A PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe and its preparation method and application

Abstract

The present invention belongs to the technical field of magnetic thermal therapy, and particularly relates to a PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe and its preparation method and application. The PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe comprises a nanocarrier, a PD-L1 polypeptide fluorescein conjugate and an RS09 polypeptide; the PD-L1 polypeptide fluorescein conjugate and the RS09 polypeptide are conjugated to the surface of the nanocarrier, and the nanocarrier is a carboxylated PEG-modified magnetic iron oxide nanoparticles PEG-COOH-Fe3O4. This PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe simultaneously has the functions of inhibiting the immune escape of tumor cells and enhancing the release of tumor-killing cytokines by immune cells, and significantly enhances the immune activation performance of SPIO on tumors in MHT.

Description

Technical Field

[0001] The present invention belongs to the field of magnetic thermal therapy technology, and specifically relates to a method based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe and its preparation method and application. Background Art

[0002] Magnetic hyperthermia therapy (MHT) is an emerging thermal therapy technology that has attracted widespread attention due to its minimal invasiveness, high efficiency and good tissue penetration. It provides a new approach for the precise molecular-level treatment of malignant tumors with high efficiency and low adverse reactions, and has become a new research direction in the field of tumor treatment.

[0003] MHT uses superparamagnetic iron oxide nanoparticles (SPIO) to generate heat under the action of an external alternating magnetic field (AMF), and through relaxation loss and hysteresis loss mechanisms, it raises the tissue temperature of the local lesion site to above 42°C, effectively inducing tumor cell ablation and promoting the maturation and activation of dendritic cells and cytotoxic T cells, thereby activating the body's immune system. However, the existing MHT technology still has certain limitations in activating the immune system in the tumor area.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe and its preparation method and application; This probe based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe has the function of inhibiting tumor cell immune escape and enhancing the release of tumor-killing cytokines of immune cells, which significantly enhances the immune activation performance of SPIO on tumors in MHT.

[0006] In order to overcome the deficiencies of the prior art, the present invention provides the following technical solutions:

[0007] A PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe, including nanocarrier, PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide; the PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide are coupled to the surface of the nanocarrier, and the nanocarrier is a carboxylated PEG-modified magnetic ferroferric oxide nanoparticle PEG-COOH-Fe 3 O 4 .

[0008] Furthermore, the weight ratio of the nanocarrier, the PD-L1 polypeptide-fluorescein conjugate, and the RS09 polypeptide is (5-8):(1-2):1.

[0009] Furthermore, the sequence of the PD-L1 polypeptide fluorescein conjugate is 5-FITC-(Acp)-SGQYASYHCWCWRDPGRSGGSK;

[0010] And / or, the structural formula of the PD-L1 polypeptide-fluorescein conjugate is as follows:

[0011] .

[0012] In addition, the present invention also provides a method based on PD-L1@Fe as described above. 3 O 4 The preparation method of the @LPS tumor magnetic thermal therapy probe comprises the following steps:

[0013] S1. PEG-COOH-Fe 3 O 4 The nanoparticles were resuspended in PBS buffer and the carboxyl groups on the surface of magnetic ferroferric oxide nanoparticles were activated under EDC / NHS conditions to obtain activated PEG-COOH-Fe 3 O 4 Solution;

[0014] S2, dissolving the PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide in the activated PEG-COOH-Fe 3 O 4 The solution is then placed on a shaker and protected from light for a period of time to obtain a reaction product;

[0015] S3, the reaction product obtained in step S2 is centrifuged, the supernatant is removed, and the particles are collected; the collected particles are then resuspended in PBS buffer, centrifuged, the supernatant is removed, and the particles are collected; and the nano-solution system is obtained, which is the PD-L1@Fe based system. 3 O 4 @LPS tumor magnetic thermal therapy probe.

[0016] Furthermore, in step S1, the concentration of the PBS buffer is 1 times; wherein, 6 mg of PEG-COOH-Fe 3 O 4 Nanoparticles.

[0017] Further, in step S1, activating the carboxyl groups on the surface of the magnetic ferroferric oxide nanoparticles under EDC / NHS conditions comprises: dissolving EDC and NHS in a solution containing PEG-COOH-Fe3 O 4 The nanoparticles were prepared in PBS buffer, wherein 22 mg of EDC and 5 mg of NHS were added to each 6 mL of PBS buffer; after being mixed evenly, the mixture was placed on a shaker and reacted at 80-100 rpm and 37-38 °C for 30 min to obtain activated PEG-COOH-Fe 3 O 4 Solution.

[0018] Furthermore, in step S2, the rotation speed of the shaking table is 80-100 rpm; and / or, the conditions of the light-shielding reaction are: reaction temperature 37-38° C., reaction time 6-6.5 h.

[0019] Furthermore, in step S3, the centrifugation condition is: centrifugation at 10,000 rpm for 15 min.

[0020] Further, in step S3, the concentration of the PBS buffer is 1 times;

[0021] And / or, in the nano-solution system, the concentration of PD-L1@Fe3O4@LPS is 3~5 mg / mL.

[0022] In addition, the present invention also provides the above-mentioned PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe or PD-L1@Fe based probe prepared by the above preparation method 3 O 4 @Application of LPS tumor magnetic thermal therapy probe in the preparation of tumor diagnosis and / or treatment products.

[0023] Compared with the prior art, the technical solution of the present invention has at least the following technical effects:

[0024] 1. The present invention is based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe has the function of inhibiting tumor cell immune escape and enhancing the release of tumor-killing cytokines of immune cells;

[0025] 2. The present invention is based on PD-L1@Fe 3 O 4 @LPS tumor magnetic hyperthermia probe significantly enhances the immune activation performance of SPIO on tumors in MHT;

[0026] 3. The present invention is based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe has good biocompatibility and excellent water solubility. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Among them:

[0028] Figure 1 Based on PD-L1@Fe in Example 1 3 O 4 @Schematic diagram of the preparation of LPS tumor magnetic thermal therapy probe;

[0029] Figure 2 PEG-COOH-Fe 3 O 4 (Left) and PD-L1@Fe 3 O 4 @ TEM morphology of LPS (right);

[0030] Figure 3 PEG-COOH-Fe 3 O 4 、PD-L1@Fe 3 O 4 , Fe 3 O 4 @LPS and PD-L1@Fe 3 O 4 @LPS hydrated particle size distribution diagram;

[0031] Figure 4 PEG-COOH-Fe 3 O 4 、PD-L1@Fe 3 O 4 , Fe 3 O 4 @LPS and PD-L1@Fe 3 O 4 @LPS surface potential map;

[0032] Figure 5 PEG-COOH-Fe 3 O 4 (Black) and PD-L1@Fe 3 O 4 @Magnetization curve of LPS (gray);

[0033] Figure 6 PD-L1@Fe 3 O 4 @Schematic diagram of the heating temperature of LPS and saline changing with time in AFM;

[0034] Figure 7 From left to right: saline + AFM group, PD-L1@Fe 3 O 4 @LPS without AFM group and PD-L1@Fe 3 O 4 @Schematic diagram of infrared imaging of the whole body temperature of mice in the LPS+AFM group 5 minutes after treatment;

[0035] Figure 8 For the saline + AFM group and PD-L1@Fe 3 O 4 @Bioluminescence images of tumor tissues at different time points in the LPS+AFM group mice after receiving MHT;

[0036] Fig. 9 The control group and PD-L1@Fe 3 O 4 @Schematic diagram of the distribution of INOS-positive cells and CD8-positive cells in tumor tissues of the LPS+AFM treatment group;

[0037] Fig.10 The control group and PD-L1@Fe 3 O 4 @Schematic diagram of the quantification of the positive area of ​​INOS-positive cells and CD8-positive cells in the tumor tissue of the LPS+AFM treatment group. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution in the embodiment of the present invention will be explained clearly and completely in combination with the embodiments of the present invention.

[0039] The present invention is described in detail. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The process parameters of the following examples that do not specify specific conditions are usually based on conventional conditions.

[0040] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0041] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0042] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0043] The professional terms involved in the present invention are explained as follows:

[0044] The term "PD-L1 polypeptide fluorescein conjugate" is synthesized by Qiangyao Biotechnology Co., Ltd. Its amino acid sequence is the same as the conventional PD-L1 polypeptide. The difference is that the PD-L1 polypeptide fluorescein conjugate is connected with a fluorescent molecule, namely 5-FITC-(Acp), to achieve fluorescent labeling of the polypeptide molecule by PD-L1.

[0045] The term "RS09 polypeptide" is a LPS peptide mimetic with a CAS number of 1449566-36-2 and a molecular formula of C 31 H 49 N 9 O 9 , are a new class of TLR4 agonist adjuvants that can increase antibody production in a vaccine setting.

[0046] The term "carboxylated PEG-modified magnetic ferroferric oxide nanoparticles PEG-COOH-Fe 3 O 4 ”: It is a conventional commercial product, the core of which is magnetic ferroferric oxide nanoparticles Fe 3 O 4 , with special magnetic properties such as superparamagnetism, can show good magnetic responsiveness under the action of magnetic field, and can be used for magnetic separation, magnetic targeting and other applications. The outer layer is modified with carboxylated polyethylene glycol PEG-COOH. Polyethylene glycol is a polymer with good water solubility, biocompatibility and stability. Carboxylated polyethylene glycol can react chemically with other substances through carboxyl groups, such as covalently connecting with biological molecules, drug molecules, etc., thereby achieving further functional modification of nanoparticles.

[0047] The term "EDC / NHS conditions" refers to the reaction conditions under which 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) act together. EDC is a commonly used carbodiimide condensation agent that can react with carboxyl groups in aqueous solution to form an unstable active intermediate O-acylurea; NHS can further react with the active intermediate to form a more stable active ester, which can react efficiently with nucleophilic groups such as amino groups, thereby achieving covalent coupling between carboxyl groups and amino groups.

[0048] According to a first aspect of the present invention, there is provided a method based on PD-L1@Fe 3 O 4 @LPS tumor magnetic thermal therapy probe, including nanocarrier, PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide; the PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide are coupled to the surface of the nanocarrier, and the nanocarrier is a carboxylated PEG-modified magnetic ferroferric oxide nanoparticle PEG-COOH-Fe 3 O 4 .

[0049] A large number of studies have found that in addition to providing magnetothermal properties, SPIO can be given more functions by modifying the surface of superparamagnetic iron oxide SPIO and coupling it with peptides or antibodies. PD-L1, as an important immunomodulatory molecule, plays a key role in tumor immune escape. Immune checkpoint inhibitors targeting PD-L1 have been widely used in clinical tumor treatment. These inhibitors block the interaction between PD-L1 and PD-1, relieve the inhibitory state of T cells, thereby reactivating T cells and enhancing their anti-tumor function. This activation can promote T cell proliferation and cytokine production, thereby enhancing the killing effect on tumor cells. Therefore, coupling anti-PD-L1 peptides on the surface of SPIO can further enhance the activation effect of MHT on the tumor immune microenvironment. In addition, RS09 peptide, as an LPS peptide mimetic, is usually used as a vaccine adjuvant and is used in the vaccine preparation process to enhance antibody production. RS09 peptide promotes the maturation and activation of antigen presenting cells (APCs) by activating specific receptors on immune cells, such as TLR4, thereby enhancing the immunogenicity of the vaccine. This activation can promote the production of proinflammatory cytokines, thereby triggering a stronger immune response. Therefore, the present invention can significantly enhance the activation effect of MHT on the tumor immune microenvironment, improve the body's ability to kill tumor cells, significantly inhibit tumor growth, and activate the body's immune response to tumors, thereby improving the magnetic thermal effect. The RS09 polypeptide in the present invention is preferably synthesized by Qiangyao Company, and its sequence structure is the same as the conventional commercially available RS09, which will not be repeated here.

[0050] In the above-mentioned PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe, as a preferred embodiment, the weight ratio of the nanocarrier, the PD-L1 polypeptide fluorescein conjugate, and the RS09 polypeptide is (5-8): (1-2): 1. If the addition ratio of the PD-L1 polypeptide fluorescein conjugate to the RS09 polypeptide is too low, the CD8 cell killing performance is low. Conversely, when the addition ratio of the RS09 polypeptide to the PD-L1 polypeptide fluorescein conjugate is too low, it is not conducive to the activation and maturation of APCs, resulting in a decrease in the antigen presentation effect.

[0051] In the above-mentioned PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe, as a preferred embodiment, the sequence of the PD-L1 polypeptide fluorescein conjugate is 5-FITC-(Acp)-SGQYASYHCWCWRDPGRSGGSK;

[0052] The structural formula of the PD-L1 polypeptide fluorescein conjugate is as follows:

[0053] .

[0054] According to a second aspect of the present invention, a method for preparing the above-mentioned PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe is provided, comprising the following steps:

[0055] S1. PEG-COOH-Fe 3 O 4 The nanoparticles were resuspended in PBS buffer and the carboxyl groups on the surface of magnetic ferroferric oxide nanoparticles were activated under EDC / NHS conditions to obtain activated PEG-COOH-Fe 3 O 4 Solution;

[0056] S2, dissolving the PD-L1 polypeptide fluorescein conjugate and RS09 polypeptide in the activated PEG-COOH-Fe 3 O 4 The solution is then placed on a shaker and protected from light for a period of time to obtain a reaction product;

[0057] S3. Centrifuge the reaction product obtained in step S2, remove the supernatant, and collect the particles; then resuspend the collected particles in PBS buffer solution, centrifuge, remove the supernatant, and collect the particles; and then resuspend in PBS buffer solution to obtain a nano solution system, which is the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe.

[0058] The preparation method of the present invention is simple and easy to operate. The prepared tumor magnetic thermal therapy probe exhibits excellent tumor immune microenvironment activation performance, good monodispersity and stability, and stable magnetic thermal performance and the like.

[0059] In the above preparation method, as a preferred embodiment, in step S1, the concentration of the PBS buffer is 1 times, that is, 1×PBS buffer; wherein, 6 mg of PEG-COOH-Fe is added to each 6 mL of 1×PBS buffer. 3 O 4 Nanoparticles.

[0060] In the above preparation method, as a preferred embodiment, in step S1, activating the carboxyl groups on the surface of magnetic ferroferric oxide nanoparticles under EDC / NHS conditions comprises: dissolving EDC and NHS in a solution containing PEG-COOH-Fe 3 O 4 The nanoparticles were prepared in PBS buffer, wherein 22 mg of EDC and 5 mg of NHS were added to each 6 mL of PBS buffer; after being mixed evenly, the mixture was placed on a shaker and reacted at 80-100 rpm and 37-38 °C for 30 min to obtain activated PEG-COOH-Fe3 O 4 Solution.

[0061] In the above preparation method, as a preferred embodiment, in step S2, the rotation speed of the shaking table is 80-100 rpm; the conditions of the light-proof reaction are: reaction temperature 37-38°C, reaction time 6-6.5 h.

[0062] In the above preparation method, as a preferred embodiment, in step S3, the centrifugation condition is: centrifugation at 10000 rpm for 15 min.

[0063] Optionally, in step S3, the concentration of the PBS buffer is 1 times;

[0064] In the nano-solution system, the concentration of PD-L1@Fe3O4@LPS is 3~5 mg / mL.

[0065] According to the third aspect of the present invention, provided is the use of the above-mentioned PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe or the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe prepared by the above-mentioned preparation method in the preparation of tumor diagnosis and / or treatment products.

[0066] The present invention will be described in detail below in conjunction with embodiments of the present invention, and each example is provided by way of explanation of the present invention without limiting the present invention. In fact, it will be clear to those skilled in the art that modifications and variations may be made in the present invention without departing from the scope or spirit of the present invention. For example, a feature shown or described as a part of an embodiment may be used in another embodiment to produce yet another embodiment. Therefore, it is desired that the present invention includes such modifications and variations within the scope of the appended claims and their equivalents.

[0067] In the embodiments of the present invention, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0068] The main raw materials used in this embodiment are described as follows:

[0069] PEG-COOH-Fe 3 O 4 Nanoparticles were selected from synomag-D Catalog No.: 104-56-701;

[0070] RS09 peptide and PD-L1 peptide fluorescein conjugates were both synthesized by Qiangyao Biotechnology Co., Ltd.

[0071] Example 1

[0072] This embodiment provides a method for preparing a tumor magnetic thermal therapy probe based on PD-L1@Fe3O4@LPS, which specifically includes the following steps:

[0073] (1) PEG-COOH-Fe 3 O 4 Activation of nanoparticles

[0074] Step 1: 6 mg of PEG-COOH-Fe 3 O 4 The nanoparticles were dispersed in 6 mL of 1× PBS buffer solution and mixed to form a uniform light yellow solution.

[0075] Step 2: Dissolve 22 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) in the above-mentioned PEG-COOH-Fe 3 O 4 The nanoparticles were added to a 1× PBS buffer solution and mixed to form a homogeneous transparent solution.

[0076] Step 3: Place the homogenous solution in step 2 on a shaker at 80 rpm for 30 min at 37 °C to obtain activated PEG-COOH-Fe 3 O 4 Solution.

[0077] (2) Activated PEG-COOH-Fe 3 O 4 Conjugation of solution with PD-L1 peptide-fluorescein conjugate and RS09 peptide

[0078] Step 1: Add 1 mg of PD-L1 peptide-fluorescein conjugate and 0.5 mg of RS09 peptide to the above activated PEG-COOH-Fe 3 O 4 The solution was mixed to form a brown-yellow transparent solution.

[0079] Step 2: Place the homogenous solution of step 1 on a shaker at 80 rpm and react at 37 °C for 6 h.

[0080] Step 3: Centrifuge the product of step 2 at 10,000 rpm for 15 min and collect the precipitate.

[0081] Step 4: Resuspend the precipitate collected in step 3 in 10 mL of 1×PBS buffer solution, sonicate for 5 min, and mix to form a uniform brown-yellow transparent solution; centrifuge the mixed brown-yellow transparent solution at 10,000 rpm for 15 min, and collect the precipitate.

[0082] Step 6: Repeat step 5 twice, resuspend the collected precipitate in 1 mL of 1×PBS buffer solution to obtain the PD-L1@Fe3O4@LPS-based tumor magnetic thermal therapy probe of this example.

[0083] Figure 1 Schematic diagram of the preparation of the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe in this embodiment;

[0084] Figure 2 PEG-COOH-Fe 3 O 4 (Left) and PD-L1@Fe 3 O 4 @ TEM morphology of LPS (right);

[0085] Figure 5 PEG-COOH-Fe 3 O 4 (Black) and PD-L1@Fe 3 O 4 @Magnetization curve of LPS (grey).

[0086] It can be seen from the above figure that the particle size of the magnetic nanoparticles modified with polypeptides is slightly larger than that of the unmodified magnetic nanoparticles, and the hysteresis effect of the magnetic nanoparticles modified with polypeptides is slightly enhanced compared with that of the unmodified magnetic nanoparticles at the same concentration.

[0087] Comparative Example 1

[0088] This comparative example provides a method based on PD-L1@Fe 3 O 4 The method for preparing a tumor magnetic thermal therapy probe specifically comprises the following steps:

[0089] (1) PEG-COOH-Fe 3 O 4 Activation of nanoparticles

[0090] Step 1: 6 mg of PEG-COOH-Fe 3 O 4 The nanoparticles were dispersed in 6 mL of 1× PBS buffer solution and mixed to form a uniform light yellow solution.

[0091] Step 2: Dissolve 22 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) in the above-mentioned PEG-COOH-Fe 3 O 4 The nanoparticles were added to a 1× PBS buffer solution and mixed to form a homogeneous transparent solution.

[0092] Step 3: Place the homogenous solution in step 2 on a shaker at 80 rpm for 30 min at 37 °C to obtain activated PEG-COOH-Fe 3 O 4 Solution.

[0093] (2) Activated PEG-COOH-Fe 3 O 4 Solution conjugation with PD-L1 peptide-fluorescein conjugate

[0094] Step 1: Add 1 mg of PD-L1 peptide-fluorescein conjugate to the above activated PEG-COOH-Fe 3 O 4 The solution was mixed to form a brown-yellow transparent solution.

[0095] Step 2: Place the homogenous solution of step 1 on a shaker at 80 rpm and react at 37 °C for 6 h.

[0096] Step 3: Centrifuge the product of step 2 at 10,000 rpm for 15 min and collect the precipitate.

[0097] Step 4: Resuspend the precipitate collected in step 3 in 10 mL of 1×PBS buffer solution, sonicate for 5 min, and mix to form a uniform brown-yellow transparent solution; centrifuge the mixed brown-yellow transparent solution at 10,000 rpm for 15 min, and collect the precipitate.

[0098] Step 6: Repeat step 5 twice, resuspend the collected precipitate in 1 mL of PBS buffer solution to obtain the PD-L1@Fe 3 O 4 Tumor magnetic hyperthermia therapy probe.

[0099] Comparative Example 2

[0100] This comparative example provides a Fe 3 O 4 The preparation method of the @LPS tumor magnetic thermal therapy probe specifically includes the following steps:

[0101] (1) PEG-COOH-Fe 3 O 4 Activation of nanoparticles

[0102] Step 1: 6 mg of PEG-COOH-Fe 3 O 4 The nanoparticles were dispersed in 6 mL of 1× PBS buffer solution and mixed to form a uniform light yellow solution.

[0103] Step 2: Dissolve 22 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) in the above-mentioned PEG-COOH-Fe 3 O 4 The nanoparticles were added to a 1× PBS buffer solution and mixed to form a homogeneous transparent solution.

[0104] Step 3: Place the homogenous solution in step 2 on a shaker at 80 rpm for 30 min at 37 °C to obtain activated PEG-COOH-Fe 3 O 4 Solution.

[0105] (3) Activated PEG-COOH-Fe 3 O 4 Coupling of solution with RS09 peptide

[0106] Step 1: Add 0.5 mg of RS09 peptide to the activated PEG-COOH-Fe 3 O 4 The solution was mixed to form a brown-yellow transparent solution.

[0107] Step 2: Place the homogenous solution of step 1 on a shaker at 80 rpm and react at 37 °C for 6 h.

[0108] Step 3: Centrifuge the product of step 2 at 10,000 rpm for 15 min and collect the precipitate.

[0109] Step 4: Resuspend the precipitate collected in step 3 in 10 mL of 1×PBS buffer solution, sonicate for 5 min, and mix to form a uniform brown-yellow transparent solution; centrifuge the mixed brown-yellow transparent solution at 10,000 rpm for 15 min, and collect the precipitate.

[0110] Step 6: Repeat step 5 twice, resuspend the collected precipitate in 1 mL of 1× PBS buffer solution to obtain the Fe-based 3 O 4 @LPS tumor magnetic thermal therapy probe.

[0111] Figure 3 PEG-COOH-Fe 3 O 4 、PD-L1@Fe 3 O 4 , Fe 3 O 4 @LPS and PD-L1@Fe 3 O 4 @LPS hydrated particle size distribution diagram;

[0112] Figure 4 PEG-COOH-Fe 3 O 4 、PD-L1@Fe 3 O 4 , Fe 3 O 4 @LPS and PD-L1@Fe 3 O 4 @LPS surface potential map;

[0113] from Figure 3 , 4 It can be seen that the PD-L1 peptide and RS09 peptide were successfully coupled to the Fc 3 O 4 On the nanoparticles, unmodified Fe 3 O 4 The potential of the nanoparticles was negatively charged. After modification with the PD-L1 peptide, the potential increased to positive charge. After modification with the RS09 peptide, the potential decreased compared to the previous charge. After modification with the PD-L1 peptide and RS09 peptide, the potential increased to positive charge, but the degree of charge increase was less than that of the single PD-L1 peptide modification. 3 O 4 The hydrated particle size of @LPS nanoparticles is larger than that of PD-L1@Fe 3 O 4 Nanoparticles, Fe 3 O 4 @LPS nanoparticles and Fe 3 O 4 The nanoparticles were slightly enlarged.

[0114] Experimental example

[0115] In order to evaluate the tumor immune activation of the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe in MHT, this experimental example conducted a mouse tumor-bearing experiment. The specific experimental method is as follows:

[0116] ①2*10 6 4T1 tumor cells were injected subcutaneously into the right leg of mice to construct a subcutaneous tumor model. When the tumor volume grew to 150 mm 3 Start treatment around .

[0117] ②The tumor-bearing mice were divided into a control group and an experimental group. The control group was treated with saline + AFM, and the experimental groups were treated with PD-L1@Fe 3 O 4 @LPS without AFM group, PD-L1@Fe 3 O 4@LPS+ AFM group, 7 in each group. (AFM is a magnetic field that changes with time. In MHT research, it is the key factor in stimulating the heat generated by the magnetic hyperthermia probe. The same alternating magnetic field as the experimental group was applied to the control group to ensure that when observing the experimental results, it is possible to clearly distinguish whether it is the effect of the magnetic hyperthermia probe itself or the effect of the alternating magnetic field alone, so as to more accurately evaluate the effect of MHT).

[0118] ③ 50 μL of normal saline and PD-L1@Fe with an iron concentration of 1 mg / mL prepared by dilution in Example 1 were injected into the tumors of mice in the control group and the experimental group respectively. 3 O 4 @LPS solution. One day later, the tumor-bearing mice were placed under AFM (354 kHz; 30 A) for 7 min. Four mice in each group were euthanized two days after MHT treatment, and the tumor tissues of the mice were taken for immunofluorescence detection. The bioluminescence of the tumor tissues of the remaining three mice in each group was collected every 5 days to evaluate the changes in tumor load.

[0119] Immunofluorescence staining was performed on tumor tissues to evaluate the distribution of M1 tumor-associated macrophages (INOS) and CD8-positive T cells in mouse tumor tissues.

[0120] Figure 6 PD-L1@Fe 3 O 4 @Schematic diagram of the heating temperature of LPS and saline changing with time in AFM;

[0121] Figure 7 From left to right: saline + AFM group, PD-L1@Fe 3 O 4 @LPS without AFM group and PD-L1@Fe 3 O 4 @Schematic diagram of infrared imaging of the whole body temperature of mice in the LPS+AFM group 5 minutes after treatment;

[0122] Figure 8 For the saline + AFM group and PD-L1@Fe 3 O 4 @Bioluminescence images of tumor tissues at different time points in the LPS+AFM group mice after receiving MHT;

[0123] Fig. 9 The control group and the 3 O 4 @Schematic diagram of the distribution of INOS-positive cells and CD8-positive cells in tumor tissues of the LPS+AFM treatment group;

[0124] Fig.10The control group and the 3 O 4 @Schematic diagram of the quantification of the positive area of ​​INOS-positive cells and CD8-positive cells in the tumor tissue of the LPS+AFM treatment group.

[0125] From the above figure, we can see that PD-L1@Fe 3 O 4 @LPS particles have good magnetothermal properties both in vivo and in vitro. 3 O 4 @After LPS+AFM treatment, tumor tissue growth was inhibited. Immunohistochemistry results showed that PD-L1@Fe 3 O 4 @The infiltration of M1 tumor-associated macrophages (INOS) and CD8-positive T cells in the tumor tissue of the LPS+AFM group was significantly increased compared with that of the control group. Therefore, the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe of the present invention can enhance the activation effect of MHT on the tumor immune microenvironment, thereby improving the efficacy of MHT.

[0126] The above describes and evaluates the efficacy of some embodiments of the present invention. It should be understood that the present invention is not limited to the above specific implementations. Any technician familiar with the art can use the above disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still falls within the scope of protection of the technical solutions of the present invention.

Claims

1. A tumor magnetic thermal therapy probe based on PD-L1@Fe3O4@LPS, characterized in that: It comprises a nanocarrier, a PD-L1 polypeptide-fluorescein conjugate and an RS09 polypeptide; the PD-L1 polypeptide-fluorescein conjugate and the RS09 polypeptide are coupled to the surface of the nanocarrier, and the nanocarrier is a carboxylated PEG-modified magnetic ferrosoferric oxide nanoparticle PEG-COOH-Fe3O4; the weight ratio of the nanocarrier, the PD-L1 polypeptide-fluorescein conjugate and the RS09 polypeptide is (5~8):(1~2):

1.

2. The PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe according to claim 1, characterized in that: The sequence of the PD-L1 polypeptide fluorescein conjugate is 5-FITC-(Acp)-SGQYASYHCWCWRDPGRSGGSK; And / or, the structural formula of the PD-L1 polypeptide-fluorescein conjugate is as follows: 。 3. A method for preparing a PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1, resuspending the PEG-COOH-Fe3O4 nanoparticles in PBS buffer, activating the carboxyl groups on the surface of the magnetic ferroferric oxide nanoparticles under EDC / NHS conditions to obtain an activated PEG-COOH-Fe3O4 solution; S2, dissolving the PD-L1 polypeptide fluorescein conjugate and the RS09 polypeptide in the activated PEG-COOH-Fe3O4 solution, and then placing them on a shaker, and reacting them in the dark for a period of time to obtain a reaction product; S3. Centrifuge the reaction product obtained in step S2, remove the supernatant, and collect the particles; then resuspend the collected particles in PBS buffer, centrifuge, remove the supernatant, and collect the particles; and resuspend them in PBS buffer to obtain a nano solution system, which is the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe.

4. The preparation method according to claim 3, characterized in that: In step S1, the concentration of the PBS buffer is 1 times; wherein, 6 mg of PEG-COOH-Fe3O4 nanoparticles are added to every 6 mL of PBS buffer.

5. The preparation method according to claim 4, characterized in that: In step S1, activating the carboxyl groups on the surface of magnetic ferroferric oxide nanoparticles under EDC / NHS conditions includes: dissolving EDC and NHS in a PBS buffer solution containing PEG-COOH-Fe3O4 nanoparticles, wherein 22 mg of EDC and 5 mg of NHS are added to each 6 mL of PBS buffer solution; after mixing evenly, placing on a shaker, reacting at 80-100 rpm and 37-38°C for 30 min to obtain an activated PEG-COOH-Fe3O4 solution.

6. The preparation method according to claim 3, characterized in that: In step S2, the rotation speed of the shaking table is 80-100 rpm; and / or, the conditions of the light-proof reaction are: reaction temperature 37-38° C., reaction time 6-6.5 h.

7. The preparation method according to claim 3, characterized in that: In step S3, the centrifugation condition is: centrifugation at 10,000 rpm for 15 min.

8. The preparation method according to claim 3, characterized in that: In step S3, the concentration of the PBS buffer is 1 times; And / or, in the nano-solution system, the concentration of PD-L1@Fe3O4@LPS is 3~5 mg / mL.

9. Use of the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe as described in any one of claims 1-2 or the PD-L1@Fe3O4@LPS tumor magnetic thermal therapy probe prepared by the preparation method according to any one of claims 3-8 in the preparation of tumor diagnosis and / or treatment products.

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