Application of PD-L1 targeting affinity-nano-drug conjugate in preparation of drugs for diagnosing and treating colon cancer

Abstract

The invention discloses application of a PD-L1 targeting affinity-nano-drug conjugate in preparation of drugs for diagnosing and treating colon cancer, and belongs to the technical field of biological medicines. The PD-L1 targeting affinity body-nano drug conjugate is prepared from dopamine hydrochloride (DA), hollow mesoporous Prussian blue (HMPB) and a PD-L1 affinity body (ZPD-L1), and the PD-L1 targeting affinity body-nano drug conjugate is prepared from the dopamine hydrochloride (DA), the hollow mesoporous Prussian blue (HMPB) and the PD-L1 affinity body (ZPD-L1). According to the conjugate provided by the invention, the drug uptake of cells is improved through receptor-mediated endocytosis under the guidance of an affinity. As an excellent photo-thermal conversion agent, prussian blue can be used for photo-thermal imaging diagnosis under near-infrared laser excitation, and also can realize colon cancer photo-thermal treatment. The affinity-nano-drug conjugate disclosed by the invention has low immunogenicity, high tumor targeting property and photo-thermal conversion effect, and realizes a photo-thermal treatment effect on PD-L1 positive expression colon cancer under the guidance of photo-thermal imaging.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of PD-L1 targeted affinity body-nano drug conjugates in the preparation of drugs for diagnosing and treating colon cancer. Background Art

[0002] Colon cancer is a common malignant tumor of the digestive system, and its incidence and mortality are increasing year by year worldwide. Traditional treatments include surgical resection, radiotherapy, and chemotherapy. However, due to the heterogeneity of colon cancer and the complexity of the tumor microenvironment, conventional treatments are often limited in effect and prone to various side effects. Therefore, there is an urgent need to develop new treatment strategies to improve the treatment effect of colon cancer and the quality of life of patients. PD-L1 (Programmed cell death 1ligand 1) is a transmembrane protein that is positively expressed in colon cancer.

[0003] In recent years, photothermal therapy (PTT), as an emerging tumor treatment method, has attracted much attention due to its ability to achieve targeted heating and kill tumor cells. Photothermal therapy usually uses photosensitive materials to generate heat under specific wavelength light irradiation, leading to apoptosis or necrosis of tumor cells. Nanomaterials have also become a research hotspot in photothermal therapy due to their excellent photothermal conversion efficiency and good biocompatibility.

[0004] Prussian blue is a new type of near-infrared photothermal conversion agent that can generate heat under the irradiation of near-infrared lasers for photothermal therapy of tumors. However, whether this agent can be effectively used for targeted treatment of colon cancer and the preparation method of the drug have not been published. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides the preparation of PD-L1 targeted affinity body-nano drug conjugates and their application in the photothermal diagnosis and treatment of colon cancer, which has good photothermal imaging diagnosis and photothermal treatment effect under the guidance of photothermal imaging for colon cancer with positive expression of PD-L1. In order to achieve the above purpose, the present invention provides the following technical solutions: The present invention provides an application of a PD-L1 targeted affinity body-nano drug conjugate in the preparation of a drug for diagnosing and treating colon cancer. The preparation method of the PD-L1 targeted affinity body-nano drug conjugate comprises the following steps: Step 1, adding polyvinyl pyrrolidone (PVP) and potassium ferrocyanide (K3Fe[CN]·3H2O) to a dilute hydrochloric acid solution and stirring to fully dissolve them; Step 2, heating the solution obtained in step 1 to react to obtain a blue precipitate; Step 3, the blue precipitate prepared in step 2 is repeatedly washed by centrifugation with deionized water and anhydrous ethanol, and then vacuum dried to prepare Prussian blue nanoparticles; Step 4, adding polyvinyl pyrrolidone and the Prussian blue nanoparticles obtained in step 3 to the hydrochloric acid solution, and stirring to fully dissolve them; Step 5, transferring the solution obtained in step 4 to a reactor and heating it to obtain a blue precipitate; Step 6, the blue precipitate prepared in step 5 is repeatedly washed by centrifugation with deionized water and anhydrous ethanol, and then vacuum dried to prepare hollow mesoporous Prussian blue nanoparticles HMPB NPs; Step 7, dispersing the hollow mesoporous Prussian blue nanoparticles obtained in step 6 into Tris-HCl buffer, adding dopamine hydrochloride, stirring, polymerizing it on the surface of the hollow mesoporous Prussian blue nanoparticles to form a polydopamine PDA bionic layer, and finally washing it thoroughly by centrifugation with deionized water to obtain bionic hollow mesoporous Prussian blue nanoparticles HMPB / PDANPs; Step 8, dispersing the biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs obtained in step 7 into Tris-HCl buffer, adding PD-L1 affinity body Z PD-L1 , slowly stirred to connect it to the biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs, and finally washed thoroughly by centrifugation with deionized water to obtain the PD-L1 targeted affinity body-nano drug conjugate HMPB / PDA-Z PD-L1 NPs.

[0006] Preferably, the PD-L1 affinity body Z PD-L1 The amino acid sequence is shown in the sequence listing SEQ ID NO.2.

[0007] Preferably, the concentration of the dilute hydrochloric acid solution in step 1 is 0.01 mol / L, the concentration of polyvinyl pyrrolidone in the dilute hydrochloric acid solution is 75 mg / mL, and the concentration of potassium ferrocyanide in the dilute hydrochloric acid solution is 2.8725 mg / mL.

[0008] In step 1, the stirring method is magnetic stirring, the stirring speed is 400 rpm-600 rpm, and the stirring time is 0.5 h.

[0009] The heating method in step 2 is sealed heating, the heating temperature is 80°C, and the heating time is 20 h.

[0010] The centrifugation speed in step 3 is 14000 rpm and the centrifugation time is 20 min.

[0011] In step 4, the concentration of hydrochloric acid in the obtained solution is 1 mol / L, the concentration of polyvinyl pyrrolidone in the hydrochloric acid solution is 5 mg / mL, and the concentration of Prussian blue nanoparticles in the hydrochloric acid solution is 1 mg / mL.

[0012] The heating temperature in step 5 is 140°C and the heating time is 4 h.

[0013] The centrifugation speed in step 6 is 14000 rpm and the centrifugation time is 20 min.

[0014] In step 7, the concentration of hollow mesoporous Prussian blue nanoparticles HMPB in Tris-HCl buffer is 0.5 mg / mL, and the concentration of dopamine hydrochloride in Tris-HCl buffer is 0.2 mg / mL. The process of forming the polydopamine PDA bionic layer requires stirring. The stirring method is aerobic magnetic stirring, the stirring speed is 500 rpm, and the stirring time is 1 h. The speed of thorough centrifugation and washing with deionized water is 16000 rpm, and the single centrifugation time is 20 min.

[0015] In step 8, the concentration of biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs in Tris-HCl buffer is 0.5 mg / mL, and the PD-L1 affinity body Z PD-L1 The concentration of PD-L1 Affibody Z was 0.01 mg / mL in Tris-HCl buffer. PD-L1 It is prepared by inducing expression and purifying Z in an E. coli expression system through genetic engineering. PD-L1 The protein has an amino acid sequence as shown in the sequence table SEQ ID NO. 2. The power of the ultrasonic cleaning machine was set to 45 KHz, 40 W, the time was 2 min, the stirring speed was 300 rpm, the stirring time was 12 h, and the speed of the thorough centrifugal cleaning with deionized water was 16000 rpm, and the single centrifugation time was 20 min.

[0016] The PD-L1 targeted affinity body-nano drug conjugate provided by the present invention can be used as a precise photothermal diagnosis and treatment drug for colon cancer based on the excellent photothermal conversion effect of Prussian blue.

[0017] The present invention explores HMPB / PDA-Z PD-L1 The therapeutic effect of NPs in the colon cancer mouse tumor model. PD-L1 The treatment with NPs inhibited the growth of tumors and had a beneficial effect on the treatment of colon cancer. Therefore, it can be used as a drug for the treatment of colon cancer.

[0018] The present invention combines PD-L1 targeting molecules with photothermal therapy, which can generate heat at specific targets under the irradiation of near-infrared (NIR) lasers, thereby improving the therapeutic effect. By constructing PD-L1 targeted affinity body nanodrug conjugates, it can effectively bind to tumor cells and achieve precise treatment under near-infrared (NIR) laser irradiation, thus providing a more effective treatment strategy for patients with colon cancer.

[0019] In order to solve the above problems, the present invention focuses on developing a nano-drug conjugate with a PD-L1 targeting affinity body, combined with photothermal therapy of tumors, aiming to improve the therapeutic effect of colon cancer. The nano-drug conjugate can accurately recognize and bind to PD-L1 on the surface of tumor cells, promoting targeted delivery of drugs. At the same time, efficient killing of tumor cells is achieved through photothermal effect.

[0020] The drug contains PD-L1 targeting affinity body-nano drug conjugate HMPB / PDA-ZPD-L1 NPs as an active ingredient and is in the form of an injection.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an application of a PD-L1 targeted affinity body-nano drug conjugate in photothermal diagnosis and treatment of colon cancer. The PD-L1 targeted affinity body-nano drug conjugate utilizes dopamine hydrochloride (DA), hollow mesoporous Prussian blue (HMPB) and PD-L1 affinity body (Z PD-L1 ). By tail vein injection, the PD-L1 targeted affinity body-nano drug conjugate can reach the tumor site under the guidance of the affinity body, and improve the cell's uptake of the drug through receptor-mediated endocytosis, playing a role in the diagnosis of PD-L1 positive expression of colon cancer under photothermal imaging and photothermal treatment under photothermal guidance.

[0022] 2. The present invention uses hollow mesoporous Prussian blue (HMPB) as a matrix, and coats it with polydopamine (PDA) and PD-L1 affinity body (Z PD-L1 ) is modified, the preparation method is simple, the raw materials used are low-priced, the production efficiency is high, and it is suitable for large-scale production applications.

[0023] 3. PD-L1 targeted affinity body-nano drug conjugate HMPB / PDA-Z prepared by the present invention PD-L1 NPs have excellent biocompatibility, low immunogenicity, significant photothermal conversion effect, and excellent tumor targeting for PD-L1-positive colon cancer.

[0024] 4. The present invention utilizes PD-L1 affinity body (Z PD-L1)-modified polydopamine (PDA)-encapsulated hollow mesoporous Prussian blue nanoparticles (HMPB / PDA NPs) were used to prepare HMPB / PDA-Z with stable solid-state nanostructures. PD-L1 NPs. HMPB / PDA-Z was achieved by binding to PD-L1 positive colon cancer cells. PD-L1 Targeted delivery of NPs to colon cancer tumors. HMPB / PDA-Z accumulated in tumor tissues PD-L1 NPs, through the excitation of near-infrared (NIR) lasers, can be used for photothermal imaging for diagnosis and can also achieve photothermal treatment of colon cancer.

[0025] 5. The present invention develops a photothermal therapy nanodrug conjugate with a PD-L1 targeting affinity body, which can not only improve the therapeutic effect of colon cancer, but also provide a new idea and method for tumor treatment, and has important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 PB, HMPB, HMPB / PDA and HMPB / PDA-Z in Example 1 PD-L1 Transmission electron microscopy images of NPs. A is the transmission electron microscopy image of PB, B is the transmission electron microscopy image of HMPB, C is the transmission electron microscopy image of HMPB / PDA, and D is the transmission electron microscopy image of HMPB / PDA-Z PD-L1 Transmission electron microscopy images of NPs.

[0028] Figure 2 For example 2 to verify HMPB / PDA-Z PD-L1 A series of characterization images of the successful preparation of NPs. A is the SEM image of PB, HMPB, and HMPB / PDA NPs, and B is the SEM image of HMPB / PDA-Z PD-L1 Mapping diagram of NPs, C is HMPB, HMPB / PDA and HMPB / PDA-Z PD-L1 Dynamic light scattering (DLS) particle size and polymer dispersibility index (PDI) of NPs, D for HMPB, HMPB / PDA and HMPB / PDA-Z PD-L1 Zeta potential of NPs.

[0029] Figure 3 HMPB / PDA-Z measured by thermocouple in Example 3 PD-L1The photothermal conversion effect diagram of NPs. A is the temperature rise effect diagram of different NPs concentrations at the same optical density, B is the temperature rise effect diagram of different optical densities at the same NPs concentration, and C is the photothermal stability effect diagram of NPs.

[0030] Figure 4 HMPB / PDA-Z in Example 4 PD-L1 The results of NPs targeting PD-L1 positive colon cancer cells. A is the cell targeting HMPB / PDA and HMPB / PDA-Z measured by ICP-OES. PD-L1 A is the result of NPs uptake, B is the temperature rise measured by the thermal imaging system within the same time period, and C is the temperature change diagram measured by the thermal imaging system.

[0031] Figure 5 This is a diagram showing the killing effect of photothermal conversion on cancer cells in Example 5. A is the cell survival rate measured using a CCK-8 kit, and B is an immunofluorescence detection diagram using a Calcein / PI cell activity and cytotoxicity detection kit.

[0032] Figure 6 HMPB / PDA-Z in Example 6 PD-L1 Figure 1 shows the results of the biocompatibility study of NPs. A represents HMPB / PDA and HMPB / PDA-Z PD-L1 A is a diagram showing the hemolysis effect of NPs; B is a statistical analysis of the hemolysis rates of the two groups of materials; C is a statistical analysis of the uptake of the two materials by RAW264.7.

[0033] Figure 7 HMPB / PDA-Z in Example 7 PD-L1 Figure 1 shows the in vivo targeting effect of NPs in a tumor cell-bearing model. A is the result of in vivo imaging of small animals; B is the statistical analysis of the local fluorescence intensity of the tumor at each time point in A; C is the fluorescence distribution in various ex vivo tissues of tumor-bearing mice, including the brain, heart, liver, spleen, lung, kidney, muscle, and tumor, 12 hours after drug administration; D is the statistical analysis of the fluorescence intensity in each ex vivo tissue in C, with muscle tissue as a reference; E is the thermal imaging effect of near-infrared light (NIR) irradiating the local tumor; F is the change in local tumor temperature within 5 minutes.

[0034] Figure 8 HMPB / PDA-Z in Example 8 PD-L1NPs-mediated in vivo PTT significantly inhibits the growth of PD-L1-positive MC-38 tumors. A is the tumor growth curve of MC-38 tumor-bearing mice in the PBS, NIR, HP / PZ, HP / P+NIR and HP / P-Z+NIR treatment groups; B is the weight curve of tumor-bearing mice in each group; C is the weight of the ex vivo tumor tissue; D is a photo of the ex vivo tumor tissue. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] Materials and reagents: polyvinylpyrrolidone (PVP), potassium ferrocyanide (K3Fe[CN]·3H2O), concentrated hydrochloric acid (HCl), dopamine hydrochloride (DA), Tris-HCl buffer, PD-L1 affinity PD-L1 ), anhydrous ethanol.

[0037] Since the high-polymerization state can significantly improve the specific affinity of the affinity body, this study designed to add a cysteine ​​residue to the C-terminus of the PD-L1-specific ZM1 affinity body and construct the PD-L1-specific dimer affinity body ZM1 by disulfide bond. PD-L1 Then the synthetic expression gene was optimized and induced to express in the E. coli expression system through genetic engineering, and purified to prepare Z PD-L1 protein.

[0038] Z PD-L1 The nucleotide sequence is: 5-gcagaggccaagtacgcgaaggagcacatgatggctgcttccgaaattctgcagctgccgaatctgaccttcatccaaaaattcgtgttt atcagcaaactctcggatgatccgagccaaagcagcgagctgctgtccgaggcgaagaagttgaatgatagccaggcaccgaaaggtagttgc-3(SEQ ID NO.1);Z PD-L1 The amino acid sequence is: NH2-AEAKYAKEHMMAASEILQLPNLTFIQKFVFISKLSDDPSQSSELLSEAKKLNDSQAPKGSC-COOH (SEQ ID NO. 2).

[0039] Example 1, PD-L1 targeted affinity body-nano drug conjugate HMPB / PDA-Z PD-L1 Preparation of NPs: (1) Use a measuring cylinder to measure 160 mL of deionized water and pour it into a narrow-necked bottle. Use a pipette to take 133.4 μL of HCl and add it to the narrow-necked bottle to prepare 160 mL of solution to obtain a dilute hydrochloric acid solution with a concentration of 0.01 mol / L.

[0040] (2) Accurately weigh 12 g of PVP into a 250 mL beaker, add 150 mL of the HCl solution prepared in (1), and stir at room temperature to dissolve the PVP.

[0041] (3) Accurately weigh 452.4 mg K3Fe[CN]·3H2O into a 50-mL beaker and add 10 mL of the HCl solution prepared in (1). After complete dissolution, transfer the solution to the solution in (2) using a pipette. Stir with a sealed magnetic stirrer at 400-600 rpm for 30 min at room temperature to allow complete dissolution.

[0042] (4) After stirring, remove the rotor, ensure that it is tightly sealed, transfer it to an electric oven, seal it and heat it at 80 °C, and react for 20 h to obtain a blue precipitate.

[0043] (5) After the reaction, the blue precipitate was cooled to room temperature, centrifuged at 14,000 rpm for 20 min, and then repeatedly rinsed with deionized water and anhydrous ethanol. It was then placed in a vacuum drying oven at 35 °C and dried overnight to prepare Prussian blue (PB) nanoparticles.

[0044] (6) Use a measuring cylinder to measure 150 mL of deionized water and pour it into a narrow-necked bottle. Use a pipette to take 15 mL of HCl and add it to the narrow-necked bottle to prepare 165 mL of solution.

[0045] (7) Accurately weigh 900 mg of PVP and add it to a 500 mL beaker. Add the HCl solution prepared in (6) and stir at room temperature to dissolve the PVP.

[0046] (8) Weigh 180 mg of PB nanoparticles, disperse them in 15 mL of deionized water, and ultrasonicate them at 45 kHz for 3 min. Transfer them to the solution in (7) using a pipette and stir them with a sealed magnetic stirrer at 600 rpm for 2 h at room temperature. The hydrochloric acid concentration in the solution obtained in this step is 1 mol / L.

[0047] (9) Transfer the solution of (8) into a reactor, make sure the reactor is tightened, transfer it to an electric oven, heat it at 140 °C, and react for 4 h to obtain a blue precipitate.

[0048] (10) After the reaction, the reactor was cooled to room temperature, the liquid in the reactor was centrifuged at 14,000 rpm for 20 min, and then repeatedly rinsed with deionized water and anhydrous ethanol. It was then placed in a vacuum drying oven at 35 °C and dried overnight to obtain hollow mesoporous Prussian blue (HMPB) nanoparticles.

[0049] (11) Weigh 10 mg of HMPB nanoparticles, add 20 mL of Tris-HCl and disperse them in a 50 mL flask, and sonicate at 45 kHz for 2–3 min.

[0050] (12) Weigh 4 mg of DA and add it to the flask in (11). Stir the mixture with aerobic magnetic stirring at 500 rpm for 1 h.

[0051] (13) The liquid in the flask (12) was centrifuged at 16,000 rpm for 20 min, the supernatant was discarded, and the mixture was washed twice with deionized water at 16,000 rpm for 20 min each time to obtain washed HMPB / PDA NPs.

[0052] (14) The HMPB / PDA NPs in (13) were dispersed with 2 mL of Tris-HCl. The HMPB NPs and HMPB / PDA NPs were quickly quantified using ICP-OES. A total of 5 mg of HMPB was extracted from HMPB / PDA NPs, which was made up to 10 mL of Tris-HCl. The mixture was dispersed in a 25 mL flask and sonicated at 45 kHz for 2–3 min.

[0053] (15) Add 100 μg PD-L1 affibody (Z PD-L1 ) into the flask of (14), ultrasonically treated at 45 kHz and 40 W for 2 min, and then stirred at 300 rpm for 12 h.

[0054] (16) The liquid obtained in (15) was centrifuged at 16000 rpm for 20 min, the supernatant was discarded, and the purified HMPB / PDA-Z was obtained by thorough centrifugation and washing with deionized water. PD-L1 NPs.

[0055] (17) Transmission electron microscopy (TEM) was used to characterize the morphology and size of each product. Figure 1 As shown in A, B, C, and D, each nanoparticle has a relatively uniform shape and size. TEM images of PB, HMPB, HMPB / PDA, and HMPB / PDA-Z PD-L1 The average sizes are 186.34 nm, 131.82 nm, 179.99 nm, and 155.60 nm, respectively. Example 2, PD-L1 targeted affinity body-nano drug conjugate HMPB / PDA-Z PD-L1 The successful preparation of NPs was verified.

[0056] like Figure 2 As shown in Figure A, the present invention uses a scanning electron microscope (SEM) to compare the surface features of PB, HMPB, and HMPB / PDA, intuitively proving the successful preparation of HMPB and the successful encapsulation of PDA. The present invention uses an energy dispersive X-ray spectrometer (EDS) to perform X-ray energy spectrum analysis on the particles, as shown in Figure 1. Figure 2 As shown in B, it is the concentration and distribution of C, N, O, S, and Fe at the mapping level. The presence of S element proves that Z PD-L1 The hydrodynamic size and polymer dispersibility index (PDI) of the nanoparticles were then measured. Figure 2 C), Zeta potential ( Figure 2 D), where HMPB is denoted as HP, HMPB / PDA is denoted as HP / P, and HMPB / PDA-Z PD-L1 Denoted as HP / PZ; HMPB / PDA-Z PD-L1 The PDI and Zeta potential of NPs indicated that they could form stable dispersed solutions and possessed stable colloidal dispersion.

[0057] Example 3, HMPB / PDA-Z was studied PD-L1 Photothermal conversion properties of NPs The present invention uses a near-infrared laser generator and a thermocouple to verify this. Figure 3 Medium A, at 0.5 W / cm 2 Under NIR irradiation with high power density, H2O, 1 μg / mL, 10 μg / mL, 50 μg / mL, and 200 μg / mL of HMPB / PDA-Z were detected in the same time. PD-L1 It also shows different heating effects, and the greater the concentration of nanoparticles, the more obvious the heating effect and the higher the photothermal conversion efficiency; Figure 3 Medium B, 50 μg / mL HMPB / PDA-Z PD-L1 NPs, at 0.5 W / cm 2 , 1 W / cm 2 , 1.5 W / cm 2 , 2 W / cm 2 Under the irradiation of four different power density NIRs, the greater the laser power density in the same time, the more obvious the heating effect; Figure 3 Middle C, 50 μg / mL HMPB / PDA-Z PD-L1, at 0.5 W / cm 2 Under the irradiation of NIR with high power density, it can maintain its heating effect in the same time and has good photothermal stability. PD-L1 NPs have good photothermal conversion properties.

[0058] Example 4, HMPB / PDA-Z was studied PD-L1 Targeting of NPs to PD-L1-positive colon cancer cells.

[0059] The present invention uses an inductively coupled plasma optical emission spectrometer (ICP-OES) and a thermal imaging system to analyze the effects of MC-38 on HMPB / PDA NPs, HMPB / PDA-Z PD-L1 The uptake of NPs was studied and the Z PD-L1 First, the present invention uses ICP-OES to quantitatively analyze the characteristic element Fe, such as Figure 4 A, HMPB / PDA is recorded as HP / P, HMPB / PDA-Z PD-L1 Denoted as HP / PZ, MC-38 vs. HMPB / PDA-Z PD-L1 The uptake of NPs was significantly higher than that of HMPB / PDANPs, and the uptake of HMPB / PDA-Z PD-L1 The uptake of NPs was significantly lower than that of the non-blocking group on HMPB / PDA-Z PD-L1 The uptake of NPs can explain the HMPB / PDA-Z PD-L1 NPs have a certain targeting effect on PD-L1 positively expressed colon cancer cells. At the same time, the present invention uses a thermal imaging system to verify its targeting effect, such as Figure 4 In B and C, HMPB / PDA is denoted as HP / P, HMPB / PDA-Z PD-L1 Denoted as HP / PZ, according to the conclusion of Example 3, the magnitude of the temperature change can reflect the amount of NPs taken up by MC-38. The temperature change of HP / PZ group>HP / P group>Blocking group, that is, the amount of NPs taken up by MC-38 in HP / PZ group>HP / P group>Blocking group, which shows that HMPB / PDA-Z PD-L1 NPs have a certain targeting effect on colon cancer cells.

[0060] Example 5, exploring HMPB / PDA-Z PD-L1 Photothermal conversion of NPs kills cancer cells.

[0061] The present invention used near-infrared (NIR) laser to irradiate MC-38 cells co-incubated with HMPB / PDA (denoted as HP / P in the figure) NPs and HMPB / PDA-ZPD-L1 (denoted as HP / PZ in the figure) NPs, and then used CCK-8 kit ( Figure 5 A) and Calcein / PI cell viability and cytotoxicity assay kit ( Figure 5 B) The cell survival rates were tested respectively. The cell survival rates of the two NIR-treated experimental groups decreased significantly, and the HMPB / PDA-Z PD-L1 The cell survival rate in the NPs group was also significantly lower than that in the HMPB / PDA group after NIR application, indicating that HMPB / PDA-Z PD-L1 The photothermal effect of NPs can effectively kill cancer cells.

[0062] Example 6, HMPB / PDA-Z was studied PD-L1 Biocompatibility of NPs.

[0063] Using different concentrations of HMPB / PDA (denoted as HP / P in the figure) and HMPB / PDA-Z PD-L1 (indicated as HP / PZ in the figure) NPs were subjected to hemolysis experiment ( Figure 6 A), HMPB / PDA-Z at 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. PD-L1 The hemolysis rates of NPs were all lower than 5%, and were lower than those of the HMPB / PDA group ( Figure 6 The present invention also uses RAW264.7 cells to detect HMPB / PDA (denoted as HP / P in the figure) and HMPB / PDA-Z PD-L1 The uptake experiment of HMPB / PDA-Z was carried out by RAW264.7. PD- L1 The uptake of NPs was lower than that of HMPB / PDA ( Figure 6 Middle C), HMPB / PDA-Z after modification with affinity body PD- L1 The immunogenicity of NPs is reduced. Therefore, the inventors believe that HMPB / PDA-Z PD-L1 NPs have good biocompatibility.

[0064] Example 7, HMPB / PDA-Z was studied PD-L1 In vivo targeting effect of NPs in a tumor cell-bearing model.

[0065] The present invention constructed an MC-38 tumor-bearing mouse model, and used Cy5.5 to label HMPB / PDA NPs (HMPB / PDA-Cy5.5 NPs) and HMPB / PDA-Z PD-L1 NPs(HMPB / PDA-Z PD-L1 -Cy5.5 NPs), injected through the tail vein, and using the IVIS imaging system, the present invention investigated the HMPB / PDA-Cy5.5 NPs (denoted as HP / PZ in the figure) and HMPB / PDA-Z at 0 hours, 2 hours, 4 hours and 6 hours after administration. PD-L1 -Distribution of Cy5.5 NPs (denoted as HP / P in the figure) in mice, e.g. Figure 7 As shown in A, after administration of HMPB / PDA-Cy5.5 NPs and HMPB / PDA-Z PD-L1 -Cy5.5 NPs, HMPB / PDA-Z PD-L1 -Cy5.5 NPs group HMPB / PDA-Z PD-L1 -Cy5.5 NPs accumulated more in the tumor site than the HMPB / PDA-Cy5.5NPs group. At the same time, the fluorescence intensity of the local tumor was compared and statistically analyzed, such as Figure 7 As shown in middle B, HMPB / PDA-Z in the tumor 2 hours, 4 hours and 6 hours after drug administration PD-L1 -Cy5.5 NPs had a higher average fluorescence intensity than HMPB / PDA-Cy5.5. In addition, the Cy5.5 fluorescence signal in the brain of each group decreased significantly from 2 hours, 4 hours, and 6 hours, indicating that the Cy5.5 fluorescence signal in each group decreased similarly over time. The present invention also investigated the effect of HMPB / PDA-Cy5.5 NPs and HMPB / PDA-Z on the fluorescence intensity of the Cy5.5 NPs and HMPB / PDA-Z at 12 h after administration. PD-L1 -Distribution of Cy5.5 NPs in various ex vivo tissues of tumor-bearing mice including brain, heart, liver, spleen, lung, kidney, muscle and tumor ( Figure 7 C), and the fluorescence intensity in the isolated tissues was compared and statistically analyzed ( Figure 7 (D) The analysis showed that HMPB / PDA-Cy5.5 NPs and HMPB / PDA-Z PD-L1 - There was no significant difference in the accumulation of Cy5.5 NPs in the brain, heart, liver, spleen, lung, and muscle, but HMPB / PDA-Z PD-L1 -Cy5.5 NPs accumulated significantly more and less in tumors and kidneys than HMPB / PDA-Cy5.5 NPs, respectively.

[0066] The present invention uses near-infrared (NIR) laser to irradiate the local tumor of tumor-bearing mice, and uses thermal imaging technology to detect the temperature change of the local tumor. The temperature change reflects the HMPB / PDA NPs and HMPB / PDA-Z PD-L1 The accumulation of NPs in the local tumor ( Figure 7 At the same time, the present invention also conducts statistical analysis on the temperature change detected by thermal imaging, such as Figure 7 Middle F, under the same light source, same time and power density local irradiation, HMPB / PDA-Z PD-L1 The temperature change of the local tumor in the NPs group was significantly greater than that in the HMPB / PDA NPs group, and was also greater than that in the PBS blank control group, indicating that the HMPB / PDA-Z PD-L1 The accumulation of NPs in the local tumor was significantly greater than that in the HMPB / PDA NPs group.

[0067] Through the above experiments, whether using small animal in vivo fluorescence imaging or thermal imaging technology, HMPB / PDA-Z PD-L1 The accumulation of NPs in the local tumor was significantly higher than that of HMPB / PDA NPs, indicating that HMPB / PDA-Z PD-L1 NPs showed good in vivo targeting effect in MC-38 tumor-bearing mouse model.

[0068] Example 8, HMPB / PDA-Z was studied PD-L1 The killing effect of NPs on tumors in a tumor cell-bearing model.

[0069] To explore HMPB / PDA-Z PD-L1 NPs-mediated photothermal therapy (PTT) anti-tumor therapeutic effect. A mouse subcutaneous transplantation model of MC-38 was established. When the tumor volume reached about 100 mm 3 At the same time, the tumor-bearing mice were equally divided into 5 groups (5 mice / group): ①PBS; ②NIR; ③HP / PZ; ④HP / P+NIR and ⑤HP / P-Z+NIR group.

[0070] ①PBS group: blank control group, tumor-bearing mice were injected with the same amount of PBS via tail vein, but no 808 nm NIR irradiation was given.

[0071] ②NIR group: The same amount of PBS was injected into the tail vein of tumor-bearing mice. 48 hours later, 808 nm NIR (2W / cm 2 ) for 10 min for photothermal therapy.

[0072] ③HP / PZ group: HMPB / PDA-Z was injected into the tail vein of tumor-bearing mice PD-L1NPs (8 mg / kg), without 808 nm NIR (2 W / cm 2 ) 10 min photothermal therapy.

[0073] ④HP / P+NIR group: HMPB / PDA NPs (8 mg / kg) were injected into the tail vein of tumor-bearing mice. 48 h later, 808 nm NIR (2 W / cm 2 ) for 10 min for photothermal therapy.

[0074] ⑤HP / P-Z+NIR group: HMPB / PDA-Z was injected into the tail vein of tumor-bearing mice PD-L1 NPs (8 mg / kg), 48 h later, 808 nm NIR (2 W / cm 2 ) for 10 min for photothermal therapy.

[0075] Tumor-bearing mice were injected with HMPB / PDA NPs or HMPB / PDA-Z via the tail vein. PD-L1 NPs (8 mg / kg) were administered to the tumor site for 48 h. 2 ) for 10 min for photothermal therapy. The tumor volume and mouse weight were monitored and recorded every day to explore the inhibitory effect of HP / PZ-mediated in vivo PTT on tumor growth. After the treatment, the mice were anesthetized and killed, and the tumor tissues were weighed and photographed. Figure 8 As shown in Figure A, tumor growth was inhibited in both the HP / P+NIR and HP / P-Z+NIR treatment groups, but the inhibitory effect was stronger in the HP / P-Z+NIR group. Compared with the PBS control group, tumor growth in the NIR and HP / PZ groups was not significantly inhibited ( Figure 8 Middle A). After treatment ( Figure 8 In Figure C, the tumor weight of the HP / P-Z+NIR treatment group was only 0.14 g, which was significantly lower than that of the other groups (PBS: 0.51 g; NIR: 0.47 g; HP / PZ: 0.38 g; HP / P+NIR: 0.28 g). Figure 8 As shown in D, the tumor volume of the HP / P-Z+NIR treatment group was significantly smaller than that of the other groups. In addition, the body weight of mice in all treatment groups did not change significantly compared with the control PBS group during the entire treatment process ( Figure 8 Middle B), indicating HMPB / PDA-Z PD-L1 NPs have good short-term biosafety. These experimental results show that HMPB / PDA-Z PD-L1 NPs-mediated PTT in vivo has a strong anti-tumor therapeutic effect and is an effective tumor-targeted PTT therapeutic nanomedicine.

[0076] In summary, the PD-L1 targeted affinity body-nano drug conjugate provided by the present invention has excellent biocompatibility, low immunogenicity, significant photothermal conversion effect, excellent tumor targeting for PD-L1 positively expressed colon cancer, and plays an outstanding role in the diagnosis and treatment of colon cancer.

[0077] Example 9, a drug for diagnosing and treating colon cancer The PD-L1 targeting affinity body-nano drug conjugate HMPB / PDA-ZPD-L1 NPs provided in Example 1 is used as an active ingredient to prepare a drug for diagnosing and treating colon cancer. The drug is in an injectable dosage form and can be administered to a desired subject (such as a human and a non-human mammal) by intravenous injection or the like.

[0078] The medicament may further comprise excipients.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of PD-L1 targeted affinity body-nano drug conjugate in the preparation of drugs for diagnosis and treatment of colon cancer, characterized in that: The method for preparing the PD-L1 targeted affinity body-nano drug conjugate comprises the following steps: Step 1, adding polyvinyl pyrrolidone and potassium ferrocyanide to a dilute hydrochloric acid solution and stirring to fully dissolve them; Step 2, heating the solution obtained in step 1 to react to obtain a blue precipitate; Step 3, the blue precipitate prepared in step 2 is repeatedly washed by centrifugation with deionized water and anhydrous ethanol, and then vacuum dried to prepare Prussian blue nanoparticles; Step 4, adding polyvinyl pyrrolidone and the Prussian blue nanoparticles obtained in step 3 to the hydrochloric acid solution, and stirring to fully dissolve them; Step 5, transferring the solution obtained in step 4 to a reactor and heating it to obtain a blue precipitate; Step 6, the blue precipitate prepared in step 5 is repeatedly centrifuged and washed with deionized water and anhydrous ethanol, and then vacuum dried to prepare hollow mesoporous Prussian blue nanoparticles HMPB NPs; Step 7, dispersing the hollow mesoporous Prussian blue nanoparticles obtained in step 6 into Tris-HCl buffer, adding dopamine hydrochloride, stirring, polymerizing it on the surface of the hollow mesoporous Prussian blue nanoparticles to form a polydopamine PDA bionic layer, and finally washing it thoroughly by centrifugation with deionized water to obtain biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs; Step 8, dispersing the biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs obtained in step 7 into Tris-HCl buffer, adding PD-L1 affinity body Z PD-L1 , slowly stirred to connect it to the biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs, and finally washed by centrifugation with deionized water to obtain the PD-L1 targeted affinity body-nano drug conjugate HMPB / PDA-Z PD-L1 NPs.

2. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: PD-L1 Affibody Z PD-L1 The amino acid sequence is shown in the sequence listing SEQ ID NO.

2.

3. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: The concentration of the dilute hydrochloric acid solution in step 1 is 0.01 mol / L, the concentration of polyvinyl pyrrolidone in the dilute hydrochloric acid solution is 75 mg / mL, and the concentration of potassium ferrocyanide in the dilute hydrochloric acid solution is 2.8725 mg / mL.

4. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: In step 1, the stirring method is magnetic stirring, the stirring speed is 400 rpm-600 rpm, and the stirring time is 0.5 h; The heating method in step 2 is sealed heating, the heating temperature is 80°C, and the heating time is 20 h; The centrifugation speed in step 3 is 14000 rpm and the centrifugation time is 20 min.

5. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: In step 4, the concentration of hydrochloric acid in the obtained solution is 1 mol / L, the concentration of polyvinyl pyrrolidone in the hydrochloric acid solution is 5 mg / mL, and the concentration of Prussian blue nanoparticles in the hydrochloric acid solution is 1 mg / mL.

6. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: The heating temperature in step 5 is 140°C and the heating time is 4 h.

7. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: The centrifugation speed in step 6 is 14000 rpm and the centrifugation time is 20 min.

8. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: In step 7, the concentration of hollow mesoporous Prussian blue nanoparticles HMPB in Tris-HCl buffer is 0.5 mg / mL, and the concentration of dopamine hydrochloride in Tris-HCl buffer is 0.2 mg / mL. The process of forming the polydopamine PDA bionic layer requires stirring. The stirring method is aerobic magnetic stirring, the stirring speed is 500 rpm, and the stirring time is 1 h. The speed of thorough centrifugation and washing with deionized water is 16000 rpm, and the single centrifugation time is 20 min.

9. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: In step 8, the concentration of biomimetic hollow mesoporous Prussian blue nanoparticles HMPB / PDA NPs in Tris-HCl buffer is 0.5 mg / mL, and the PD-L1 affinity body Z PD-L1 The concentration in Tris-HCl buffer was 0.01 mg / mL; the power of the ultrasonic cleaning machine was set to 45 KHz, 40 W, the time was 2 min, the stirring speed was 300 rpm, the stirring time was 12 h, and the centrifugal speed was 16000 rpm, and the time was 20 min.

10. The use of the PD-L1 targeted affinity body-nano drug conjugate according to claim 1 in the preparation of drugs for diagnosing and treating colon cancer, characterized in that: The drug comprises a PD-L1 targeting affinity body-nano drug conjugate HMPB / PDA-Z PD-L1 NPs as active ingredients.

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