An immune-assisted patch for preventing postoperative tumor recurrence and its preparation method

Abstract

The present invention discloses an immune-assisted patch for preventing postoperative tumor recurrence and a preparation method thereof. The patch uses hydrophilic chitosan as a matrix, and the immune active components are dissolved, dispersed and mixed with the matrix, and then placed in a mold for solidification and molding; the immune active components are immune antibodies and immune adjuvants. The immune-assisted patch prepared by the present invention uses hydrophilic medical chitosan to electrostatically adsorb positively charged LDHs functional nanomaterials with acid regulation properties and anti-tumor immune adjuvant properties, and electrostatically adsorbs therapeutic antibodies that carry rich amino groups and can reverse tumor immunosuppression, and finally assembles into a biofunctional patch that can be disassembled under physiological conditions of the wound. The present invention has the characteristics of safe application and excellent performance, and is expected to serve as a new postoperative immune-assisted solution to promote tumor cure and benefit patients, and improve the long-term survival rate and quality of life of postoperative patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofunctional materials, and in particular relates to an immune-assisted patch for resisting postoperative tumor recurrence and a preparation method thereof. Background Art

[0002] Surgery is the primary treatment option for most patients with solid tumors. According to statistics from the 2015 Lancet Oncology Commission, over 80% of cancer patients worldwide require surgical treatment. A recent "2018 to 2040" annual projection model indicates that this demand will continue to grow by 52% by 2040. However, surgery alone is not enough to completely eliminate cancer cells from the body. The natural healing of wounds after surgery forces the surrounding tissue into a state of immunosuppression. This state easily stimulates the growth and spread of residual cancer cells while suppressing the activity of anti-cancer cells (such as cytotoxic T lymphocytes, natural killer cells, and antigen-presenting cells), leading to severe recurrence and metastasis within five years.

[0003] In the natural postoperative wound microenvironment, the immune system will prioritize repairing surgical trauma. This selective behavior forces the postoperative wound to rapidly accumulate a large number of M2 phenotype macrophages (also known as M2-TAMs). M2-TAMs are immunosuppressive cells that can stimulate angiogenesis, promote the migration, invasion, or migration of cancer cells into blood vessels, thereby suppressing the body's ability to defend against cancer cells and accelerating the occurrence, deterioration, and spread of cancer. Recent studies have found that the acidic microenvironment of the tumor is primarily responsible for the polarization of the M2 phenotype of macrophages. Neutralizing the slightly acidic environment of surgical wounds has been shown to reshape macrophages in the wound into immunostimulatory types (M1-TAMs), attacking residual cancer cells and significantly inhibiting tumor recurrence.

[0004] However, it cannot be ignored that in addition to the immunosuppressive surgical wound, the lymphatic system also plays an important role in promoting the survival and metastasis of residual cancer cells. The current postoperative research has not paid attention to solving this key problem. A large number of studies have confirmed that compared with direct blood metastasis, cancer cells are more inclined to invade remotely through nearby draining lymph nodes. This is because, unlike the blood environment, the draining lymph nodes have low iron ion content and are rich in oily substances. Cancer cells can adaptively improve their antioxidant capacity in this relatively safe environment and arm themselves against blood iron death. In addition, before metastasis occurs, cancer cells will preferentially use the peripheral drainage lymphatic vessels to immunosuppress anti-tumor killer T cells, antigen-presenting cells, etc. in the lymph nodes to ensure their own safe entry and achieve systemic metastasis. Summary of the Invention

[0005] Given that surgery is difficult to completely remove cancer cells from a patient's body, and that postoperative wounds can inhibit the body's anti-cancer immune response and synergize with draining lymph nodes to promote the proliferation and metastasis of residual cancer cells, the present invention provides an immune-assisted patch and its preparation method, which can be applied to postoperative wounds, respond to the wound microenvironment, and slowly and sustainably release immunotherapy components. The released immune antibodies and immune adjuvants utilize efficient synergistic effects to reshape surgical wounds and inhibit the cancer-promoting effects caused by trauma; at the same time, the two, by virtue of their size advantage, utilize the network connection between tumors and draining lymph nodes to enrich in the draining lymph nodes, fully activating the rich anti-tumor immune cells inside, removing residual cancer cells, awakening the body's long-term anti-tumor immune memory, and effectively preventing postoperative tumor recurrence.

[0006] The immune auxiliary patch for preventing postoperative tumor recurrence uses hydrophilic chitosan as a matrix, and the immune active components are dissolved, dispersed and mixed evenly with the matrix, and then placed in a mold for solidification and molding; the immune active components are immune antibodies and immune adjuvants.

[0007] The hydrophilic chitosan is one or more of carboxylated chitosan, carboxymethyl chitosan and methacryloyl carboxymethyl chitosan.

[0008] The immunoadjuvant is hydrotalcite, having a particle size of less than 100 nm. The divalent metal ions in the hydrotalcite layer are one or more of magnesium ions, calcium ions, manganese ions, nickel ions, copper ions, and zinc ions, and the trivalent metal ions are one or more of iron ions, aluminum ions, and cobalt ions. The molar ratio of the divalent metal ions to the trivalent metal ions is 1-20:1.

[0009] The molar ratio of the immune antibody and immune adjuvant to the hydrophilic chitosan is 1-20:1.

[0010] The specific conditions for the solidification molding are: vacuum drying at 4-60° C. for 1-24 hours.

[0011] The immune antibody is one or more of anti-programmed death protein-1 antibody, anti-programmed death ligand-1 antibody, anti-cytotoxic T lymphocyte-associated antigen 4 antibody, anti-lymphocyte activation gene 3 antibody, anti-T cell immunoglobulin mucin 3 antibody, anti-CD47 antibody, and anti-NKG2A antibody.

[0012] The preparation method of the immune adjuvant comprises: preparing a mixed metal salt solution by mixing divalent metal nitrate and trivalent metal nitrate in a molar ratio of 1-20:1, then rapidly mixing the mixed metal salt solution with a sodium hydroxide aqueous solution to precipitate nuclei, performing a crystallization reaction at 20-200° C. for 5 min-24 h, and finally centrifuging and washing at 4,000-15,000 rpm, and dispersing the precipitate in deionized water.

[0013] The molar ratio of the sum of the moles of the divalent metal nitrate and the trivalent metal nitrate to the sodium hydroxide is 1:1-100.

[0014] The use of the above-mentioned immune auxiliary patch in the preparation of an immune auxiliary patch for preventing postoperative tumor recurrence.

[0015] The immunoadjuvant patch prepared by the present invention utilizes hydrophilic medical chitosan to electrostatically adsorb positively charged LDHs-functionalized nanomaterials with acid-regulating and anti-tumor immune adjuvant properties. This electrostatically adsorbs therapeutic antibodies rich in amino groups that can reverse tumor immunosuppression, ultimately forming a biofunctional patch that disintegrates under physiological wound conditions. When the patch is implanted in situ at the postoperative wound site, the released immune antibodies can enter the tumor-draining lymph nodes and restore the function of immunosuppressive killer T cells. The released LDHs immune adjuvant modulates the wound's slightly acidic conditions, reversing the immunosuppressive to immunostimulatory state of tumor surgical trauma. The LDHs can also migrate to the tumor-draining lymph nodes, assisting the immune antibodies and enhancing the anti-tumor immune response. The immunoadjuvant patch of the present invention is designed to maintain and enhance the patient's own anti-cancer immune function, triggering a powerful, long-term anti-cancer effect. It leverages the patient's own immune system to combat residual cancer cells and inhibit tumor recurrence. With its safe application and excellent performance, the present invention holds promise as a novel postoperative immunoadjuvant solution, promoting tumor cure and benefiting patients, improving their long-term survival and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Schematic diagram of the structure of the immune-assisted patch that prevents postoperative tumor recurrence (a); Schematic diagram of the patch acting on the wound site after tumor resection in a mouse model (b).

[0017] Figure 2 : Schematic diagram of the location of the patch action site, tumor-draining lymph nodes, and contralateral lymph nodes in the mouse model (a); after the patch was attached to the postoperative wound, the enrichment amount of the patch-loaded immune active components in the tumor-draining lymph nodes and contralateral lymph nodes at different time periods (b) and the enrichment fluorescence image (c).

[0018] Figure 3 Figure 5: PD-1 expression (a, d), CD206 expression (b, e), and co-expression of CD80 and CD86 (c, f) on macrophages within the wound microenvironment on day 5 of patch-assisted immunotherapy. The Untreated group received no subsequent treatment after tumor resection; the CC group received a pure chitosan matrix patch after tumor resection; the aPD-1@CC group received a patch containing only the PD-1 antibody active component; and the LDH@aPD-1@CC group received a patch containing both the PD-1 antibody and the LDHs immune adjuvant active component.

[0019] Figure 4 : The levels of tumor-specific killer T cells (a, b), the proliferation level of killer T cells (c), and the expression level of toxic proteins (d) in the tumor-draining lymph nodes on the 5th and 30th days of patch-assisted immunotherapy. Figure 3 illustrate.

[0020] Figure 5 : Glutathione (GSH, a), oxidized glutathione (GSSG, b) and their ratio in tumor-draining lymph nodes on day 30 of patch-assisted immunotherapy (c); Regulatory T cell levels in tumor-draining lymph nodes on day 5 and day 30 of patch-assisted immunotherapy (d). Figure 3 illustrate.

[0021] Figure 6 :After the patch immune adjuvant effect, the tumor recurrence (a, b) and mouse survival rate (c) of each group of mice. Figure 3 illustrate.

[0022] Figure 7 :collect Figure 6 In the experiment, mice without relapse were implanted with the same type of tumor cells for the second time to determine the tumor-specific immune effect of the mice (a, b). Figure 3 illustrate. DETAILED DESCRIPTION

[0023] To ensure that the purpose, technical solutions, and technical uniqueness of the present invention are more clearly understood, the inventors have elaborated on the contents of the present invention in detail with reference to specific embodiments. The described embodiments only include some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] Example 1

[0025] Dissolve 4 mmol of magnesium nitrate and 1 mmol of ferric nitrate in 5 ml of deionized water to obtain a mixed salt solution A. Quickly pour solution A into 20 ml of sodium hydroxide aqueous solution (containing 10 mmol of sodium hydroxide) to obtain a mixed suspension B. After rapidly stirring suspension B at room temperature for half an hour, a light brown precipitate is obtained. Repeatedly centrifuge and wash the precipitate with a large amount of deionized water at 5,000 rpm for 5 minutes. The obtained precipitate is dispersed in 20 ml of deionized water to obtain a 15 mg / ml aqueous solution of LDHs nanoparticles, which is the immune adjuvant aqueous solution. The obtained LDHs are measured to have a hexagonal plate structure with a diameter of 20 to 40 nm and a charge of 30 to 38 mV.

[0026] Preparation of immune-adjuvant patch: Commercial carboxylated chitosan (CC) was dissolved in deionized water to prepare a CC aqueous solution with a concentration of 60 mg / ml. The 48-well plate cover required for the cell experiment was selected as the patch preparation template. After ultrasonic mixing of 200 μl of immune adjuvant aqueous solution, 50 μg of PD-1 antibody and 100 μl of CC aqueous solution, they were titrated into the cover plate holes and vacuum dried at 40 ° C for 1 hour to obtain a 10 mm diameter patch carrying PD-1 antibody and LDHs nanoparticles (LDH@aPD-1@CC). Its final morphology and components are shown in Figure 2. Figure 1 As shown in a.

[0027] Comparative Example 1

[0028] The patch preparation method was the same as that in Example 1, but without adding the immune adjuvant aqueous solution and PD-1, to obtain a circular CC patch with a diameter of 10 mm.

[0029] Comparative Example 2

[0030] The patch preparation method was the same as that in Example 1, but without adding the immune adjuvant aqueous solution, to obtain a 10 mm diameter patch carrying PD-1 antibodies (aPD-1@CC).

[0031] Application Example 1

[0032] To establish an orthotopic breast cancer model, 2×10 6 Murine 4T1 breast cancer cells were implanted in the right fourth mammary cavity of BALB / c female mice. Tumor growth was monitored, and tumor volume was calculated as 4 / 3π × (x / 2)^2 × (y / 2), where x is the tumor length and y is the tumor width perpendicular to x. Unless otherwise specified, mice were euthanized if their tumors reached 2 cm in length.

[0033] Patch surgery is performed when the tumor grows to about 500mm 3 Time period. Figure 1 As shown in b, after anesthetizing the mouse, the breast tumor was surgically removed and the LDH@aPD-1@CC patch in Example 1 was applied in situ. The immune-assisted patch was able to fit perfectly with the postoperative wound.

[0034] Example 2

[0035] Inserting fluorescently labeled peptides between the LDHs nanoparticle layers can achieve LDHs labeling and track the whereabouts of LDHs released after the patch disintegrates in the mouse body. 1 mg of the peptide SPSYVYHQF carrying the fluorescent group Cyanine-5.5 was selected and dissolved in 100 μl of DMSO solution. The obtained fluorescent peptide solution was dispersed in 2 ml of the LDHs nanoparticle aqueous solution prepared in Example 1. After 1 hour of stirring reaction, the fluorescent peptide was able to insert into the LDHs interlayer to obtain LDH-Cy5.5 nanoparticles. After repeated centrifugation (10,000 rpm, 5 min) and washing, the obtained particles were dispersed in deionized water to obtain a 15 mg / ml LDH-Cy5.5 nanoparticle aqueous solution.

[0036] As in Example 1, LDH was replaced with LDH-Cy5.5 to obtain the LDH-Cy5.5@aPD-1@CC patch.

[0037] As described in Application Example 1, the LDH-Cy5.5@aPD-1@CC patch was applied to the wound in situ after surgery. Before the patch application and on days 1, 2, 4, 8, 12, 24, and 36 after the patch application, the fourth tumor-draining lymph node on the right side of the mouse and the corresponding contralateral lymph node ( Figure 2 a, 4 mice at each time point). After digestion, the Fe content in the lymph nodes was determined by ICP-MS. One week after the patch was applied, the mice were surgically dissected, and the LDHs enrichment in the fourth tumor-draining lymph node was monitored by fluorescence imaging. Five weeks after the patch was applied, the mice were surgically dissected, and the LDHs enrichment in the lymph node contralateral to the fourth tumor-draining lymph node was monitored by fluorescence imaging. The results are shown in Figure 2. Figure 2 As shown in Figures 2b and 2c, during the first week after patch application, the active components released from the patch were concentrated in the tumor-draining lymph nodes. After the fifth week, some of the active components migrated to the distant contralateral lymph nodes. Analysis of the experimental results in Example 3 shows that the active components released from the patch can enter the peripheral draining lymph nodes.

[0038] Application Example 2

[0039] The tumors were surgically removed as described in Application Example 1, leaving 1‰ (w / w) of the tumor at the postoperative site to simulate postoperative residual tissue. The mice were divided into four groups of five mice each. The first group received no postoperative treatment and is labeled "Untreated"; the second group had the CC patch applied to their wounds; the third group had the aPD-1@CC patch applied to their wounds; and the fourth group had the LDH@aPD-1@CC patch applied to their wounds.

[0040] On the 5th day after surgery, tissues from the postoperative area of ​​mice in each group were collected and digested to obtain monodispersed cells. After fluorescent antibody staining and labeling, the phenotypic differentiation of macrophages in the postoperative wound was analyzed by flow cytometry. Figure 3 As shown in a, 3b, 3d and 3e, postoperative wound macrophages (TAMs) without any treatment or treated with CC patches showed high expression of PD-1 and CD206. These two are typical characteristics of immunosuppressive M2-TAMs. After treatment with the aPD-1@CC patch, the released PD-1 antibody can target PD-1 on the surface of M2-TAMs and alleviate its immunosuppressive properties (i.e., reduce CD206 expression). After treatment with the LDH@aPD-1@CC patch, the levels of PD-1 and CD206 on the surface of M2-TAMs were further significantly downregulated. Combined Figure 3 The results of c and 3f show that the LDH@aPD-1@CC patch caused a significant increase in CD80 and CD86 on the surface of TAMs (both of which are typical characteristics of immune-promoting M1-TAMs). We can conclude that the LDH@aPD-1@CC patch reversed the M2-TAMs in the postoperative wound into M1-TAMs, reshaped macrophages and exerted anti-tumor effects.

[0041] Application Example 3

[0042] The tumor was surgically resected as described in Application Example 1, leaving 1‰ (w / w) of the tumor at the postoperative site to simulate postoperative residual tissue. The mice were divided into four groups of 10 mice each. The first group received no postoperative treatment and is labeled "Untreated"; the second group had the CC patch applied to their wound; the third group had the aPD-1@CC patch applied to their wound; and the fourth group had the LDH@aPD-1@CC patch applied to their wound.

[0043] On the 5th and 30th days after surgery, 5 mice were randomly selected from each group, and their tumor-draining lymph nodes were collected. After digestion, monodispersed cells were obtained. After fluorescent antibody staining, the CD8 T cell stimulation results in the lymph nodes were analyzed by flow cytometry. Figure 4 As shown in a and 4b, under the action of aPD-1@CC or LDH@aPD-1@CC patch, tumor-specific CD8 + T cells (SPSYVYHQF-pentamer + CD8 + Compared with simple PD-1 antibody treatment, the addition of LDHs immune adjuvant further enhances the tumor-specific CD8 + The content of T cells increased to three times of the original level. On the 30th day after surgery, the above significant increase disappeared, which was due to the activation of tumor-specific CD8 + T cells can completely eliminate the residual cancer cells in the body in a short period of time. After the elimination is completed, these functional cells will retire. These speculations can be made by measuring CD8 +The proliferation of T cells (Ki67) and the expression level of cytotoxic protein (GzmB) were confirmed. Figure 4 As shown in c and 4d, under the action of LDH@aPD-1@CC patch, the results on the 5th and 30th days after surgery were compared. + T cells showed higher Ki67 in the former + and GzmB + level.

[0044] Application Example 4

[0045] The tumor was surgically removed as described in Application Example 1, leaving 1‰ (w / w) of the tumor at the postoperative site to simulate postoperative residual tissue. The mice were divided into four groups, each consisting of 15 mice. The first group received no postoperative treatment and is labeled "Untreated"; the second group had the CC patch applied to their wound; the third group had the aPD-1@CC patch applied to their wound; and the fourth group had the LDH@aPD-1@CC patch applied to their wound.

[0046] On the 30th day after surgery, 5 mice were randomly selected from each group, and their tumor-draining lymph nodes were collected and placed on ice, cryopreserved, and ultrasonically broken up. The levels of GSH and GSSG (oxidized glutathione) in the lymph nodes were measured using a glutathione (GSH) kit. Figure 5 As shown in a-5c, the presence of LDHs immune adjuvant in the patch can oxidize part of GSH to GSSG, downregulate the GSH / GSSG ratio, and increase the oxidative pressure in the lymph nodes. This change can downregulate the expression of immunosuppressive regulatory T cells (Foxp3 + CD4 + T).

[0047] On the 5th and 30th day after surgery, 5 mice were randomly selected from each group, and their tumor-draining lymph nodes were collected. After digestion, monodispersed cells were obtained. After fluorescent antibody staining, the expression of Foxp3 in the lymph nodes was analyzed by flow cytometry. + CD4 + T cell level. The results are as follows Figure 5 As shown in d, compared with the PD-1 antibody to Foxp3 + CD4 + LDHs immune adjuvant showed a more potent down-regulation function compared with the mild effect on T cells.

[0048] Application Example 5

[0049] The tumor was surgically removed as described in Application Example 1, leaving 1‰ (w / w) of the tumor at the postoperative site to simulate postoperative residual tissue. The mice were divided into four groups of six each. The first group received no postoperative treatment and is labeled "Untreated"; the second group had the CC patch applied to their wound; the third group had the aPD-1@CC patch applied to their wound; and the fourth group had the LDH@aPD-1@CC patch applied to their wound.

[0050] The tumor recurrence of mice in different groups was monitored. Figure 6 As shown, within 40 days after surgery, the tumors in mice without any treatment or treated with CC patches relapsed severely; the PD-1 antibody was able to inhibit the recurrence of 50% of mice; and the LDH@aPD-1@CC patch showed the best postoperative inhibitory effect, with only one of the six mice relapsing and the rest all cured.

[0051] Application Example 6

[0052] The cured / non-relapsed mice in each group of Application Example 5 were collected for secondary tumor implantation (1 in the second group, 3 in the third group, and 5 in the fourth group) to evaluate the tumor-specific immune activation of the mice. 6 Murine 4T1 breast cancer cells were implanted in the left fourth mammary gland of BALB / c female mice. Tumor growth was monitored, and tumor volume was calculated as 4 / 3π × (x / 2)^2 × (y / 2), where x is the tumor length and y is the tumor width perpendicular to x. Unless otherwise specified, mice were euthanized if their tumors reached 2 cm in length.

[0053] The tumor growth of mice in different groups was monitored. Figure 7 As shown, within 80 days after tumor implantation, only mice treated with the LDH@aPD-1@CC patch displayed robust tumor-specific immune activity, with 60% of the mice successfully resisting tumor cell growth, achieving tumor prevention. Combined with the tumor recurrence inhibition efficacy demonstrated in Example 7, the patch of the present invention effectively demonstrated its proposed goal of "preventing and treating" postoperative tumors.

[0054] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An immune-adjuvant patch for in situ implantation in a postoperative wound of a subject suffering from a tumor, characterized in that: The patch is made of hydrophilic chitosan as a matrix, and the immune active components are dissolved, dispersed and mixed evenly with the matrix, and then placed in a mold for solidification and molding; the immune active components are immune antibodies and immune adjuvants; The hydrophilic chitosan is carboxylated chitosan; The immune adjuvant is hydrotalcite, and its particle size is less than 100nm; The immune antibody is one or more of anti-programmed death protein-1 antibody, anti-programmed death ligand-1 antibody, anti-cytotoxic T lymphocyte-associated antigen 4 antibody, anti-lymphocyte activation gene 3 antibody, anti-T cell immunoglobulin mucin 3 antibody, anti-CD47 antibody, and anti-NKG2A antibody; The molar ratio of the immune antibody and immune adjuvant to the hydrophilic chitosan is 1-20:1; The specific conditions for the solidification molding are: vacuum drying at 4-60° C. for 1-24 hours.

2. The immune assist patch according to claim 1, characterized in that: The divalent metal ions in the hydrotalcite layer are one or more of magnesium ions, calcium ions, manganese ions, nickel ions, copper ions, and zinc ions; the trivalent metal ions are one or more of iron ions, aluminum ions, and cobalt ions; and the molar ratio of the divalent metal ions to the trivalent metal ions is 1-20:

1.

3. The immune assist patch according to claim 1, characterized in that: The hydrophilic chitosan is carboxymethyl chitosan.

4. The immune assist patch according to claim 1, characterized in that: The hydrophilic chitosan is methacrylated carboxymethyl chitosan.

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