Intestinal organ and application thereof in intestinal injury drug screening

Abstract

The invention relates to the field of drug analysis, in particular to an intestinal organ and application thereof in screening of intestinal injury drugs. The invention provides an application of nano-particles based on a lactobacillus rhamnosus source in preparation of a drug analysis system, and is characterized in that the drug analysis system comprises the nano-particles based on the lactobacillus rhamnosus source and constructed intestinal organs; the intestinal organ comprises a normal intestinal organ; the passage generations of the intestinal organs are from P6 generation to P15 generation; the medicine comprises a platinum medicine. The drug analysis system provided by the invention is high in controllability and accuracy, can better simulate the dynamic environment of the intestinal organ of the patient, and provides help for objectively guiding the personalized medication scheme of the patient.

Description

[0001] This application claims priority to Chinese invention patent application 202411194770.4 filed on August 28, 2024, “A drug analysis system based on nanoparticles derived from Lactobacillus rhamnosus”, which is incorporated by reference in its entirety. Technical Field

[0002] The present invention relates to the field of drug analysis, and in particular to an intestinal organoid and its application in intestinal injury drug screening. Background Art

[0003] Organoid 3D culture is an emerging in vitro research system for studying the growth, differentiation, and organ formation of adult stem cells. The 3D culture of intestinal organoids involves implanting isolated intestinal crypts or stem cells into a matrix gel containing a variety of growth factors, and generating micro hollow spheres with intestinal epithelial-like structures under the 3D support of the matrix. These spheres are called intestinal organoids. In addition, intestinal tumor organoids can also be obtained using tumor stem cells or tumor precursor cells. These intestinal organoids are widely used in the study of various intestinal diseases such as inflammatory bowel disease, intestinal injury regeneration, and colorectal cancer.

[0004] Currently, many cells used as cell lines are immortalized cells, that is, non-aging cell lines that can proliferate indefinitely in vitro under external stimuli such as physical and chemical stimulation, gene mutation, and viral infection. Therefore, although two-dimensionally cultured cell lines have the advantages of high operability, short culture cycle, and high throughput, when they are applied to drug screening, the sensitivity of a specific cell line to a certain drug is difficult to represent other cell lines, let alone human tissues. In addition, two-dimensionally cultured cell lines cannot simulate the three-dimensional growth environment of normal tissues and tumor tissues, so it is difficult to simulate the infiltration process of drugs. This also shows that the reference value of drug screening results produced by two-dimensionally cultured cell lines is low.

[0005] Compared with traditional two-dimensional cell culture, organoids are derived from the body's own tissues or stem cells. They are three-dimensional (3D) structural models with original tissues and organs formed through in vitro 3D culture. They are highly similar to the source tissues in terms of tissue structure, cell type and function, that is, organoids can show cell composition and physiological functions close to the physiological state. Therefore, organoids show unique advantages in drug screening and can more accurately evaluate the effects of drugs in the body and the toxic and side effects of drugs, thereby improving the efficiency of drug development and the success rate of drug clinical trials. In addition, organoid technology can realize the construction of separate libraries for patients' cancer and adjacent or normal tissues to further carry out more personalized drug screening for the patient.

[0006] Although organoids have shown unique advantages in drug screening, their development still faces huge challenges, such as the uncontrollable problems of organoid stability and accuracy. Summary of the invention

[0007] In a first aspect, the present invention provides an application of nanoparticles derived from Lactobacillus rhamnosus in the preparation of a drug analysis system, characterized in that the drug analysis system comprises nanoparticles derived from Lactobacillus rhamnosus and constructed intestinal organoids; In some embodiments, the intestinal organoid comprises a normal intestinal organoid.

[0008] In some embodiments, the intestinal organoids are passaged from P6 to P15.

[0009] In some embodiments, the drug analysis system further includes a drug.

[0010] In some embodiments, the drug comprises a platinum drug.

[0011] In some embodiments, the platinum drug comprises cisplatin or oxaliplatin.

[0012] In some embodiments, the concentration of the platinum drug is 0.01 – 1000 μM.

[0013] In some embodiments, the concentration of the platinum drug comprises 0.01 – 1 μM.

[0014] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles comprises 5 - 20 μg / mL.

[0015] In some embodiments, the constructed intestinal organoid is in the form of an organoid cell suspension.

[0016] In some embodiments, the cell density of the organoid cell suspension comprises 1 - 5×10 4 intestinal organoid cells / mL.

[0017] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids before or simultaneously with the platinum drug.

[0018] In some embodiments, the average particle size of the Lactobacillus rhamnosus-derived nanoparticles is 50-120 nm.

[0019] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoid is 1 – 5 μg / 1000 intestinal organoid cells.

[0020] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoids is 1 μg / 1000 intestinal organoid cells.

[0021] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles is 10 μg / mL.

[0022] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are prepared by the following method: S101 When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10-15 minutes to obtain supernatant A; S102 centrifuging the supernatant A at 5000 g for 20-30 minutes to obtain supernatant B; S103 centrifuging the supernatant B at 10000 g for 30-40 minutes to obtain supernatant C; S104 filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000-150,000 g for 70 minutes to obtain a precipitate D; S105 The precipitate D is resuspended and filtered using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus-derived nanoparticles.

[0023] In some embodiments, the organoid cell suspension is obtained by digesting the constructed intestinal organoid.

[0024] In some embodiments, the normal intestinal organoids are derived from intestinal stem cells or crypts in healthy intestinal tissue.

[0025] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for at least 24 hours.

[0026] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids 6-8 hours before the platinum drug.

[0027] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for 72-120 hours.

[0028] In a second aspect, the present invention provides a drug analysis system based on nanoparticles derived from Lactobacillus rhamnosus, characterized in that the drug analysis system comprises nanoparticles derived from Lactobacillus rhamnosus and constructed intestinal organoids; In some embodiments, the intestinal organoid comprises a normal intestinal organoid.

[0029] In some embodiments, the intestinal organoids are passaged from P6 to P15.

[0030] In some embodiments, the drug analysis system is used for drug screening of platinum drugs.

[0031] In some embodiments, the drug analysis system further comprises a platinum drug.

[0032] In some embodiments, the platinum drug comprises cisplatin or oxaliplatin.

[0033] In some embodiments, the concentration of the platinum drug is 0.01 – 1000 μM.

[0034] In some embodiments, the concentration of the platinum drug comprises 0.01 – 1 μM.

[0035] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles comprises 5 - 20 μg / mL.

[0036] In some embodiments, the constructed intestinal organoid is in the form of an organoid cell suspension.

[0037] In some embodiments, the cell density of the organoid cell suspension comprises 1 - 5×10 4 intestinal organoid cells / mL.

[0038] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids before or simultaneously with the platinum drug.

[0039] In some embodiments, the average particle size of the Lactobacillus rhamnosus-derived nanoparticles is 50-120 nm.

[0040] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoid is 1 – 5 μg / 1000 intestinal organoid cells.

[0041] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoids is 1 μg / 1000 intestinal organoid cells.

[0042] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles is 10 μg / mL.

[0043] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are prepared by the following method: S101 When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10-15 minutes to obtain supernatant A; S102 centrifuging the supernatant A at 5000 g for 20-30 minutes to obtain supernatant B; S103 centrifuging the supernatant B at 10000 g for 30-40 minutes to obtain supernatant C; S104 filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000-150,000 g for 70 minutes to obtain a precipitate D; S105 The precipitate D is resuspended and filtered using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus-derived nanoparticles.

[0044] In some embodiments, the organoid cell suspension is obtained by digesting the constructed intestinal organoid.

[0045] In some embodiments, the normal intestinal organoids are derived from intestinal stem cells or crypts in healthy intestinal tissue.

[0046] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for at least 24 hours.

[0047] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids 6-8 hours before the platinum drug.

[0048] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for 72-120 hours.

[0049] In a third aspect, the present invention provides a method for drug screening based on intestinal organoids, characterized in that it comprises the following steps: constructing intestinal organoids, wherein the intestinal organoids include normal intestinal organoids; obtaining an organoid cell suspension, wherein the organoid cell suspension is obtained by digesting the constructed intestinal organoid; adding Lactobacillus rhamnosus-derived nanoparticles and a drug to the organoid cell suspension, wherein the Lactobacillus rhamnosus-derived nanoparticles are added before or simultaneously with the drug; evaluating the effect of the drug on the viability of the intestinal organoid cells; The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 5-20 μg / mL, and the cell density of the organoid cell suspension is 1-5×10 4 intestinal organoid cells / mL; the drugs include platinum drugs.

[0050] In some embodiments, the intestinal organoids are passaged from P6 to P15.

[0051] In some embodiments, the drug comprises a platinum drug.

[0052] In some embodiments, the platinum drug comprises cisplatin or oxaliplatin.

[0053] In some embodiments, the concentration of the platinum drug is 0.01 – 1000 μM.

[0054] In some embodiments, the concentration of the platinum drug comprises 0.01 – 1 μM.

[0055] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles comprises 5 - 20 μg / mL.

[0056] In some embodiments, the cell density of the organoid cell suspension comprises 1 - 5×10 4 intestinal organoid cells / mL.

[0057] In some embodiments, the average particle size of the Lactobacillus rhamnosus-derived nanoparticles is 50-120 nm.

[0058] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoid is 1 – 5 μg / 1000 intestinal organoid cells.

[0059] In some embodiments, the ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoids is 1 μg / 1000 intestinal organoid cells.

[0060] In some embodiments, the concentration of the Lactobacillus rhamnosus-derived nanoparticles is 10 μg / mL.

[0061] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are prepared by the following method: S101 When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10-15 minutes to obtain supernatant A; S102 centrifuging the supernatant A at 5000 g for 20-30 minutes to obtain supernatant B; S103 centrifuging the supernatant B at 10000 g for 30-40 minutes to obtain supernatant C; S104 filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000-150,000 g for 70 minutes to obtain a precipitate D; S105 The precipitate D is resuspended and filtered using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus-derived nanoparticles.

[0062] In some embodiments, the normal intestinal organoids are derived from intestinal stem cells or crypts in healthy intestinal tissue.

[0063] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for at least 24 hours.

[0064] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids 6-8 hours before the platinum drug.

[0065] In some embodiments, the Lactobacillus rhamnosus-derived nanoparticles, the platinum drug, and the constructed intestinal organoids are co-cultured for 72-120 hours.

[0066] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: Although the prior art has made certain progress in using organoids for drug screening, it still faces problems such as uncontrollable stability and accuracy of organoids and difficulty in establishing a microenvironment. The experiments of the present invention show that the cause of the above problems may be that organoids are easily limited by the passage number and have an adverse effect on the results of drug screening. Although the selection of low-passage organoids (for example, primary to P5 generations) can overcome this problem to a certain extent, this inevitably causes a great waste of organoid resources and increases the cost of constructing organoids, and is seriously deviated from the actual use of organoids for high-throughput drug screening.

[0067] When using organoids from the same individual for high-throughput screening of therapeutic drugs, intestinal stem cells or crypts from a specific patient can be collected to construct normal intestinal organoids for analysis and testing of whether the patient is suitable for platinum drugs and the recommended drug concentration. When using organoids for drug screening, it is often high-throughput, for example, different drugs at different concentrations need to be screened at the same time. In this scenario, a large number of organoid cells are usually required, and when amplified to the target amount, the generation of the organoid may be higher.

[0068] Specifically, the present invention has found that as the passage number of normal intestinal organoids increases, normal intestinal organoids will show toxicity to a certain type of drug (such as platinum drugs) that low-passage normal intestinal organoids do not show, so in high-throughput drug screening based on high-passage normal intestinal organoids from the same source, the drug is likely to be screened out. Specifically, if normal intestinal organoids have already shown a certain toxicity to a lower concentration of added platinum drugs, when compared with the toxicity caused by other drugs (such as non-platinum drugs), the false positive results brought about by such high-passage organoids may exclude the therapeutic drugs (such as cisplatin or oxaliplatin) that are potentially suitable for the patient, which is very unfavorable for the realization of personalized drug administration.

[0069] In addition, if the toxicity of the same drug is evaluated using normal intestinal organoids from different sources, combined with the reality that organoid resources are limited, it may not be possible to use organoids of the same generation and lower generations for drug screening. It is very obvious that high-passage organoids may show amplified toxicity to low concentrations (e.g., 0.01-1 μM) of a certain class of drugs (e.g., platinum drugs). If a large proportion of high-passage organoids are used, it is likely that the conclusion that the drug is toxic to most organoids will be drawn, which may exclude platinum drugs with therapeutic prospects.

[0070] The stability and accuracy of the organoid-based drug screening system are actually very important. For the above-mentioned application scenarios, the drug analysis system provided by the present invention (such as the addition of nanoparticles derived from Lactobacillus rhamnosus at a specific concentration (e.g., 5-20 μg / mL)) can just restore the normal intestinal organoids that are affected by the passage generation and cause the toxicity of platinum drugs to be amplified to the level of normal intestinal organoids of low passage generations (e.g., primary to P5 generations), thereby more realistically restoring the response of intestinal organoids to drugs, so that the actual efficacy of the platinum drugs to be analyzed in the patient's body can be more accurately reflected, and the intestinal organoids with passage generations of P6 and above can be suitable for drug screening of platinum drugs. However, excessively high concentrations of nanoparticles derived from Lactobacillus rhamnosus will bring about the problem of false negatives to the intestinal organoids in the drug analysis system (i.e., not the level of intestinal organoids of low passage generations), which makes the drug toxicity presented by the drug to be analyzed also distorted, and thus cannot accurately reflect the actual efficacy of the drug to be analyzed in the patient's body.

[0071] That is to say, the drug analysis system provided by the present invention can not only analyze and screen different types (for example, choosing between cisplatin and oxaliplatin) and different drug concentrations for a certain patient, but can also cooperate with the conventional drug screening system to screen out therapeutic drugs that are more suitable for the patient. Since the drug analysis system provided by the present invention avoids the problem of false positives for platinum drugs, the analysis and screening results of platinum drugs obtained based on it are more accurate, thereby screening out therapeutic drugs that are more suitable for the patient (for example, selecting drugs with lower concentrations based on similar organoid viability, and selecting drugs with higher organoid viability based on similar concentrations).

[0072] In the drug analysis system provided by the present invention, by controlling the ratio of nanoparticles derived from Lactobacillus rhamnosus to intestinal organoid cells, the nanoparticles derived from Lactobacillus rhamnosus will not promote the proliferation of intestinal organoid cells, and will not bring additional cytotoxicity to the intestinal organoids, nor will it excessively affect the cytotoxicity of the drugs to be analyzed in the drug analysis system provided by the present invention, thereby ensuring the controllability and accuracy of the drug analysis system. Therefore, the drug analysis system provided by the present invention can be used to accurately evaluate the intestinal toxicity of the drugs to be analyzed based on the cell viability of the intestinal organoid cells. In addition, after testing, the above ratio is suitable for testing of drugs of different concentrations (e.g., 0.01-1000 μM) and intestinal organoids of different passages, meeting the needs of actual drug screening, and will not cause false negatives and false positives.

[0073] In summary, compared with conventional drug screening systems and methods, the controllability and accuracy of the drug analysis system provided by the present invention are significantly improved, and the application prospects of intestinal organoids in drug evaluation are expanded. It can better simulate the dynamic environment of the patient's intestinal organs, and provide help for early prediction of the physiological response of drugs in the in vivo environment and objective guidance of patients' personalized medication plans.

[0074] As used herein, "nanoparticles" refers to material particles with a diameter between 1 and 150 nanometers. As used herein, "Lactobacillus rhamnosus-derived nanoparticles" refers to nanoscale extracellular lipid bivesicles derived from the culture supernatant of Lactobacillus rhamnosus. In the present invention, Lactobacillus rhamnosus-derived nanoparticles and Lactobacillus rhamnosus exosomes (abbreviated as LGG exosomes or exosomes) have the same meaning unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0076] Figure 1 A representative transmission electron microscope image of nanoparticles derived from Lactobacillus rhamnosus obtained in Example 1 of the present invention; Figure 2 Zeta potential diagram of nanoparticles derived from Lactobacillus rhamnosus obtained in Example 1 of the present invention; Figure 3 This is a bright field image of a normal intestinal organoid constructed in Example 3 of the present invention; Figure 4 This is a graph showing the results of organoid cell viability for evaluating drug intestinal toxicity in Example 4 of the present invention; Figure 5 A heat map showing the response of normal intestinal organoids at different passages to 1 μM doxorubicin; Figure 6 This is a graph showing the organoid cell viability results of normal intestinal organoids of different generations in response to intestinal toxicity of different drugs. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0078] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0079] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0080] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0081] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0082] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0083] Example 1: Extraction and identification of nanoparticles derived from Lactobacillus rhamnosus The Lactobacillus rhamnosus (LGG) strain used in this example is the LGG strain with the strain number BNCC136673 of Beina Biotechnology.

[0084] S101 LGG strain was cultured in MRS broth. When the strain density of Lactobacillus rhamnosus culture suspension reached 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10-15 minutes to obtain supernatant A; S102 centrifuging the supernatant A at 5000 g for 20-30 minutes to obtain supernatant B; S103 centrifuging the supernatant B at 10000 g for 30-40 minutes to obtain supernatant C; S104 filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000-150,000 g for 70 minutes to obtain a precipitate D; S105 The precipitate D is resuspended and filtered using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus-derived nanoparticles.

[0085] The obtained Lactobacillus rhamnosus-derived nanoparticles were stained with 1% phosphotungstic acid for 1-2 min at room temperature and observed using a JEM-1400FLASH transmission electron microscope. Figure 1 The representative transmission electron microscope image of the obtained Lactobacillus rhamnosus-derived nanoparticles. The particle size and potential of the Lactobacillus rhamnosus-derived nanoparticles were measured in a particle size analyzer. The average particle size of the Lactobacillus rhamnosus-derived nanoparticles was 50-120 nm, and each milliliter of the nanoparticle solution contained 3.02×10 11 The zeta potential of nanoparticles is -16.87±2.68 mV. Figure 2 shown.

[0086] Example 2: Determination of protein concentration of nanoparticles derived from Lactobacillus rhamnosus The obtained Lactobacillus rhamnosus-derived nanoparticles were quantified using a BCA protein assay kit, and the specific steps are as follows: (1) Dilute the protein standard with sterile PBS buffer to a final concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively; take 20 μL of each concentration of protein standard and add it to the standard well of a 96-well plate to prepare a standard curve; (2) Dissolving the Lactobacillus rhamnosus-derived nanoparticles prepared in Example 1 in a sterile PBS buffer to obtain a nanoparticle solution; taking 20 μL of the nanoparticle solution and adding it to the sample wells of a 96-well plate; (3) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 min. (4) Determine the A562 value or the absorbance at a wavelength of 540-595 nm using an ELISA reader; (5) Calculate the protein concentration in the sample based on the standard curve.

[0087] The protein concentration of the nanoparticle solution obtained by the present invention was finally determined to be 0.9-1.1 μg / μL.

[0088] Example 3: Construction of human normal intestinal organoids Healthy intestinal mucosal tissue is obtained from colonoscopy biopsy or surgery. The tissue is washed with PBS buffer to remove impurities and blood, and then the tissue is cut into small pieces of 1 mm³. These small pieces are digested in tissue digestion solution to separate crypt stromal cells. After digestion is complete, the digestion process is terminated with a basal medium containing serum. The cell pellet is collected by low-speed centrifugation, the supernatant is discarded, and the cell pellet is resuspended with matrix gel to form a mixture. The mixture is inoculated into a 48-well culture plate at a volume of 25 μL / well and incubated in a 37°C incubator for 30 minutes to solidify the matrix gel. After that, preheated organoid culture medium is added for primary culture. As the organoids grow to a certain size, passaging operations are performed, which include separating the organoids from the matrix gel, enzymatically splitting them, and culturing them again to expand the organoids to the number and size required for the experiment. The constructed primary cultured normal intestinal organoids are as follows Figure 3 shown.

[0089] Example 4: Drug analysis system based on nanoparticles derived from Lactobacillus rhamnosus First, normal intestinal organoids of primary and various passages (P0-P15) were collected and treated with cell digestion solution to disperse them into single cells or uniform cell clusters. Subsequently, the digested cells were counted to ensure that the number of cells in each experimental group was consistent. The cells were divided into the desired density and number (1×10 4 Cells / mL, 1000 cells per well) were distributed into different culture plates, and the experimental group was treated with 1 μM doxorubicin, while the control group was not treated with drugs. Five days after treatment, the viability of the organoids in the control and experimental groups was detected by ATP method, and the survival rate of the organoids in the experimental group after drug treatment was calculated with the control group as 100% viability value.

[0090] Figure 5 This is a heat map of the drug response of four normal organoids from different sources to 1 μM doxorubicin at each generation. The results in the figure show that the four normal organoids in the primary generation and the first five generations did not show obvious toxic response to 1 μM doxorubicin, but starting from the 6th generation (P6), all four normal organoids began to show toxic response to 1 μM doxorubicin.

[0091] Organoids have attracted much attention because of their ability to simulate the complex structure and function of in vivo tissues. They can usually maintain a high biological similarity in primary culture and early generations, which is crucial for drug screening and toxicity assessment. The above results of the present invention show that the response of organoids to drugs is not static. As the generations increase, the sensitivity of organoids to drugs may change, that is, the stability and accuracy of organoids decrease. However, if only primary or early generations (such as P1-P5) of organoids are used for drug toxicity and effectiveness assessment, this will actually shorten the use cycle of organoids for drug evaluation, be unfavorable for high-throughput screening of organoids for drugs (which usually requires a large number of organoids, and when expanded to the target amount, the generation of organoids may be higher), and increase the dependence on the source of organoids, thereby limiting the application prospects of organoids.

[0092] Next, according to the drug treatment information and Lactobacillus rhamnosus-derived nanoparticle concentration information in Table 1, the intestinal toxicity of the drug was evaluated using the P8 generation organoids. The specific method is as follows: First, normal intestinal organoids at passage P8 were collected and treated with cell digestion solution to disperse them into single cells or uniform cell clusters. Subsequently, the digested cells were counted to ensure that the number of cells in each experimental group was consistent. According to the experimental protocol in Table 1, cells were plated at the required density and number (1×10 4 cells / mL, 1000 cells per well) were distributed into different culture plates.

[0093] Next, for the experimental group that needed to use Lactobacillus rhamnosus-derived nanoparticles, the Lactobacillus rhamnosus-derived nanoparticles were prepared into a solution of a specific concentration and added to the culture plate to ensure that the Lactobacillus rhamnosus-derived nanoparticles were in full contact with the normal intestinal organoid cells. For the control group, an equal volume of culture medium without Lactobacillus rhamnosus-derived nanoparticles was added to simulate untreated conditions.

[0094] Intestinal toxic drug treatment: After 6 to 8 hours of pretreatment with Lactobacillus rhamnosus-derived nanoparticles, specific drugs were introduced into the corresponding normal intestinal organoids according to the experimental scheme in Table 1 (the concentrations of drugs and Lactobacillus rhamnosus-derived nanoparticles in Table 1 are the final concentrations in the drug screening system after their addition). For the control group without drug treatment, an equal volume of culture medium was added accordingly to maintain the consistency of the culture environment. All experimental groups continued to be cultured in the incubator for 72 hours to fully observe and record the effects of drugs and Lactobacillus rhamnosus-derived nanoparticles on the viability and morphology of normal intestinal organoid cells at the P8 generation.

[0095] Cell viability assay: After 72 hours of culture, the intestinal organoid cell viability of all experimental groups was quantitatively analyzed by ATP colorimetry. In the experiment, the ATP level of the untreated blank control group was set as a 100% viability benchmark. Subsequently, the cell viability of each experimental group relative to the blank control group was calculated and compared to evaluate the effects of drugs and Lactobacillus rhamnosus-derived nanoparticles on the viability of normal intestinal organoid cells.

[0096] Table 1 The results are as follows Figure 4 As shown in the low-dose control group of doxorubicin, 1 μM doxorubicin produced a significant inhibitory effect on normal intestinal organoids at P8. However, it is reported that doxorubicin is usually used at ≥20 mg / m 2 In other words, 1 μM doxorubicin (equivalent to 2.5 mg / m 2 , see: Rodrigues D, Coyle L, Füzi B, et al. Unravelling Mechanisms of Doxorubicin-Induced Toxicity in 3D Human Intestinal Organoids. Int J Mol Sci. 2022;23(3):1286.) In fact, the intestinal toxicity caused to the human body is negligible, which is consistent with the experimental results of this embodiment. In other words, the conventional drug screening system using normal organoids of passage number P6 and above will amplify the toxicity of low-dose drugs to organoids, making their sensitivity to drugs significantly deviate from the intestinal organoids of low passage number. Therefore, the conventional organoid-based drug screening system is limited by the passage number.

[0097] Figure 4 The results also showed that no intestinal drug toxicity was identified in the low-dose doxorubicin experimental group, indicating that the addition of 10 μg / mL of nanoparticles derived from Lactobacillus rhamnosus restored the drug sensitivity of normal organoids at passage P8 to that of normal intestinal organoids at low passages, which can more realistically restore the response of normal intestinal organoids to drugs and avoid the amplification of drug toxicity and false positive identification of drug toxicity assessment.

[0098] Figure 4The results also showed that the survival rate of the high-dose doxorubicin experimental group was only slightly higher than that of the high-dose doxorubicin control group, and the addition of 10 μg / mL of Lactobacillus rhamnosus-derived nanoparticles did not have a significant effect on the toxic response of the drug (i.e., the addition of 10 μg / mL of Lactobacillus rhamnosus-derived nanoparticles was only reflected in the restoration of the sensitivity of high-passage normal organoids to the drug, rather than providing a therapeutic effect), which indicates that the addition of Lactobacillus rhamnosus-derived nanoparticles did not reduce the accuracy of the drug evaluation system of the present invention, and would not cause false negative identification of drug toxicity assessment.

[0099] Organoids are cells derived from stem cells or organ tissues. After three-dimensional culture in vitro, they can differentiate and self-organize into cell clusters with some specific functions and structures of the corresponding organs in the human body. They can well simulate the development process and physiological and pathological states of organ tissues.

[0100] Example 5 First, normal intestinal organoids of primary and P8 passages were collected and treated with cell digestion solution to disperse them into single cells or uniform cell clusters. Subsequently, the digested cells were counted to ensure that the number of cells in each experimental group was consistent. The cells were divided into the desired density and number (1×10 4 cells / mL, 1000 cells per well) were distributed into different culture plates.

[0101] Next, for the experimental group that needed to use Lactobacillus rhamnosus-derived nanoparticles, the Lactobacillus rhamnosus-derived nanoparticles were prepared into a solution of a specific concentration and added to the culture plate to ensure that the Lactobacillus rhamnosus-derived nanoparticles were in full contact with the normal intestinal organoid cells. For the control group, an equal volume of culture medium without Lactobacillus rhamnosus-derived nanoparticles was added to simulate untreated conditions.

[0102] Treatment of enterotoxic drugs: After 6 to 8 hours of pretreatment with Lactobacillus rhamnosus-derived nanoparticles, Figure 6 The experimental scheme ( Figure 6 The concentrations of the drugs in the test tubes are all the final concentrations in the drug screening system after they are added), and specific drugs are introduced into the corresponding normal intestinal organoids. The drugs used in this embodiment include cisplatin (0.1 μM), oxaliplatin (0.2 μM), trastuzumab (0.1 μM), lenvatinib (0.5 μM), bevacizumab (1 μM), and imatinib (0.5 μM). For the control group without drug treatment, an equal volume of culture medium was added accordingly to maintain the consistency of the culture environment. All experimental groups continued to be cultured in the incubator for 72 hours in order to fully observe and record the effects of drugs and Lactobacillus rhamnosus-derived nanoparticles on the viability and morphology of normal intestinal organoid cells at the P8 generation.

[0103] Cell viability assay: After 72 hours of culture, the intestinal organoid cell viability of all experimental groups was quantitatively analyzed by ATP colorimetry. In the experiment, the ATP level of the untreated blank control group was set as a 100% viability benchmark. Subsequently, the cell viability of each experimental group relative to the blank control group was calculated and compared to evaluate the effects of drugs and Lactobacillus rhamnosus-derived nanoparticles on the viability of normal intestinal organoid cells.

[0104] Figure 6 The middle horizontal axis shows the tested drugs and their concentrations. Each drug is a group, and the group includes the organoid survival rate test results of three groups: primary, P8, and P8+nanoparticles. The results of cisplatin and oxaliplatin showed that the primary organoids did not show obvious toxicity to platinum drugs, while the P8-generation organoids showed obvious toxicity to platinum drugs. After adding nanoparticles derived from Lactobacillus rhamnosus at a dose of 10μg / mL, the P8-generation organoids did not show obvious toxicity to platinum drugs, that is, the nanoparticles derived from Lactobacillus rhamnosus restored the sensitivity of the organoids to low concentrations of platinum drugs. For trastuzumab, lenvatinib, bevacizumab, and imatinib, the addition of nanoparticles derived from Lactobacillus rhamnosus did not restore the sensitivity of the P8-generation organoids to these four drugs to the primary level. The above results show that the drug evaluation system of the present invention is suitable for the screening of platinum drugs.

[0105] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. An application of nanoparticles derived from Lactobacillus rhamnosus in the preparation of a drug analysis system, characterized in that: The drug analysis system includes nanoparticles derived from Lactobacillus rhamnosus and constructed intestinal organoids; the intestinal organoids include normal intestinal organoids; the intestinal organoids are passaged from P6 to P15; and the drugs include platinum drugs; The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 5-20 μg / mL, the constructed intestinal organoids are in the form of organoid cell suspensions, and the cell density of the organoid cell suspensions is 1-5×10 4 intestinal organoid cells / mL; the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids before or simultaneously with the drug.

2. The use according to claim 1, characterized in that The ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoids is 1 – 5 μg / 1000 intestinal organoid cells.

3. The use according to claim 1, characterized in that The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 10 μg / mL.

4. The use according to claim 1, characterized in that The concentration of the platinum drug includes 0.01 – 1000 μM.

5. A drug analysis system based on nanoparticles derived from Lactobacillus rhamnosus, characterized in that: The drug analysis system comprises nanoparticles derived from Lactobacillus rhamnosus and constructed intestinal organoids, wherein the intestinal organoids comprise normal intestinal organoids; the intestinal organoids are passaged from P6 to P15; and the drug analysis system is used for drug screening of platinum drugs; The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 5-20 μg / mL, the constructed intestinal organoids are in the form of organoid cell suspensions, and the cell density of the organoid cell suspensions is 1-5×10 4 intestinal organoid cells / mL; the Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids before or simultaneously with the drug.

6. The system according to claim 5, characterized in that The ratio of the Lactobacillus rhamnosus-derived nanoparticles to the intestinal organoids is 1 – 5 μg / 1000 intestinal organoid cells.

7. The system according to claim 5, characterized in that The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 10 μg / mL.

8. A method for drug screening based on intestinal organoids, characterized in that: The following steps are involved: Constructing intestinal organoids, wherein the intestinal organoids include normal intestinal organoids, and the passage number of the intestinal organoids is P6 to P15; obtaining an organoid cell suspension, wherein the organoid cell suspension is obtained by digesting the constructed intestinal organoid; adding Lactobacillus rhamnosus-derived nanoparticles and a drug to the organoid cell suspension, wherein the Lactobacillus rhamnosus-derived nanoparticles are added before or simultaneously with the drug; evaluating the effect of the drug on the viability of the intestinal organoid cells; The concentration of the Lactobacillus rhamnosus-derived nanoparticles is 5-20 μg / mL, and the cell density of the organoid cell suspension is 1-5×10 4 intestinal organoid cells / mL; the drugs include platinum drugs.

9. The method according to claim 8, characterized in that The Lactobacillus rhamnosus-derived nanoparticles are added to the constructed intestinal organoids 6-8 hours before the platinum drugs.

10. The method according to claim 8, characterized in that The Lactobacillus rhamnosus-derived nanoparticles, the drug and the organoid cell suspension are co-cultured for at least 24 hours.

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