A macromolecular photosensitizer targeting macrophages and mediating NK cell therapy for solid tumors, and its preparation method and application

Abstract

The present invention relates to a macromolecular photosensitizer targeting macrophages and NK cell therapy for mediating solid tumors, and a preparation method and application thereof, and belongs to the field of medical technology. The macromolecular photosensitizer of the present invention includes a hydrophilic group A, a hydrophilic main chain B and a hydrophobic group C; the hydrophilic group A is mannose, the hydrophilic main chain B is a hyaluronic acid with a molecular weight between 4kDa and 40kDa, and the hydrophobic group C is a photosensitizer; the mannose and the photosensitizer are respectively connected to the hyaluronic acid. The present invention designs a simple and easy-to-obtain biocompatible preparation mHA-Ce6 to target and reset the TAM phenotype, thereby activating NK cells, while promoting NK cell proliferation with the aid of light, and finally realizing the powerful immune function of NK cells to kill tumor cells naturally. This can supplement the existing cell immunotherapy and provide a promising approach for the combined immunotherapy of tumors.

Description

Technical Field

[0001] The invention relates to a macromolecular photosensitizer for targeting macrophages and mediating NK cell therapy for solid tumors, and a preparation method and application thereof, belonging to the technical field of medicine. Background Art

[0002] As the first line of defense against hematological and solid malignancies, natural killer cells (NK cells) play a key role because their killing activity is not MHC-restricted and does not rely on antibodies to quickly identify and lyse malignant cells. This natural ability to kill cells directly after recognition has made them increasingly popular in the field of immunotherapy [1]. In recent decades, immunotherapy research has made significant progress in immune checkpoint inhibitors (ICI) such as PD-L1, PD-1 therapy [2], and cell therapy such as chimeric antigen receptor T lymphocytes and macrophages (CAR-T, CAR-M) [3]. Some have achieved great success from laboratory to clinical, but there are also some problems, such as immune checkpoint inhibitors being restricted by the poor tumor microenvironment and CAR-T therapy causing clinical side effects such as cytokine syndrome [4]. NK cells are a type of natural killer cells. Compared with T cells, NK cells do not require tumor cell antigen presentation, have the advantages of higher feasibility, lower risk of side effects, faster response, stronger cytokine release ability, and communication with other immune cells [5,6]. Among them, the ability of NK cells to communicate with other immune cells is of great interest. NK cells initiate activation by receiving soluble factors from the tumor microenvironment and interacting with receptors on their potential target cells. Several immune cell types, including M1-like polarized macrophages (M1), dendritic cells (DCs), and T cells, promote NK cell activation by producing cytokines such as IL-18, IL-12, IL-15, and IL-2 [7]. These cytokines can induce NK cells to secrete IFN-γ or TNF-α. They also increase the killing potential of NK cells by enhancing the expression of effector molecules (perforin and granzymes). Therefore, emerging NK-targeted immunotherapy provides an alternative approach for cancer patients who are not suitable for T cell-based therapies. However, despite the great success of NK adoptive immunotherapy in hematological cancers, the microenvironment of solid cancers severely impairs NK-mediated cytotoxicity by reducing infiltration, impairing target cell recognition, inhibiting activation, and impairing NK immunoregulatory and cytotoxic functions [8]. Therefore, restoring NK-mediated immune surveillance would provide a promising therapeutic target for patients with solid tumors. Currently, immunotherapies targeting NK cells, including traditional cytokine administration, bispecific or trispecific killer cell engagers (BiKEs or TriKEs), and recently developed transgenic NK cells (such as CAR-NKs), have been developed to restore the immune function of NK cells in an immunosuppressive microenvironment [9]. However, in fact, more attention should be paid to the effect of the tumor microenvironment on NK cell function. The tumor immunosuppressive microenvironment not only hinders the activation and proliferation of NK cells, the secretion of cytokines, the expression of activation receptors, and the lytic cell activity, but also affects the interaction between immune cells and NK cells in the tumor environment [10,11].Solid tumors recruit a large number of immune cells such as macrophages, dendritic cells and T cells to activate NK cells in different ways, but the tumor microenvironment limits the function of these immune cells. Among them, macrophages (TAMs) are the main type of immune cells in the tumor environment, accounting for 50% of the tumor mass

[12] . TAMs are roughly divided into anti-inflammatory phenotype M2 and pro-inflammatory phenotype M1, among which the M2 phenotype is the main phenotype of macrophages in tumor tissues. Because TAMs overexpress growth factors that promote angiogenesis, proteases that promote metastasis, and inhibitory molecules that inhibit adaptive immune responses, the infiltration of TAMs in tumor tissues and the expression of different phenotypes are associated with poor prognosis in a variety of human cancers [13,14]. Therefore, M2-like TAMs are new targets for the development of anticancer therapy. Although there is a lot of evidence that TAMs play an important role in regulating the tumor microenvironment and TAM-based therapies have been developed, such as direct killing or resetting phenotypes to kill tumors

[15] , there are few studies specifically developing a preparation to reset TAMs to restore the natural killing function of NK cells. Interleukin 10 (IL-10) secreted by TAMs can inhibit the production of local interleukin 12 (IL-12), which is one of the important cytokines that enables NK cells to exert cytotoxicity [16,17]. In addition, a variety of immunosuppressive factors released by TAMs, including TGF-β, indoleamine 2,3-dioxygenase and PGE2, can affect the killing activity of NK cells in tumors

[18] . This shows that TAMs can reduce the functional activity of NK cells in the tumor immunosuppressive environment. However, there are literatures showing that M1 macrophages can secrete IL-12 and interleukin 18 (IL-18), which can activate resting NK cells, thereby promoting NK cells to secrete some tumor-killing cytokines such as TNF-α and IFN-γ, which is completely opposite to the effect of M2-like TAMs [19,20]. Previous related studies have also shown that M1-like TAMs can indeed secrete IL-18, a factor that activates NK cells. In summary, different phenotypes of TAMs have different effects on NK cell function, and M1-like TAMs can activate NK cells whose natural killer activity is suppressed due to the influence of the tumor immunosuppressive microenvironment. In addition, previous studies unexpectedly found that under the action of light, the administration of a small amount of photosensitive substances can promote the proliferation of NK cells. This may be related to reactive oxygen species (ROS), a highly reactive chemical substance containing oxygen free radicals including peroxides, singlet oxygen, and hydroxyl free radicals. It has been reported that ROS can act as both an anti-tumor agent and a promoter

[21] . Increased ROS levels can inhibit tumor growth by inhibiting the cell cycle or induce cell apoptosis through intrinsic and extrinsic pathways, and can also induce cell proliferation, cell migration, and epithelial-mesenchymal transition through the activation of mitogen-activated protein kinase (MAPK) and Ras-ERK [22,23].Since normal cells have a higher threshold for ROS than tumor cells, regulating ROS within a certain range can cause tumor cells to die before normal cells. In short, whether ROS promotes cell proliferation or kills cells depends on the strength of the antioxidant system of different cells[24,25].

[0003] Based on the above background, it is necessary to develop a special preparation to reset TAM to restore the natural killing function of NK cells.

[0004] References

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[0028] 25. Zhang, Y., et al., Upregulation of Antioxidant Capacity and Nucleotide Precursor Availability Suffices for Oncogenic Transformation. CellMetabolism, 2021.33(1):p.94-109.e8. Summary of the invention

[0029] In order to overcome the deficiencies of the prior art, the present invention provides a macromolecular photosensitizer targeting macrophages and NK cell therapy mediating solid tumors, as well as a preparation method and application thereof.

[0030] In the present invention, the inventors have designed a photosensitive nanoparticle mHA-Ce6 (mHC) that can target TAMs. On the one hand, it can target M2-like TAMs and polarize M2 to M1 to activate NK cells. On the other hand, it can promote the in situ proliferation of NK cells through light action, thereby exerting the tumor-killing effect mediated by NK cells. Therefore, the research of the present invention reveals a simple and easy-to-obtain biocompatible macromolecular drug to target and reset the TAM phenotype, thereby activating NK cells, while promoting NK cell proliferation with the aid of light, and finally realizing the powerful natural killing tumor cell function of NK cells. This can supplement the existing cell immunotherapy and provide a promising approach for combined immunotherapy of tumors.

[0031] The technical solution of the present invention to solve the technical problem is as follows:

[0032] In a first aspect of the present invention, a macromolecular photosensitizing drug is provided that targets macrophages and mediates NK cell therapy of solid tumors.

[0033] The macromolecular photosensitizer includes a hydrophilic group A, a hydrophilic main chain B and a hydrophobic group C; the hydrophilic group A is mannose, the hydrophilic main chain B is hyaluronic acid with a molecular weight between 4kDa and 40kDa, and the hydrophobic group C is a photosensitizer; the mannose and the photosensitizer are respectively connected to the hyaluronic acid.

[0034] Mannose, because of the hydroxyl group in its structural formula, can form an ester bond with the carboxyl group on hyaluronic acid through the hydroxyl group;

[0035] The photosensitizer is preferably a hydrophobic photosensitizer containing a carboxyl group, such as chlorin Ce6, verteporfin, hematoporphyrin, pyropheophorbide a, etc. The hydrophobic photosensitizer containing a carboxyl group can be connected to the hydroxyl group on the hyaluronic acid by forming an ester bond through the carboxyl group.

[0036] Furthermore, in the macromolecular photosensitizer, the hydrophilic group A is mannose, the hydrophilic main chain B is hyaluronic acid with a molecular weight of 40 kDa, and the hydrophobic group C is the photosensitizer dihydrochlorin Ce6; the mannose forms an ester bond with the carboxyl group on the hyaluronic acid through a hydroxyl group; the photosensitizer dihydrochlorin Ce6 forms an ester bond with the hydroxyl group on the hyaluronic acid through a carboxyl group.

[0037] In a preferred embodiment of the present invention, in the macromolecular photosensitizer, the molar ratio of hyaluronic acid HA with a molecular weight of 40 kDa, mannose Man and photosensitizer Ce6 is 200:20:1.

[0038] In a preferred embodiment of the present invention, the particle size of the macromolecular photosensitizing drug is 233±10 nm.

[0039] In the second aspect of the present invention, a method for preparing the macromolecular photosensitizing drug as described in the first aspect is provided.

[0040] The method is directly obtained by two-step esterification reaction of the carboxyl group and hydroxyl group of hyaluronic acid with the hydroxyl group of mannose and the carboxyl group of Ce6, and specifically comprises the following steps:

[0041] 1) Preparation of mannose-modified mHA:

[0042] First, hyaluronic acid HA was dissolved in deionized water, EDC and NHS were added, and activated at room temperature for 2 hours. Then, mannose Man was added to the activated hyaluronic acid, reacted overnight for 24 hours, and freeze-dried to obtain mHA foam solid.

[0043] 2) Preparation of mannose-modified mHA-Ce6

[0044] First, the mHA prepared in step 1) is dissolved in deionized water to obtain a mannose-modified hyaluronic acid solution; then, Ce6 is taken, EDC and HOBT are added, and activated at room temperature for 2 hours, and then the activated Ce6 is added to the above mannose-modified hyaluronic acid solution, reacted overnight for 24 hours, and freeze-dried to obtain mHA-Ce6 foamy solid.

[0045] Furthermore, in the step 1), the molar ratio of hyaluronic acid HA to mannose Man is 10:1; in the step 2), the molar ratio of mHA to Ce6 is 200:1.

[0046] Furthermore, in the step 1), the molar ratio of Man:EDC:NHS is 1:1:1;

[0047] Furthermore, in the step 2), the molar ratio of Ce6:EDC:HOBT is 1:1:1.

[0048] In the third aspect of the present invention, there is provided the use of the macromolecular photosensitizer as described in the first aspect or the macromolecular photosensitizer prepared by the method described in the second aspect.

[0049] There are two main types of applications:

[0050] The first one is the application in the preparation of a drug for inducing polarization of M2 macrophages to M1 macrophages. This application is based on the fact that the macromolecular photosensitizer of the present invention has the ability to induce the transformation of M2-like macrophages to M1-like macrophages.

[0051] The second is its application in the preparation of drugs for enhancing the ability of NK cells to kill tumor cells. This application is based on the fact that the macromolecular photosensitizer of the present invention has the performance of enhancing the ability of NK cells to kill tumor cells under light conditions, acting alone or in combination with M1 macrophages.

[0052] The present invention is based on a relatively new concept of restoring the natural killer function of NK cells by resetting tumor-related giant cells to activate NK cells and promoting their proliferation with the help of light: hyaluronic acid polarizes M2 macrophages to M1 type, thereby secreting cytokine IL-18, and IL-18 is used to activate NK cells to secrete tumor-killing cytokines such as TNF-α and IFN-γ. At the same time, an appropriate amount of photosensitizer dihydrochlorin (Ce6) promotes the proliferation of NK cells under the action of light. NK cells are activated from two aspects, thereby exerting the immune killing effect mediated by NK cells.

[0053] The material hyaluronic acid involved in the present invention can increase the probability of targeting M2-like TAM after modification with mannose. The present invention uses low molecular weight (40kDa) hyaluronic acid, which has the ability to reset tumor-associated macrophages, that is, to transform M2-like macrophages toward M1-like macrophages. Then M1-like macrophages secrete IL-18 cytokines, which can activate NK cells to secrete TNF-α and IFN-γ that are toxic to tumors. Here, hyaluronic acid acts both as a carrier and a drug to regulate the phenotype of tumor-associated macrophages.

[0054] When the tumor-associated macrophages regulated by the present invention are M1, they will secrete IL-18, which can activate NK cells. Compared with the NK cell and tumor cell co-incubation group, the M2 macrophage group treated with the preparation in advance was given, and the ability of NK cells to kill tumors was significantly enhanced.

[0055] The photosensitive substance Ce6 involved in the present invention can generate ROS under the action of light. According to previous research results and the different levels of oxidative stress in different cells, by adjusting the appropriate Ce6 concentration to generate ROS, the tumor can be put in a dying state and promote the proliferation of NK cells, thereby enhancing the ability of NK cells to kill tumor cells.

[0056] The preparation process of the present invention mainly involves two steps of simple esterification reaction to construct an amphiphilic material, and a polymer spherical structure is self-assembled after stirring in water for 24 hours. The materials involved have good biocompatibility. In animal experiments, the administration method adopted is mouse tail vein injection, and light treatment is given the next day.

[0057] The present invention has the following technical effects:

[0058] 1) The present invention designs a simple and readily available biocompatible preparation mHA-Ce6 to target and reset the TAM phenotype, thereby activating NK cells, while promoting NK cell proliferation with the aid of light, and ultimately achieving the powerful immune function of NK cells to naturally kill tumor cells. This can supplement existing cell immunotherapy and provide a promising approach for combined immunotherapy of tumors. As can be seen from Example 3, the mHA-Ce6 preparation of the present invention has a good polarization effect, and the polarization effect of converting M2 to M1 is as high as 85.7%, while the polarization effect of the only preparation currently prepared with hyaluronic acid is about 60% according to investigations.

[0059] 2) As an immune adjuvant, the hyaluronic acid in the mHA-Ce6 preparation of the present invention can not only be used as a carrier to graft photosensitive substances, but also can be used to prepare mHA-Ce6 preparations, polarize M2 macrophages into M1 macrophages, and secrete IL-18, an important factor that activates NK cell activity.

[0060] 3) The photosensitive substance Ce6 in the mHA-Ce6 preparation of the present invention has a dual role. On the one hand, it can put the vitality of tumor cells in a fragile state, and on the other hand, it promotes the proliferation of NK cells, thereby helping NK cells to kill tumor cells more easily.

[0061] 4) The present invention has carried out pharmacodynamic tests on animals in compliance with animal ethics regulations, achieving a relatively ideal effect of delaying tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the preparation principle of preparation mHA-Ce6.

[0063] Figure 2 The particle size and electron microscope image of the preparation mHA-Ce6.

[0064] Figure 3 This is a flow cytometry analysis chart of the experiment in which the mHA-Ce6 preparation in Example 2 polarized M2-like macrophages into M1-like macrophages. Among them, *p<0.05, **p<0.01, ***p<0.001, n=3. Here, n means that there are 3 parallel groups.

[0065] Figure 4 This is a flow cytometry analysis chart of the experiment of resetting TAM activation of NK cells by mHA-Ce6, a preparation in Example 3. Among them, *p<0.05, **p<0.01, ***p<0.001, n=3. Here n means that there are 3 parallel groups.

[0066] Figure 5This is a flow cytometry analysis chart of the experiment on the ability of mHA-Ce6, a preparation in Example 4, to enhance NK cell killing tumors. Among them, *p<0.05, **p<0.01, ***p<0.001, n=3. Here n means that there are 3 parallel groups.

[0067] Figure 6 This is a graph showing the particle size of the preparation mHA-Ce6 in Example 5, which remains stable for 24 hours at the in vivo hyaluronidase concentration.

[0068] Figure 7 The cytotoxicity bar graph of the mHA-Ce6 preparation in Example 6, wherein n=3, where n means that there are 3 parallel groups.

[0069] Figure 8 The pharmacodynamic evaluation results of the preparation mHA-Ce6 in Example 7 at the animal level. DETAILED DESCRIPTION

[0070] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0071] Experimental materials and sources

[0072]

[0073] Example 1 Preparation of mHA-Ce6

[0074] 1. Prescription screening, screening the appropriate feed ratio

[0075] The preparation is directly obtained by two-step esterification reaction of the carboxyl and hydroxyl groups of hyaluronic acid with the hydroxyl groups of mannose and the carboxyl groups of Ce6. The inventor screened the prescription and selected the appropriate feed ratio to obtain the appropriate particle size.

[0076] The specific steps include:

[0077] 1) Preparation of mannose-modified mHA

[0078] First, hyaluronic acid HA was dissolved in deionized water, EDC and NHS were added, and activated at room temperature for 2 hours. Then, mannose Man was added to the activated hyaluronic acid, reacted overnight for 24 hours, and freeze-dried to obtain mHA foamy solid.

[0079] There are two options for the molar ratio of hyaluronic acid (HA) and mannose (Man), a and b, a = 10:1, b = 20:1, Man: EDC: NHS = 1:1:1 (molar ratio). The specific dosage is shown in Table 1:

[0080] Table 1

[0081]

[0082] 2) Preparation of mannose-modified mHA-Ce6

[0083] First, the mHA prepared in step 1) is dissolved in deionized water to obtain a mannose-modified hyaluronic acid solution; then, Ce6 is taken, EDC and HOBT are added, and activated at room temperature for 2 hours, and then the activated Ce6 is added to the above mannose-modified hyaluronic acid solution, reacted overnight for 24 hours, and freeze-dried to obtain mHA-Ce6 foamy solid.

[0084] There are three options for the molar ratio of mHA and Ce6, c, d and e, c = 50:1, d = 200:1, e = 500:1, Ce6: EDC: HOBT = 1:1:1 (molar ratio). See Table 2 for specific feed amounts:

[0085] Table 2

[0086]

[0087] After combining various feed ratios, the mHA-Ce6 was obtained and the measured particle size is shown in Table 3.

[0088] Table 3

[0089]

[0090] After comprehensive consideration, the combination of feed ratios a and d was selected as the ratio for the final preparation.

[0091] 2. Preparation of mHA-Ce6 with different molecular weights according to the feed ratio a and d

[0092] Preparation of 40kDa mHA-Ce6 (No. ①), 4kDa mHA-Ce6 (No. ②)

[0093] No. ①: In this experiment, 40kDa hyaluronic acid HA (100mg, 0.264mmol) was dissolved in 40ml deionized water, EDC (5.76mg, 0.03mmol) and NHS (3.45mg, 0.03mmol) were added for activation at room temperature for 2h, and then mannose Man (4.752mg, 0.0264mmol) was taken, reacted overnight for 24h, and freeze-dried to obtain mHA foam solid. mHA (50mg, 0.1319mmol) was dissolved in pure water and stirred for 2h. At the same time, dihydrochlorin Ce6 (0.4 mg, 0.00067 mmol) was taken, EDC (0.129 mg, 0.00067 mmol) and HOBT (0.102 mg, 0.00067 mmol) were added and activated at room temperature for 2 h, then added to the above mHA solution, reacted overnight for 24 h, and freeze-dried to obtain mHA-Ce6 foamy solid.

[0094] No. ②: In this experiment, 4kDa molecular weight hyaluronic acid HA (100mg, 0.264mmol) was dissolved in 40ml deionized water, EDC (5.76mg, 0.03mmol) and NHS (3.45mg, 0.03mmol) were added to activate at room temperature for 2h, and then mannose Man (4.752mg, 0.0264mmol) was taken, reacted overnight for 24h, and freeze-dried to obtain mHA foam solid. Take mHA (50mg, 0.1319mmol) and dissolve it in pure water and stir for 2h. At the same time, take dihydrochlorin Ce6 (0.4mg, 0.00067mmol), add EDC (0.129mg, 0.00067mmol), HOBT (0.102mg, 0.00067mmol) to activate at room temperature for 2h, then add it to the above mHA solution, react overnight for 24h, freeze-dry, and obtain mHA-Ce6 foam solid.

[0095] Figure 1 Schematic diagram of the preparation principle of preparation mHA-Ce6.

[0096] Figure 2 The particle size and electron microscope image of the preparation mHA-Ce6 of No. ① are shown. Figure 2It can be seen that the particle size of mHA-Ce6 is uniform, with a particle size of about 233nm. The electron microscope image shows a complete spherical structure, indicating that the material self-assembles and successfully prepares the nano-sized macromolecular photosensitizer mHA-Ce6. The obtained macromolecular photosensitizer mHA-Ce6, numbered ①, includes a hydrophilic group A, a hydrophilic main chain B and a hydrophobic group C; wherein the hydrophilic group A is mannose, the hydrophilic main chain B is a hyaluronic acid with a molecular weight of 40kDa, and the hydrophobic group C is the photosensitizer dihydrochlorin Ce6; mannose forms an ester bond with the carboxyl group on the hyaluronic acid through a hydroxyl group; the photosensitizer dihydrochlorin Ce6 forms an ester bond with the hydroxyl group on the hyaluronic acid through a carboxyl group, and the molar ratio of the hyaluronic acid HA with a molecular weight of 40kDa, mannose Man and the photosensitizer Ce6 is 200:20:1.

[0097] 3. Preparation of HA-Ce6 for comparison

[0098] Preparation of 40kDa HA-Ce6 (No. ③): In this experiment, 40kDa hyaluronic acid HA (50mg, 0.1319mmol) was dissolved in pure water and stirred for 2h. At the same time, dihydrochlorin Ce6 (0.4mg, 0.00067mmol) was taken, EDC (0.129mg, 0.00067mmol) and HOBT (0.102mg, 0.00067mmol) were added and activated at room temperature for 2h, then added to the above HA solution, reacted overnight for 24h, and freeze-dried to obtain HA-Ce6 foam solid.

[0099] Example 2: Formulation mHA-Ce6 polarizes M2-like macrophages into M1-like macrophages

[0100] In this experiment, 75% volume of tumor supernatant or 40ng / ml IL-4 was used to pretreat RAW264.7 cells for 24 hours. According to the observation of cell morphology under an ordinary optical microscope, the original M0-like macrophage round state will change to the M2-like macrophage spindle state. At the same time, flow cytometry was used to monitor the decrease in CD206 expression and the increase in CD86 expression to evaluate the generation of M2-like macrophages. Raw264.7 cells in the logarithmic growth phase were taken and made into a cell suspension. 1×10 5 The cells were plated in a 6-well plate at a density of 100 cells / mL, 1 ml of culture medium was added to each well, and then 3 / 4 volume of tumor supernatant or IL-4 was added, and the plated culture plate was placed in a CO2 incubator at 37°C for 24 hours. The culture plate was taken out, the culture medium was discarded, and the mHA-Ce6 NPs numbered ① in Example 1 were added, with two concentrations of 25ug / ml and 50ug / ml, and the mHA-Ce6 NPs numbered ② in Example 1 were also added, with two concentrations of 25ug / ml and 50ug / ml, and incubated at 37°C for 4 hours.

[0101] After 4 hours of culture, the plate was removed, the culture medium was discarded, 500 μL PBS was added to blow the cells, the cell suspension was transferred to a 1.5 ml centrifuge tube, centrifuged (1000 r / min, 3 min), the supernatant was discarded, 500 μL PBS was added to wash the cells, and centrifuged (1000 r / min, 3 min). 500 μL of paraformaldehyde cell fixative was added to each centrifuge tube and fixed for 20 minutes.

[0102] After fixation for 20 minutes, centrifuge, discard the supernatant, add 200 μL PBS solution for washing, discard the supernatant, and then add 500 μL membrane permeabilization agent to permeabilize the membrane for 20 minutes.

[0103] After 20 minutes of membrane permeabilization, centrifuge, discard the supernatant, and wash with 200μL PBS solution. Then add CD206 (1:80) and CD86 (1:160) to label the cells, label for 1 hour at 4°C, centrifuge and discard the supernatant, and wash twice with 200μL PBS. Finally, add 350ul PBS to resuspend, filter with a 400-mesh screen, and perform detection and analysis using a cell flow cytometer.

[0104] The experimental data results can be found in Figure 3 : Preparation mHA-Ce6 polarizes M2-like macrophages into M1-like macrophages. The experimental results show that CD206 has no significant increase, and the expression level of CD86 has a significant increase, that is, No. ①② in Example 1 has the ability to convert M2 macrophages into M1, and No. ① is better than No. ②, and the concentration of 50ug / ml is better than the concentration of 25ug / ml. In short, No. ① preparation prepared with hyaluronic acid with a molecular weight of 40kDa has a better effect on polarizing M2-like macrophages when administered at a concentration of 50ug / ml.

[0105] Judging from the M2 / M1 data, after administration of preparation No. ①, the ratio of the number of cells converted from M2 to M1 decreased from 35 to 5 (indicating that the ratio of M2 / M1 decreased from 35 to 5, the ratio of M2 being the percentage of M2-like macrophages in the total number of macrophages, and the ratio of M1 being the percentage of M1-like macrophages in the total number of macrophages), and the conversion rate reached 85.7%.

[0106] The conversion rate calculation formula is: (the ratio of the number of cells converted from M2 to M1 in the non-medication control group - the ratio of the number of cells converted from M2 to M1 in the medication group) ÷ the ratio of the number of cells converted from M2 to M1 in the non-medication control group × 100%.

[0107] Example 3 Preparation mHA-Ce6 resets TAM to activate NK cells

[0108] In this experiment, 75% volume of tumor supernatant or 40ng / ml IL-4 was used to pretreat RAW264.7 cells for 24 hours to produce M2 macrophages. Raw264.7 cells in the logarithmic growth phase were taken and made into a cell suspension. The cells were plated into a 6-well plate at a density of 1×105 / mL and divided into a control group, HA-Ce6, and mHA-Ce6NPs. 1ml of culture medium was added to each well, and then 3 / 4 volume of tumor supernatant or IL-4 was added, and the plated culture plate was placed in a CO2 incubator at 37°C and cultured for 24 hours.

[0109] After 24 hours of culture, HA-Ce6 No. ③ and mHA-Ce6 NPs No. ① in Example 1 were administered at a concentration of 25 ug / ml each and incubated for 24 hours.

[0110] After 24 hours of culture, NK cells were added to the macrophages in the above 6-well plate at a ratio of 1:2, and the cells were incubated for 24 hours. The cells were centrifuged (1000 r / min, 3 min), and the cell supernatant was taken. The IFN-γ concentration in the supernatant was analyzed using IFN-γ and TNF-α ELISA kits.

[0111] The experimental data results can be found in Figure 4 The experimental results showed that mHA-Ce6 could successfully polarize M2 macrophages into M1 macrophages and successfully activate NK cells to secrete the tumor-killing cytokine IFN-γ.

[0112] Example 4 Preparation mHA-Ce6 enhances the ability of NK cells to kill tumors

[0113] In this experiment, 4T1 cells were first plated with 100 μL per well in a 96-well plate and divided into 6 groups (n=3) as follows: 4T1 group alone (PBS group), NK, mHA-Ce6 NPs, mHA-Ce6 NPs+NK, NK+M1 and NK+mHA-Ce6 NPs+M1 groups, and cultured for 24 h. (mHA-Ce6 NPs are all preparations numbered ① in Example 1).

[0114] After 24 hours of culture, the supernatant was discarded and drugs were added according to the above groups, with concentrations of 25ug / ml each, NK (the ratio with 4T1 was 1:5), M1 (the volume with NK was 2:1), the total volume was 100ul, and culture was carried out for 12h.

[0115] After 12 hours of culture, the cells were illuminated for 2.5 minutes and cultured for another 12 hours.

[0116] After 12 hours of culture, 10ul of CCK-8 was added to each well. After 2 hours of culture, the cells were tested immediately and the tumor-killing ability of NK cells was evaluated according to the following formula.

[0117] The killing ability of NK cells against 4T1: [tumor control group - (experimental group - effector cell group)] / tumor control group.

[0118] The experimental data results can be found in Figure 5 The experimental results show that mHA-Ce6 can enhance the ability of NK to kill tumors alone, and can also work synergistically with M1 macrophages to enhance the ability of NK to kill tumors. The ability of NK to kill tumors in mHA-Ce6+M1+NK is higher than that of NK in mHA-Ce6+NK, and also higher than that of NK in M1 macrophages+NK.

[0119] Example 5: Preparation mHA-Ce6 can maintain stability at in vivo hyaluronidase concentrations

[0120] The concentration of hyaluronidase in the human body is between 60 and 100 ng / ml, and hyaluronidase can degrade hyaluronic acid into small molecule oligosaccharide state. In order to detect and analyze whether the preparation is degraded under the concentration of hyaluronidase in the body and whether it still maintains polarization ability, the stability and polarization ability of the preparation mHA-Ce6 are tested.

[0121] In this experiment, the preparation No. ① in Example 1 was first dissolved in 10 ml of pure water at a concentration of 25 ug / ml, and then hyaluronidase was added at a concentration of 100 ng / ml, and the mixture was stirred thoroughly, and the particle size was measured. The particle size was measured every 3 hours for 24 hours and recorded.

[0122] The experimental data results can be found in Figure 6 The experimental results showed that the particle size of the mHA-Ce6 preparation decreased from 240nm to about 150nm after 24 hours, and the process slowed down and became gentle. The preparation did not completely degrade within 24 hours and still had the particle size of nano-spherical particles.

[0123] Example 6 Cytotoxicity of formulation mHA-Ce6

[0124] In this experiment, MTT method and CCK-8 method were used to detect the in vitro cytotoxicity of Ce6, number ① and number ③ in Example 1 to RAW, 4T1 and NK cells under illumination and non-illumination, respectively.

[0125] 4T1 cells or RAW cells were seeded in a 96-well cell plate at 4×104 to 8×104 cells / well, and the corresponding culture medium was added. After culturing for 12 hours, different concentration gradients of Ce6, number ① and number ③ in Example 1 were prepared using incomplete DMEM culture medium, and incubated with the cells for 4 hours, and then illuminated or not illuminated, and cultured for another 8 hours.

[0126] The drug-containing medium was aspirated, and 15 μL of medium containing MTT (5 mg / mL) was added to each well. After incubation for 4 h, the medium containing MTT was aspirated, and 150 μL of DMSO was added to each well to dissolve the blue-purple crystals at the bottom of the 96-well plate. The absorbance at 490 nm was measured using an enzyme-labeled instrument to calculate the cell viability %.

[0127] NK cells were inoculated at 4×104 to 8×104 cells / well in a 96-well cell plate, and the corresponding culture medium was added. After culturing for 12 hours, different concentration gradients of Ce6, number ① and number ③ in Example 1 were prepared using NK-specific culture medium, and the cells were incubated for 4 hours, and then the cells were illuminated or not illuminated, and the culture was continued for 8 hours.

[0128] The drug-containing culture medium was aspirated, and 15 μL of CCK-8 solution was added to each well. After incubation for 4 h, the absorbance at 490 nm was directly measured using an ELISA reader, and the cell viability % was calculated according to formula 2-1.

[0129] Cell viability (%) = experimental group - control group / experimental group (2-1)

[0130] The experimental data results can be found in Figure 7 . Ce6 has no obvious killing effect on RAW cells in the given concentration range of 0.00-0.32ug / ml, whether illuminated or not, indicating that our preparation has no effect on the activity of RAW in this dosage range. The preparation mHA-Ce6 NPs still keeps NK cells highly active under both illumination and non-illumination, and we found that the activity of NK cells can be increased under the action of light and when the Ce6 concentration is in the range of 0.18-0.32ug / ml. We speculate that this may be because the appropriate amount of ROS produced by the appropriate Ce6 under the action of light promotes the active proliferation of NK cells.

[0131] Example 7 Results of pharmacodynamic evaluation of mHA-Ce6 on animals

[0132] Mouse breast cancer cells 4T1 in the logarithmic growth phase were digested with trypsin and diluted with culture medium or sterile PBS to a concentration of 1×10 7 The cell suspension of 100 μg / mL was stored on ice and immediately inoculated into the mammary pad near the right leg of female BALB / c mice (5-6 weeks old, weighing 18-22 g), with 120 μL injected per mouse. On the 8th day after tumor inoculation, the tumor grew to approximately 100 mm. 3The mice were randomly divided into 6 groups, 6 mice in each group: saline group (PBS group), HA group, mHA-Ce6 group, Ce6+L group, HA-Ce6 group and mHA-Ce6+L group (mHA-Ce6NPs are all numbered ① preparations in Example 1). On the 9th day after tumor implantation, drug administration began, with a Ce6 dose of 0.1 mg / kg, and the drug was injected every other day. The light group was treated with a 650nm red laser (100mW / cm 2 ) irradiated the tumor site of the mouse for 5 minutes. The tumor volume and weight of the mouse were measured every day to evaluate its anti-tumor effect. Similarly, on the 21st day of treatment, the mice were euthanized and the tumors and organs were collected and fixed in 4% paraformaldehyde.

[0133] The experimental data results can be found in Figure 8 After administration according to the above method, the combined treatment of the macromolecular photosensitizer mHA-Ce6 and light can slow down the tumor growth of mice compared with other groups, and the weight of mice has not changed significantly, indicating that mHA-Ce6 has biological safety. In short, the macromolecular photosensitizer mHA-Ce6 has a good anti-tumor effect under light.

[0134] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A macromolecular photosensitizer targeting macrophages and NK cell therapy for solid tumors, characterized in that: The macromolecular photosensitizer comprises a hydrophilic group A, a hydrophilic main chain B and a hydrophobic group C; In the macromolecular photosensitizer, the hydrophilic group A is mannose, the hydrophilic main chain B is hyaluronic acid with a molecular weight of 40 kDa, and the hydrophobic group C is the photosensitizer chlorin Ce6; the mannose forms an ester bond with the carboxyl group on the hyaluronic acid through the hydroxyl group; the photosensitizer chlorin Ce6 forms an ester bond with the hydroxyl group on the hyaluronic acid through the carboxyl group; In the macromolecular photosensitizer, the molar ratio of hyaluronic acid HA with a molecular weight of 40 kDa, mannose Man and photosensitizer Ce6 is 200:20:1; The particle size of the macromolecular photosensitizer is 233±10nm; The method for preparing the macromolecular photosensitizer specifically comprises the following steps: 1) Preparation of mannose-modified mHA: First, hyaluronic acid HA was dissolved in deionized water, EDC and NHS were added, and activated at room temperature for 2 hours. Then, mannose Man was added to the activated hyaluronic acid, reacted overnight for 24 hours, and freeze-dried to obtain mHA foam solid. 2) Preparation of mannose-modified mHA-Ce6 First, the mHA prepared in step 1) is dissolved in deionized water to obtain a mannose-modified hyaluronic acid solution; then, Ce6 is taken, EDC and HOBT are added, and activated at room temperature for 2 hours, and then the activated Ce6 is added to the above mannose-modified hyaluronic acid solution, reacted overnight for 24 hours, and freeze-dried to obtain mHA-Ce6 foamy solid.

2. The macromolecular photosensitizer according to claim 1, characterized in that: In step 1) of the preparation method, the molar ratio of hyaluronic acid HA to mannose Man is 10:1; in step 2), the molar ratio of mHA to Ce6 is 200:

1.

3. The macromolecular photosensitizer according to claim 1, characterized in that: In step 1) of the preparation method, the molar ratio of Man:EDC:NHS is 1:1:1; In step 2) of the preparation method, the molar ratio of Ce6:EDC:HOBT is 1:1:

1.

4. Use of the macromolecular photosensitizer according to any one of claims 1 to 3 in the preparation of a drug for inducing polarization of M2 macrophages to M1 macrophages.

5. Use of the macromolecular photosensitizer according to any one of claims 1 to 3 in the preparation of a drug for enhancing the ability of NK cells to kill tumor cells.

6. The use according to claim 5, characterized in that: In the application, the macromolecular photosensitizer acts under light conditions, either alone or in combination with M1 macrophages.

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