Preparation method and application of dual-targeting carrier-free delivery system for enhancing anti-tumor immunity of NKT cells

Abstract

The invention discloses a preparation method and application of a dual-targeting carrier-free delivery system for enhancing anti-tumor immunity of NKT cells, Lys-alpha-GalCer is used as a raw material, and two molecules of LGC are connected through a disulfide bond to synthesize an amphiphilic LGC-SS-LGC micromolecule prodrug; the LGC-SS-LGC micromolecule prodrug is prepared into vesicles internally loaded with an NKT cell antigen presenting molecule CD1d and externally modified with polysarcosine through electrostatic adsorption through a film dispersion-probe ultrasonic method, finally a CD1d / LGC-SS-LGC / PSar dual-targeting carrier-free delivery system is formed, and the dual-targeting carrier-free delivery system can be used for liver cancer treatment after intravenous administration. In addition, the compound can be used for preparing a preparation for enhancing NKT cell Th1 type immune response. The dual-targeting carrier-free delivery system prepared by the invention can directly or indirectly enhance the anti-tumor immune effect of the NKT cells, and has a dual-synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery system technology, specifically relating to the preparation method and application of a dual-target carrier-free delivery system for enhancing NKT cell anti-tumor immunity. Background Technology

[0002] Natural killer T cells (NKT) are a subset of T cells with specific markers that recognize glycolipid antigens and rapidly secrete immunomodulatory cytokines, which can directly or indirectly exert anti-tumor effects. This unique combination of innate and adaptive immunity makes NKT cells play an important role between the two branches of the immune system, and they have great potential in the field of anti-tumor therapy.

[0003] α-GalCer is the most representative glycolipid agonist of NKT cells. It is a type of glycolipid extracted from spongy tissue and obtained through structural optimization. It has shown good safety and great anti-tumor potential in animal models and clinical trials and has been used in many clinical trials. However, there are two bottleneck problems in the clinical anti-tumor application of α-GalCer: (1) α-GalCer stimulates NKT cells to secrete almost equal amounts of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokines. The release of Th1 cytokines can fight tumors and bacteria, while enhancing the Th2 response helps to treat autoimmune diseases. When these two cytokines are secreted at the same time, their effects are antagonistic. (2) Immune dysfunction of NKT cells. Repeated high-dose stimulation of α-GalCer can cause NKT cells to enter an immune dysfunction state within a few months, and the response to subsequent antigen stimulation is weak or non-responsive.

[0004] Nanomedicine delivery systems represent a promising drug delivery strategy, particularly in cancer therapy. Leveraging the enhanced permeability and retention effect (EPR) of tumor tissue, drug-loaded nanoparticles aggregate within the tumor to achieve anti-tumor effects. However, in practical applications, most intravenously injected nanomedicines are initially taken up by the reticuloendothelial system (RES), accumulating in the spleen, liver, and bone marrow, limiting their effective delivery to the tumor site. NKT cells, the target of α-GalCer, develop in the thymus and are mainly distributed in the spleen, liver, bone marrow, lymph nodes, skin, mucous membranes, and peripheral blood. In peripheral blood, NKT cells account for only 0.01%-0.1% of circulating T cells. Therefore, the optimal delivery destination for α-GalCer and its analogues in vivo should be the spleen and liver, especially the spleen, which contains a large number of antigen-presenting cells. Therefore, this invention fully utilizes the high permeability and retention effect of the reticuloendothelial system and tumor tissue to design and synthesize a dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity, addressing two issues in the clinical application of α-GalCer: (1) The α-GalCer analog Lys-α-GalCer (LGC) and CD1d, which efficiently induce Th1 immune responses, are targeted and transported to the spleen via RES, activating NKT cells to induce Th1 immune responses and achieving an anti-tumor effect; (2) Simultaneously, the EPR effect of tumor tissue is utilized to target and transport the α-GalCer analog Lys-α-GalCer (LGC) and CD1d, which efficiently induce Th1 immune responses, to tumor tissue, upregulating the expression of CD1d in tumor tissue, directly lysing tumor cells, and jointly enhancing the anti-tumor immune effect of NKT cells. Currently, there are no relevant literature reports on this aspect. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity. The CD1d / LGC-SS-LGC / PSar (CLLP) dual-targeting carrier-free delivery system prepared by this method can effectively solve the bottleneck problem of α-GalCer in clinical applications, enhance the anti-tumor immune effect based on NKT cells, and can be used for liver cancer treatment after intravenous administration of the prepared dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity. Furthermore, the prepared dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity can be used to prepare agents that enhance NKT cell Th1-type immune responses.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for preparing a dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity, characterized in that: using Lys-α-GalCer (LGC) as a raw material, two LGC molecules are linked by disulfide bonds to synthesize an amphiphilic LGC-SS-LGC small molecule prodrug. This LGC-SS-LGC small molecule prodrug is then used to prepare an internally loaded NKT cell antigen-presenting molecule CD1d and an externally modified polysarcosine vesicle through electrostatic adsorption using a thin-film dispersion-probe ultrasonic method, ultimately forming a CD1d / LGC-SS-LGC / PSar dual-targeting carrier-free delivery system.

[0007] Further specifying, the preparation process of the LGC-SS-LGC small molecule prodrug is as follows: Lys-α-GalCer is subjected to three steps: amidation reaction, DTT thiol reduction reaction, and thiol-disulfide bond exchange reaction, and finally connected by disulfide bonds to generate amphiphilic LGCd-SS-LGCd small molecule prodrug.

[0008] Further specifying the specific synthesis process of the LGC-SS-LGC small molecule prodrug, it is as follows: Lys-α-GalCer and succinimide 3-(2-pyridyldithio)-propionate (SPDP) are dissolved in ice-cold DMSO, and the reaction is stirred under ice bath conditions. A portion of the reaction solution is taken out for later use. Dithiothreitol (DTT) solution is added to the remaining reaction solution, and the reaction is stirred again. The previously taken-out reaction solution is then added and stirred overnight to obtain the LGC-SS-LGC small molecule prodrug.

[0009] Further specifying the preparation process of the CD1d / LGC-SS-LGC / PSar dual-target carrier-free delivery system, the following steps are taken: The LGCd-SS-LGCd small molecule prodrug is dissolved in a chloroform / methanol mixture. The mixture is placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film is formed. The mixture is then vacuum dried overnight. An aqueous solution containing CD1d is added to the pear-shaped flask, and the mixture is placed in a shaker and hydrated at 37°C for 30-50 minutes. The mixture is then sonicated under ice bath conditions with a probe and an ultrasonic power of 100-600 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. Polysarcosine is added to the obtained CD1d / LGC-SS-LGC vesicle solution, and the mixture is allowed to stand and mix thoroughly to obtain the CD1d / LGC-SS-LGC / PSar dual-target carrier-free delivery system.

[0010] Further specifying, the volume percentage of chloroform in the chloroform / methanol mixed solution is 10%-40%.

[0011] Further specified, the mass percentage of CD1d in the CD1d / LGC-SS-LGC vesicles is 10%-40%; the weight percentage of CD1d / LGC-SS-LGC to polysarcosine is 10%-60%.

[0012] The dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity described in this invention is used to prepare targeted anticancer drugs.

[0013] The dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity described in this invention is used to prepare agents that enhance NKT cell Th1-type immune responses.

[0014] Compared with the prior art, the present invention has the following beneficial effects and advantages: (1) The present invention uses the α-GalCer analog LGC, which can efficiently induce Th1 immune response, to synthesize an amphiphilic LGC-SS-LGC prodrug by connecting it with disulfide bonds. The prodrug also serves as a carrier to load CD1d molecules. During the conversion of the prodrug, the spatial structure changes and the loaded CD1d molecules are released, thus constructing a novel carrier-free self-delivery system. The outer layer is modified with polysarcosine to improve its fate in blood circulation and target the spleen and tumor tissue rich in NKT cells respectively. (2) The present invention uses glycolipids and CD1d molecules in combination to overcome the problems existing in the clinical use of α-GalCer and establishes a simple method to enhance the anti-tumor immune activity of NKT cells. Attached Figure Description

[0015] Figure 1 Particle size potential diagram of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1.

[0016] Figure 2 The determination of erythrocyte hemolysis rate using the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1.

[0017] Figure 3 The effects of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 on spleen cell toxicity and proliferation.

[0018] Figure 4 The effect of the CD1d / LGC-SS-LGC / Psar dual-targeting formulation prepared in Example 1 on the in vitro induction of NKT cell cytokine release.

[0019] Figure 5 Imaging of the tissue distribution of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 in ex vivo tissues (heart, liver, spleen, lung, kidney, and tumor) of tumor-bearing mice.

[0020] Figure 6 The amount of IFN-γ and IL-4 induced in vivo in mice by the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1.

[0021] Figure 7 The in vivo antitumor effect of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1. Detailed Implementation

[0022] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0023] References for Lys-α-GalCer (LGC), an α-GalCer analog that efficiently induces Th1 immune responses ( Bioorg Med Chem The preparation was carried out using the method reported in 2020;28(1):115141. doi:10.1016 / j.bmc.2019.115141). Example 1

[0024] Lys-α-GalCer (200 mg) and SPDP (succinimide 3-(2-pyridyldithio)-propionate, 20 mg) were dissolved in 4 mL of ice-cold DMSO. The mixture was stirred in an ice bath for 30 min. 2 mL of the reaction solution was taken out for later use. 0.5 mL of DTT (dithiothreitol) solution (final DTT concentration 50 mM) was added to the remaining reaction solution. The mixture was stirred for another 30 min. The previously taken-out reaction solution was then added back in and stirred overnight to obtain the amphiphilic LGC-SS-LGC small molecule prodrug. 100 mg of the small molecule prodrug LGCd-SS-LGCd was dissolved in a chloroform / methanol mixture (chloroform comprising 20%). The mixture was placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film was formed. The mixture was then vacuum dried overnight. 2 mL of an aqueous solution containing 10 mg of CD1d was added to the pear-shaped flask, and the mixture was placed in a shaker and hydrated at 37°C for 30 min. The solution was then sonicated under ice bath conditions at a power of 400 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. 100 μL of the CD1d / LGC-SS-LGC vesicle solution was added to polysarcosine (40 mg), and the mixture was allowed to stand and mix for 20 min to obtain a CD1d / LGC-SS-LGC / PSar dual-targeting formulation. Example 2

[0025] Lys-α-GalCer (200 mg) and SPDP (succinimide 3-(2-pyridyldithio)-propionate, 20 mg) were dissolved in 4 mL of ice-cold DMSO. The mixture was stirred in an ice bath for 30 min. 2 mL of the reaction solution was taken out for later use. 0.5 mL of DTT (dithiothreitol) solution (final DTT concentration 50 mM) was added to the remaining reaction solution. The mixture was stirred for another 30 min. The previously taken-out reaction solution was then added back in and stirred overnight to obtain the amphiphilic LGC-SS-LGC small molecule prodrug. 100 mg of the small molecule prodrug LGCd-SS-LGCd was dissolved in a chloroform / methanol mixture (10% chloroform). The mixture was placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film was formed. The mixture was then vacuum dried overnight. 2 mL of an aqueous solution containing 20 mg of CD1d was added to the pear-shaped flask, and the mixture was placed in a shaker and hydrated at 37°C for 30 min. The solution was then sonicated under ice bath conditions at a power of 400 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. 100 μL of the CD1d / LGC-SS-LGC vesicle solution was added to polysarcosine (10 mg), and the mixture was allowed to stand and mix for 20 min to obtain a CD1d / LGC-SS-LGC / PSar dual-targeting formulation. Example 3

[0026] Lys-α-GalCer (200 mg) and SPDP (succinimide 3-(2-pyridyldithio)-propionate, 20 mg) were dissolved in 4 mL of ice-cold DMSO. The mixture was stirred in an ice bath for 30 min. 2 mL of the reaction solution was taken out for later use. 0.5 mL of DTT (dithiothreitol) solution (final DTT concentration 50 mM) was added to the remaining reaction solution. The mixture was stirred for another 30 min. The previously taken-out reaction solution was then added back in and stirred overnight to obtain the amphiphilic LGC-SS-LGC small molecule prodrug. 100 mg of the small molecule prodrug LGCd-SS-LGCd was dissolved in a chloroform / methanol mixture (10% chloroform). The mixture was placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film was formed. The mixture was then vacuum dried overnight. 2 mL of an aqueous solution containing 20 mg of CD1d was added to the pear-shaped flask, and the mixture was placed in a shaker and hydrated at 37°C for 30 min. The solution was then sonicated under ice bath conditions at a power of 400 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. 100 μL of the CD1d / LGC-SS-LGC vesicle solution was added to polysarcosine (20 mg), and the mixture was allowed to stand and mix for 20 min to obtain a CD1d / LGC-SS-LGC / PSar dual-targeting formulation. Example 4

[0027] Lys-α-GalCer (200 mg) and SPDP (succinimide 3-(2-pyridyldithio)-propionate, 20 mg) were dissolved in 4 mL of ice-cold DMSO. The mixture was stirred in an ice bath for 30 min. 2 mL of the reaction solution was taken out for later use. 0.5 mL of DTT (dithiothreitol) solution (final DTT concentration 50 mM) was added to the remaining reaction solution. The mixture was stirred for another 30 min. The previously taken-out reaction solution was then added back in and stirred overnight to obtain the amphiphilic LGC-SS-LGC small molecule prodrug. 100 mg of the small molecule prodrug LGCd-SS-LGCd was dissolved in a chloroform / methanol mixture (10% chloroform). The mixture was placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film was formed. The mixture was then vacuum dried overnight. 2 mL of an aqueous solution containing 20 mg of CD1d was added to the pear-shaped flask, and the mixture was placed in a shaker and hydrated at 37°C for 30 min. The solution was then sonicated under ice bath conditions at a power of 200 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. 100 μL of the CD1d / LGC-SS-LGC vesicle solution was added to polysarcosine (20 mg), and the mixture was allowed to stand and mix for 20 min to obtain a CD1d / LGC-SS-LGC / PSar dual-targeting formulation. Example 5

[0028] Lys-α-GalCer (200 mg) and SPDP (succinimide 3-(2-pyridyldithio)-propionate, 20 mg) were dissolved in 4 mL of ice-cold DMSO. The mixture was stirred in an ice bath for 30 min. 2 mL of the reaction solution was taken out for later use. 0.5 mL of DTT (dithiothreitol) solution (final DTT concentration 50 mM) was added to the remaining reaction solution. The mixture was stirred for another 30 min. The previously taken-out reaction solution was then added back in and stirred overnight to obtain the amphiphilic LGC-SS-LGC small molecule prodrug. 100 mg of the small molecule prodrug LGCd-SS-LGCd was dissolved in a chloroform / methanol mixture (10% chloroform). The mixture was placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film was formed. The mixture was then vacuum dried overnight. 2 mL of an aqueous solution containing 20 mg of CD1d was added to the pear-shaped flask, and the mixture was placed in a shaker and hydrated at 37°C for 30 min. The solution was then sonicated under ice bath conditions at a power of 200 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. 100 μL of the CD1d / LGC-SS-LGC vesicle solution was added to polysarcosine (30 mg), and the mixture was allowed to stand and mix for 20 min to obtain a CD1d / LGC-SS-LGC / PSar dual-targeting formulation.

[0029] Figure 1The particle size potential diagram of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 shows that the average particle size and PDI value are 155.2±3.41 and 0.31±0.04, respectively, and the potential is -0.078±0.1mV.

[0030] Figure 2 The effects of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 on erythrocytes and the determination of hemolysis rate were investigated. The results showed that the hemolysis rate of the prepared dual-targeting formulation was less than 5%, and it can be used for tail vein injection in mice.

[0031] Figure 3 The effects of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 on spleen cell toxicity and proliferation were investigated. Results showed that the prepared dual-targeting formulation had no toxicity to spleen cells and could be used for targeted spleen drug delivery.

[0032] Figure 4 The effect of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 on the in vitro induction of NKT cell cytokine release was investigated. The results showed that the prepared dual-targeting formulation induced NKT cells to secrete significantly more Th1-type cytokines than Th2-type cytokines in vitro, exhibiting a strong Th1-type induction bias.

[0033] Figure 5 The CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared for Example 1 was distributed in vivo in tumor-bearing mice, and images of ex vivo tissues (heart, liver, spleen, lung, kidney, and tumor) were obtained. The results showed that the prepared dual-targeting formulation had targeting ability in both the spleen and tumor tissues.

[0034] Figure 6 The CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 was used to induce the secretion of IFN-γ and IL-4 in mice in vivo. The results showed that the prepared dual-targeting formulation induced a higher level of Th1-type factor IFN-γ than Th2-type factor IL-4 in vivo, exhibiting significant Th1 polarization.

[0035] Figure 7 The in vivo antitumor effect of the CD1d / LGC-SS-LGC / PSar dual-targeting formulation prepared in Example 1 was investigated. Results showed that the prepared dual-targeting formulation exhibited significant antitumor effects.

[0036] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity, characterized in that: Using Lys-α-GalCer (LGC) as a raw material, two LGC molecules were linked by disulfide bonds to synthesize an amphiphilic LGC-SS-LGC small molecule prodrug. The LGC-SS-LGC small molecule prodrug was prepared by thin film dispersion-probe sonication to internally load the NKT cell antigen presenting molecule CD1d and externally by electrostatic adsorption-modified polysarcosine vesicles, ultimately forming a CD1d / LGC-SS-LGC / PSar dual-target carrier-free delivery system.

2. The method for preparing the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity according to claim 1, characterized in that... The preparation process of the LGC-SS-LGC small molecule prodrug is as follows: Lys-α-GalCer is subjected to three steps: amidation reaction, DTT thiol reduction reaction, and thiol-disulfide bond exchange reaction, and finally connected by disulfide bonds to generate amphiphilic LGCd-SS-LGCd small molecule prodrug.

3. The method for preparing the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity according to claim 1, characterized in that... The specific synthesis process of the LGC-SS-LGC small molecule prodrug is as follows: Lys-α-GalCer and succinimide 3-(2-pyridyldithio)-propionate are dissolved in ice-cold DMSO and stirred under ice bath conditions. A portion of the reaction solution is taken out for later use. Dithiothreitol solution is added to the remaining reaction solution and the reaction is stirred again. Then, the previously taken-out reaction solution is added and stirred overnight to obtain the LGC-SS-LGC small molecule prodrug.

4. The method for preparing the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity according to claim 1, characterized in that... The specific preparation process of the CD1d / LGC-SS-LGC / PSar dual-target carrier-free delivery system is as follows: The LGCd-SS-LGCd small molecule prodrug is dissolved in a chloroform / methanol mixture. The mixture is placed in a pear-shaped flask and rotary evaporated at 37°C to remove the chloroform until a thin film is formed. The mixture is then vacuum dried overnight. An aqueous solution containing CD1d is added to the pear-shaped flask, and the mixture is placed in a shaker and hydrated at 37°C for 30-50 minutes. The mixture is then sonicated under ice bath conditions with a probe and an ultrasonic power of 100-600 kHz to obtain a CD1d / LGC-SS-LGC vesicle solution. Polysarcosine is added to the obtained CD1d / LGC-SS-LGC vesicle solution, and the mixture is allowed to stand and mix thoroughly to obtain the CD1d / LGC-SS-LGC / PSar dual-target carrier-free delivery system.

5. The method for preparing the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity according to claim 4, characterized in that: The volume percentage of chloroform in the chloroform / methanol mixture is 10%-40%.

6. The method for preparing the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity according to claim 4, characterized in that: The mass percentage of CD1d in the CD1d / LGC-SS-LGC vesicles is 10%-40%; the weight percentage of CD1d / LGC-SS-LGC to polysarcosine is 10%-60%.

7. The application of the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity prepared by the method according to any one of claims 1-6 in the preparation of targeted anticancer drugs.

8. The use of the dual-targeting carrier-free delivery system for enhancing NKT cell anti-tumor immunity prepared by the method according to any one of claims 1-6 in the preparation of agents that enhance NKT cell Th1 immune responses.

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