resistance, insufficient targeting precision, and immune microenvironment remodeling.

[0003] Cholesterol metabolism imbalance in the tumor microenvironment is a key characteristic of colorectal cancer development and progression, closely related to tumor cell proliferation, immune escape, and treatment resistance. On one hand, cancer cells need to maintain high cholesterol levels to meet their rapid proliferation requirements, thus maintaining cell membrane fluidity, stability, and signal transduction functions, providing a structural basis for cell division, migration, and metabolism. Simultaneously, FGF21 secreted by tumor cells can activate CD8+. + The AKT-mTORC1-SREBP1 signaling pathway in T cells promotes the expression and activity of cholesterol synthase, leading to excessive cholesterol accumulation in T cells. This, in turn, triggers T cell metabolic reprogramming, dysfunction, and exhaustion, rendering them incapable of anti-tumor killing. On the other hand, high concentrations of cholesterol in the tumor microenvironment can synergistically exacerbate immunosuppression by upregulating the expression of immune checkpoint molecules (such as PD-1 and TIM-3) and inducing endoplasmic reticulum stress and inflammatory responses, forming a vicious cycle of "cholesterol enrichment - immune exhaustion" and significantly weakening the efficacy of immunotherapy.

[0004] Lipid rafts are microdomains on the cell membrane composed of cholesterol, sphingomyelin, and specific proteins, participating in various cellular processes. Cholesterol, as a crucial component of lipid rafts and the cell membrane, interacts with sphingomyelin through its hydrophobic tail, forming ordered lipid microdomains that provide the basis for the stability and function of lipid rafts. Lipid rafts are abundant in many cancer cells, and cancer cells accumulate cholesterol within these rafts, making them more susceptible to cell death than normal cells after cholesterol depletion. Cholesterol depletion disrupts lipid raft integrity, leading to the loss of microdomain-related proteins such as integrins, FAK, and TGF-β receptors. This, in turn, blocks mechanotransduction and TGF-β-induced EMT (FAK loss further inhibits EMT), ultimately reducing the stiffness of the tumor physical barrier, promoting drug penetration, and inhibiting cancer cell growth, proliferation, invasion, and metastasis. As can be seen from the above, cholesterol regulation has great potential in restoring sensitivity to colorectal cancer treatment, remodeling the immunosuppressive microenvironment, and activating anti-tumor immune responses. It can break the vicious cycle of "cholesterol accumulation-immune depletion" and reverse CD8+ by inhibiting intracellular cholesterol synthesis in tumor cells and clearing free cholesterol from the tumor microenvironment through a dual mechanism. + T-cell depletion can achieve highly efficient anti-tumor activity. However, current technologies do not deeply integrate cholesterol regulation with immune microenvironment remodeling, and existing cholesterol regulation strategies mostly rely on chemotherapy drugs, which suffer from poor targeting and significant systemic side effects, failing to achieve a closed-loop treatment mechanism of "cholesterol clearance-immune activation-long-term memory." Therefore, how to achieve precise tumor targeting while simultaneously regulating cholesterol synergistically is a major challenge. Summary of the Invention

[0005] The purpose of this invention is to provide a cholesterol-regulated biohybrid nanoparticle formulation, its preparation method, and its application, to address the problems existing in the prior art. The biohybrid nanoparticle formulation provided by this invention can effectively combine tumor microenvironment regulation and cholesterol dual regulation. Leveraging its multi-dimensional advantages, this nanoparticle formulation achieves good tumor-suppressing effects in a colorectal cancer model through cholesterol regulation, providing an efficient, precise, and safe solution for colorectal cancer treatment.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides a method for preparing a cholesterol-regulated biohybrid nanoparticle formulation, comprising the steps of preparing a gold nanoparticle solution, mixing the gold nanoparticle solution with a photosynthetic bacteria culture to obtain a PSB@GNP complex, mixing the PSB@GNP complex with a Proteus mirabilis culture to obtain a PM-PSB@GNP core solution, and modifying the PM-PSB@GNP core solution with a coating solution and then spray-drying it to obtain the biohybrid nanoparticle formulation EC-PM-PSB@GNP.

[0008] Preferably, the gold nanoparticle solution is prepared by: adding 0.5-1.5 mL of 1-3% chloroauric acid solution to water and heating, adding 5-10 mL of 1-3% sodium citrate solution dropwise while continuously heating and stirring, and then filtering after cooling to obtain the gold nanoparticle solution.

[0009] Preferably, the PSB@GNP complex is prepared by mixing photosynthetic bacteria solution and gold nanoparticle solution at a volume ratio of 1:1 to 1:10 and allowing it to stand at 25-34°C for 90-120 minutes to obtain the PSB@GNP complex.

[0010] Preferably, the preparation method of the photosynthetic bacteria culture is as follows: Rhodopseudomonas palustris CGA009 strain is cultured to the logarithmic growth phase, 10-15 mL of the culture is taken, the cells are collected by centrifugation, the culture medium is washed away, and the concentration of the culture is adjusted to 1×10⁻⁶. 8 -1×10 9 The photosynthetic bacteria culture can be obtained at a concentration of CFU / mL.

[0011] Preferably, the PM-PSB@GNP core solution is prepared by mixing PSB@GNP complex with Proteus mirabilis bacterial solution at a volume ratio of 1:1-3:1, co-culturing at 35-37℃ and 100-150rpm for 4-6h, collecting the precipitate by centrifugation, and resuspending to obtain the PM-PSB@GNP core solution.

[0012] Preferably, the viable bacteria concentration in the PM-PSB@GNP core solution is 4 × 10⁻⁶. 8 -5×10 8 CFU / mL.

[0013] Preferably, the preparation method of the bio-hybrid nano-formulation EC-PM-PSB@GNP is as follows: prepare a coating solution, take 10-15mL of PM-PSB@GNP core solution and add 2-5g of polyethylene glycol 4000, disperse ultrasonically to form a suspension, and spray dry the suspension with the coating solution as the spray medium to obtain the bio-hybrid nano-formulation EC-PM-PSB@GNP.

[0014] Secondly, the present invention also provides a bio-hybrid nanoformulation EC-PM-PSB@GNP prepared by the preparation method described above.

[0015] Thirdly, the present invention also provides the application of the aforementioned biohybrid nanoformulation EC-PM-PSB@GNP in the preparation of drugs for treating colorectal cancer.

[0016] Fourthly, the present invention also provides a medicament for treating colorectal cancer, comprising the aforementioned biohybrid nanoformulation EC-PM-PSB@GNP.

[0017] The present invention discloses the following technical effects:

[0018] 1. Gold nanoparticles have good biocompatibility and can achieve dual anti-tumor effects of "physicochemical killing and immune activation". They are also easy to bind to microbial carriers and their functional stability in the optimized tumor microenvironment is not affected by cholesterol.

[0019] 2. Photosynthetic bacteria can metabolize waste products such as ammonia in the tumor microenvironment, inhibit cholesterol synthesis in tumor cells, and reduce the virulence of Proteus mirabilis. At the same time, they can act as secondary carriers to protect gold nanoparticles, making them suitable for oral delivery and highly biocompatible.

[0020] 3. Proteus mirabilis can respond to signals from the tumor microenvironment of colorectal cancer to achieve precise colonization. It can directly degrade free cholesterol and can also entangle photosynthetic bacterial complexes loaded with gold nanoparticles to prolong the duration of action. After modification, its toxicity is reduced, making it suitable for oral administration and exhibiting outstanding targeting.

[0021] 4. The preparation process of this invention is simple and controllable, requiring no complex and precise equipment. The oral administration method is convenient, and patient compliance is high. Compared with traditional chemical drugs and injectable preparations, it significantly reduces systemic toxic side effects and environmental pollution risks, while reducing treatment procedures and medication costs, achieving efficient, safe, and precise colorectal cancer treatment and meeting clinical application needs.

[0022] This invention provides a cholesterol-regulated biohybrid nanoformulation, EC-PM-PSB@GNP, which effectively combines tumor microenvironment regulation and cholesterol dual regulation. The particle size and potential of the products at each stage of this nanoformulation system are adapted to oral delivery requirements, and it possesses enteric coating properties, exhibiting stability in gastric acid and release in the intestine. Bacterial activity evaluation confirmed that the preparation process causes minimal damage to the activity of PSB and PM. Cholesterol regulation experiments demonstrated that PSB and PM can synergistically achieve dual regulation, namely, intracellular inhibition of tumor cell cholesterol synthesis and extracellular degradation of free cholesterol in the microenvironment. In vitro and in vivo efficacy evaluations showed that it effectively inhibited the proliferation of colorectal cancer cells in vitro without killing normal cells, and exhibited excellent tumor-suppressive effects in tumor-bearing mouse models in vivo. It also induced immune memory, reducing the risk of tumor recurrence. In biosafety evaluations, healthy mice showed stable physiological states after oral administration, and the enteric coating effectively reduced bacterial virulence, demonstrating good biocompatibility. Ultimately, this nanoformulation, leveraging its multi-dimensional advantages, achieved good tumor-suppressive effects in colorectal cancer models through cholesterol regulation, providing an efficient, precise, and safe treatment option for colorectal cancer. Attached Figure Description

[0023] 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.

[0024] Figure 1 Particle size distribution diagrams for different groups;

[0025] Figure 2 Zeta potential graphs for different groups;

[0026] Figure 3 The intracellular loading rate of PSB on GNP;

[0027] Figure 4 This represents the actual load of GNP in EC-PM-PSB@GNP;

[0028] Figure 5 Dissolution was observed in media with pH=1.2 and pH=6.8 over 0-2 hours.

[0029] Figure 6 For the determination of PSB and PM activity;

[0030] Figure 7 Cholesterol content;

[0031] Figure 8 The effects of different groups on the proliferation activity of CT26 cells;

[0032] Figure 9 The effects of different groups on the proliferation activity of NCM460 cells;

[0033] Figure 10 Bioluminescence graphs showing the antitumor effects of different groups on mice;

[0034] Figure 11 Tumor weight corresponding to different groups of mice treated;

[0035] Figure 12 The graph shows the changes in body weight of mice in different groups after treatment. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] 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.

[0041] Gold nanoparticles (GNPs) are a class of biocompatible nanomaterials that can interact with intracellular reactive oxygen species (ROS) or disrupt lysosomal stability, triggering tumor cell stress and releasing damage-associated molecular patterns (DAMPs) (such as calreticulin, ATP, and HMGB1). DAMPs are recognized and taken up by dendritic cells (DCs), activating DC maturation and subsequently activating cytotoxic T cells (CTLs). CTLs migrate to the tumor site to specifically kill tumor cells, while simultaneously generating "immune memory" to prevent tumor recurrence. They can also alleviate CD8... +T cell depletion enhances anti-tumor immune responses, but its anti-tumor ability is easily interfered with by high concentrations of cholesterol in the tumor microenvironment (non-specific adsorption destroys surface structure and dispersibility), and it lacks active targeting capabilities. Oral delivery is easily degraded by digestive enzymes and may cause toxic side effects when distributed in normal tissues. Therefore, a carrier system is needed to achieve functional protection and targeted delivery. *Proteus mirabilis* (PM) is a type of bacteria capable of colonizing the gut. PM possesses pili and adhesion factors, enabling it to adsorb other bacteria to form symbiotic complexes, achieving a physical "entanglement." PM can directly secrete enzymes to degrade free cholesterol in the tumor site, thereby disrupting the integrity of lipid rafts—leading to the loss of key cholesterol-dependent proteins in lipid rafts (such as integrins, FAK, and TGF-β receptors). This blocks the mechanotransduction of tumor cells (reducing tumor matrix stiffness) and inhibits TGF-β-induced EMT (reducing tumor proliferation and metastasis), indirectly achieving the "inactivation" of cholesterol function. Photosynthetic bacteria (PSB) are a type of microorganism with metabolic regulation and microenvironment optimization capabilities. They can regulate the local microenvironment by metabolizing metabolic waste in the tumor microenvironment to reduce the body's immune response to bacteria. PSB can inhibit cholesterol biosynthesis in tumor cells at its source by downregulating the activity of the "AKT-mTORC1-sterol regulatory element binding protein 1 (SREBP1)" signaling axis, thereby reducing the expression and activity of key cholesterol synthesis enzymes (such as Hmgcr, Hmgcs1, and Sqle) and preventing excessive intracellular cholesterol accumulation. It can also target hypoxic areas of tumors to some extent, but its tumor targeting is still not precise enough. Some bacteria may also be distributed in normal tissues, increasing the potential risk of side effects. Synergistic effects with Proteus mirabilis and gold nanoparticles are necessary to fully realize its adjuvant anti-tumor effects.

[0042] The present invention relates to an oral targeted bacterial symbiotic system for colorectal cancer—a biohybrid nanoparticle formulation—which utilizes photosynthetic bacteria, including Proteus mirabilis, to form symbiotic functional units by wrapping gold nanoparticles around them, and is further modified with an enteric coating material (EC) to construct EC-PM-PSB@GNP.

[0043] Example 1: Preparation of bio-hybrid nanoformulation EC-PM-PSB@GNP

[0044] 1. Preparation of GNP

[0045] Under magnetic stirring at 300 rpm, 0.5 mL of 1% chloroauric acid solution (v / v) was added to 50 mL of deionized water and heated to boiling. Then, 5-10 mL of 1% sodium citrate solution (v / v) was rapidly added dropwise, and the mixture was continuously stirred and heated until boiling for 10 minutes. The heating power was then turned off, and the mixture was stirred and cooled for another 15 minutes. The solution was filtered through a 0.22 μm aqueous filter membrane to remove agglomerated impurities, yielding a uniform gold nanoparticle (GNP) solution, which was stored at 4 °C protected from light for later use.

[0046] 2. Preparation of PSB@GNP

[0047] The photosynthetic bacterium strain Rhodopseudomonas palustris CGA009, which has metabolic regulatory functions, was selected and inoculated into a special culture medium for photosynthetic bacteria containing carbon and nitrogen sources such as sodium acetate and ammonium chloride. It was cultured under anaerobic conditions at 25-34℃ and 3000-4000 lx light intensity with shaking until the logarithmic growth phase (OD200). 600 =0.6-0.8); Take 10 mL of the above bacterial culture, centrifuge at 4000 g for 10 minutes, collect the photosynthetic bacteria cells, and gently wash three times with PBS buffer (pH 7.4) to remove culture medium residue; adjust the bacterial viable count concentration to 1×10⁻⁶. 9 CFU / mL. The bacterial suspension and gold nanoparticle solution were mixed at a 1:10 volume ratio and incubated at 25-34℃ for 90-120 minutes to allow the gold nanoparticles to bind to the bacterial surface via electrostatic interactions or surface adsorption. Finally, the mixture was centrifuged at 8000g for 15 minutes, the precipitate was collected, and washed three times with PBS buffer to remove unencapsulated free gold nanoparticles, yielding the PSB@GNP complex, which was stored at 4℃ for later use.

[0048] 3. Preparation of PM-PSB@GNP

[0049] Proteus mirabilis (ATCC 29906 strain) was inoculated into nutrient broth medium and cultured at 35-37℃ with shaking at 180 rpm until the logarithmic growth phase. The prepared PSB@GNP complex was mixed with the Proteus mirabilis bacterial suspension at a 2:1 volume ratio and co-cultured at 37℃ and 150 rpm for 4 hours. Subsequently, the symbiotic complex precipitate was collected by centrifugation at 6000g for 10 minutes, resuspended in PBS buffer (v / v) containing 5% mannitol, and the total viable count was adjusted to 5 × 10⁻⁶ using colony counting. 8 CFU / mL was used to obtain the PM-PSB@GNP core solution.

[0050] 4. Preparation of EC-PM-PSB@GNP

[0051] Dissolve 3g of Eudragit L100 polyacrylic acid resin, 1g of mannitol, and 2g of polyethylene glycol 4000 in 50mL of deionized water. Stir continuously at 300rpm until completely dissolved. Add 0.5g of triethyl citrate as a plasticizer and continue stirring until homogeneous and transparent coating solution is formed. Take 10mL of the PM-PSB@GNP core solution prepared above, add 2g of polyethylene glycol 4000, and place it in an ultrasonic instrument to ultrasonically disperse at 300W power and 20kHz frequency for 10 minutes to form a uniform suspension. Transfer the suspension to the feed tank of a spray dryer, set the inlet temperature to 60℃, the outlet temperature to 30℃, the atomization pressure to 0.15MPa, and the feed rate to 8mL / min. Use the prepared coating solution as the spray medium for spray coating. Collect the spray-dried microsphere formulation and place it in a vacuum drying oven. Vacuum dry at 40℃ for 4 hours to remove residual solvent and obtain the bio-hybrid nano-formulation EC-PM-PSB@GNP.

[0052] Comparative Example 1: Preparation of PSB@GNP Nanoformulation

[0053] The preparation method is the same as that used in Example 1 for PSB@GNP.

[0054] Comparative Example 2: Preparation of PM@GNP Nanoparticle Formulation

[0055] The preparation method of GNP is the same as in Example 1. The differences from Example 1 are the preparation of PM and PM@GNP, and the specific steps are as follows:

[0056] 1. Preparation of PM

[0057] Proteus mirabilis (ATCC 29906 strain) was inoculated into nutrient broth and cultured at 35-37℃ with shaking at 180 rpm until the logarithmic growth phase. 10-15 mL of the logarithmic growth phase bacterial suspension was collected by centrifugation at 8000g and 4℃ for 15 minutes. The cells were gently washed three times with pH 7.4 PBS buffer to remove residual culture medium. After washing, the suspension was resuspended in 0.9% NaCl solution, and the viability was verified by trypan blue staining to be >95%. The cells were resuspended in PBS buffer, and the viable cell count was adjusted to 2 × 10⁻⁶ using colony counting. 8 CFU / mL, for later use.

[0058] 2. Preparation of PM@GNP

[0059] Mix the adjusted concentration of PM bacterial culture with GNP solution at a volume ratio of 1:10, and incubate at 37°C and 150 rpm for 180 minutes with shaking. After incubation, centrifuge at 8000g for 15 minutes (4°C) to collect the precipitate, wash three times with PBS buffer at pH 7.4 to completely remove free GNPs not loaded by PM, and resuspend the precipitate with a small amount of PBS buffer to obtain PM@GNP nanoparticles. Store at 4°C for short-term use, or freeze after adding 5% mannitol (v / v) as a protectant.

[0060] Preparation of Comparative Example 3 PM-PSB@GNP Nanoformulation

[0061] Its preparation method is the same as that of PM-PSB@GNP in Example 1.

[0062] Experimental Example 1

[0063] Using Example 1 and Comparative Examples 1-3, as well as free GNP, as experimental materials, and PBS directly mixed with PM bacterial solution and PSB bacterial solution as control (the amount of PM bacterial solution and PSB bacterial solution added to the mixture is the same as in Example 1), the performance of each component nano-formulation was studied.

[0064] 1. Determination of particle size and potential

[0065] The particle size distribution and surface potential of GNP, PSB@GNP, PM@GNP, PM-PSB@GNP and EC-PM-PSB@GNP were detected by a dynamic light scattering particle size analyzer.

[0066] Depend on Figures 1-2 It can be seen that the average particle size of GNP is about 10 nm and the surface potential is about +40 mV; the average particle size of PSB@GNP is about 1.2 μm and the surface potential is about +15 mV; the average particle size of PM@GNP is about 2 μm and the surface potential is about +30 mV; the average particle size of PM-PSB@GNP is about 3.0 μm and the surface potential is about +5 mV; and the average particle size of EC-PM-PSB@GNP is about 200 μm and the surface potential is about 0 mV.

[0067] 2. Determination of encapsulation efficiency and loading rate of gold nanoparticles

[0068] High-performance liquid chromatography (HPLC) was used to determine the intracellular loading efficiency of PSB for GNPs and the actual GNP loading in EC-PM-PSB@GNP.

[0069] Depend on Figure 3 It can be seen that the intracellular encapsulation efficiency of PSB for GNP reaches its peak when the GNP concentration reaches 200 μg / mL. From... Figure 4It can be seen that when the GNP concentration reaches 400 μg / mL, the actual GNP loading in EC-PM-PSB@GNP reaches saturation, approximately 60 mg / g.

[0070] 3. Enteric coating performance determination

[0071] Using a drug dissolution apparatus, the dissolution rate of the formulation under different pH conditions was detected by simulating the gastrointestinal environment (pH=1.2 hydrochloric acid solution, pH=6.8 phosphate buffer).

[0072] Depend on Figure 5 It can be seen that in simulated gastric fluid (pH=1.2), the dissolution rate of the formulation is ≤5% within 2 hours, indicating that enteric coating can effectively resist gastric acid erosion and prevent internal bacteria and gold nanoparticles from being enzymatically degraded or destroyed in the stomach; in simulated intestinal fluid (pH=6.8), the dissolution rate is ≥80% within 30 minutes, achieving rapid release in the intestine and ensuring that the active ingredients can play an efficient role at the target site.

[0073] 4. Microbial activity assay

[0074] The colony counting method was used to detect the number of viable PSB and PM in the formulation (CFU / mL) and to evaluate the impact of the preparation process on the activity of the strain.

[0075] Depend on Figure 6 It can be seen that in all groups containing PSB, PSB activity remained at a high level, far exceeding 1×10⁻⁶. 8 The CFU / mL indicates that the preparation process caused minimal damage to the activity of PSB. In all groups containing PM, PM also maintained high activity. The activities of both strains in the combined formulation were close to the control group level, indicating that the preparation process caused minimal damage to the activity of the strains and that the process compatibility was good.

[0076] 5. Evaluation of cholesterol regulation effect

[0077] A tumor cell model was constructed using colorectal cancer cells (CT26). After treatment with the formulation, the amount of intracellular cholesterol synthesis was determined using a cholesterol detection kit, and the concentration of free cholesterol in the culture medium was detected by high performance liquid chromatography.

[0078] Depend on Figure 7 It is known that PSB reduces intracellular cholesterol accumulation by inhibiting the activity of key enzymes in cholesterol synthesis within tumor cells; PM directly degrades free cholesterol in the tumor microenvironment by secreting specific enzymes. The two work synergistically to achieve dual regulation of "intracellular inhibition + extracellular clearance". The total cholesterol content in cells of the EC-PM-PSB@GNP treatment group was reduced by ≥30% compared with the PBS control group, laying the microenvironmental foundation for reversing T cell exhaustion and activating anti-tumor immunity.

[0079] 6. Evaluation of in vitro antitumor activity

[0080] The CCK-8 assay was used to detect the inhibition rate of each formulation on the proliferation of CT26 cells.

[0081] Depend on Figure 8 It can be seen that the inhibitory effects of each formulation on the proliferation of CT26 cells showed a gradient change of "PBS group < free GNP group < PSB@GNP group ≈ PM@GNP group < PM-PSB@GNP group < EC-PM-PSB@GNP group"; EC-PM-PSB@GNP showed the best proliferation inhibitory effect, with a cell survival rate ≤20%, which reflects the synergistic advantage of the bio-hybrid nano-formulation prepared in this invention in inhibiting tumor cell proliferation.

[0082] 7. In vitro safety evaluation

[0083] The CCK-8 assay was used to detect the inhibition rate of each formulation on the proliferation of normal colorectal epithelial cells (NCM460).

[0084] Depend on Figure 9 It is known that EC-PM-PSB@GNP and its intermediate formulations have no significant killing effect on NCM460 cells, and the cell survival rate is close to 100%. This proves that the bio-hybrid nano-formulation prepared in this invention has a specific response to the tumor microenvironment, exerts a killing effect only on tumor cells, does not damage normal cells, and has good safety.

[0085] 8. Evaluation of in vivo antitumor efficacy

[0086] CT26 was inoculated orally into the cecum of mice. CT26-Luc tumor-bearing mice were randomly divided into four groups (PBS group, free GNP group, PSB@GNP group, PM@GNP group, PM-PSB@GNP group, EC-PM-PSB@GNP group), and administered orally on days 0, 5, and 10. The free GNP group received 12 μg GNP per mouse; all nano-formulation groups received 200 μL orally per mouse, with a single dose containing 1 × 10⁻⁶ live bacteria. 8 CFU / each (5×10) 8 CFU / mL × 0.2mL); PBS group was administered 200μL / mouse orally with pure PBS solution; all groups were administered once on the day of administration; bioluminescence images were taken on day 15, and mice were euthanized by cervical dislocation, tumors were removed, and tumor weight was measured.

[0087] Depend on Figures 10-11 It can be seen that the tumor weight in the EC-PM-PSB@GNP group was ≤0.2g, which was ≥60% lower than that in the PBS control group, and the tumor inhibition effect was significantly better than that in other groups.

[0088] 9. Biosafety assessment

[0089] After administering the preparation orally to normal mice, observe their body weight, diet, and activity status for 14 consecutive days and record changes in body weight.

[0090] Depend on Figure 12 It can be seen that the weight of mice in all groups showed a steady growth trend, with no significant difference in weight change compared with the PBS control group. No sudden drop or abnormal fluctuation in weight was observed. The mice had normal diet and activity levels, and no obvious gastrointestinal irritation or systemic discomfort, proving that all preparations had good biocompatibility.

[0091] 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 a cholesterol-regulated bio-hybrid nanoparticle formulation, characterized in that, The process includes the steps of preparing a gold nanoparticle solution, mixing the gold nanoparticle solution with a photosynthetic bacteria culture to obtain a PSB@GNP complex, mixing the PSB@GNP complex with a Proteus mirabilis culture to obtain a PM-PSB@GNP core solution, and modifying the PM-PSB@GNP core solution with a coating solution and then spray-drying it to obtain the bio-hybrid nanoformulation EC-PM-PSB@GNP. The preparation method of the bio-hybrid nano-formulation EC-PM-PSB@GNP is as follows: prepare a coating solution, take 10-15mL of PM-PSB@GNP core solution and add 2-5g of polyethylene glycol 4000, disperse ultrasonically to form a suspension, and spray dry the suspension with the coating solution as the spray medium to obtain the bio-hybrid nano-formulation EC-PM-PSB@GNP; The PSB@GNP complex is prepared by mixing photosynthetic bacteria solution and gold nanoparticle solution at a volume ratio of 1:1 to 1:10, and allowing it to stand at 25-34℃ for 90-120 min to obtain the PSB@GNP complex. The preparation method of the photosynthetic bacteria culture is as follows: Culture the photosynthetic bacteria to the logarithmic growth phase, take 10-15 mL of the culture, centrifuge to collect the bacterial cells, wash to remove the culture medium, and adjust the bacterial concentration to 1×10⁻⁶. 8 -1×10 9 The photosynthetic bacteria culture can be obtained at a concentration of CFU / mL. The preparation method of the PM-PSB@GNP core solution is as follows: PSB@GNP complex and Proteus mirabilis bacterial solution are mixed at a volume ratio of 1:1-3:1, co-cultured at 35-37℃ and 100-150rpm for 4-6h, centrifuged to collect the precipitate, and resuspended to obtain the PM-PSB@GNP core solution. The photosynthetic bacteria is the photosynthetic bacterium Rhodopseudomonas palustris CGA009 strain; The *Proteus mirabilis* strain is *Proteus mirabilis* ATCC 29906.

2. The preparation method according to claim 1, characterized in that, The gold nanoparticle solution is prepared by adding 0.5-1.5 mL of 1-3% chloroauric acid solution to water and heating, then adding 5-10 mL of 1-3% sodium citrate solution dropwise while continuously heating and stirring, and finally filtering after cooling to obtain the gold nanoparticle solution.

3. The preparation method according to claim 1, characterized in that, The viable bacteria concentration in the PM-PSB@GNP core solution was 4 × 10⁻⁶. 8 -5×10 8 CFU / mL.

4. A bio-hybrid nanoformulation EC-PM-PSB@GNP prepared by the preparation method according to any one of claims 1-3.

5. The use of the bio-hybrid nanoformulation EC-PM-PSB@GNP according to claim 4 in the preparation of a drug for treating colorectal cancer.

6. A drug for treating colorectal cancer, characterized in that, This includes the biohybrid nanoformulation EC-PM-PSB@GNP as described in claim 4.

Images