CN110585127B

CN110585127B - Preparation method of targeted PD-L1 microvesicle and application thereof in preparation of drugs for inhibiting cervical cancer

Info

Publication number: CN110585127B
Application number: CN201910636084.0A
Authority: CN
Inventors: 赵云; 刘朝奇; 马瑶
Original assignee: China Three Gorges University CTGU
Current assignee: Hefei Xingzhicheng Information Technology Co ltd; Shanghai Meiha Biotechnology Development Co ltd
Priority date: 2019-07-15
Filing date: 2019-07-15
Publication date: 2022-03-08
Anticipated expiration: 2039-07-15
Also published as: CN110585127A

Abstract

The invention relates to a preparation method of a targeted PD-L1 microbubble, and specifically relates to a preparation method of a targeted PD-L1 microbubble which is obtained by adding streptavidin to a biotinylated lipid microbubble for incubation, adding a biotinylated PD-L1 antibody, and centrifuging after incubation. The targeted PD-L1 microvesicle is applied to the preparation of the medicine for inhibiting the cervical cancer transplantation tumor, and experimental results show that the targeted PD-L1 microvesicle has higher tumor inhibition effect, wherein the volume tumor inhibition rate is 39.21%, and the mass tumor inhibition rate is 44.44%.

Description

Preparation method of targeted PD-L1 microvesicle and application thereof in preparation of drugs for inhibiting cervical cancer

Technical Field

The invention relates to a lipid microbubble, in particular to a targeted PD-L1 microbubble, which is applied to the preparation of a medicine for treating cervical cancer.

Background

Cervical cancer is one of the most common gynecological malignant tumors of women all over the world, the incidence rate is second to that of breast cancer high-lying gynecological malignant tumors, the incidence rate is high, the breast cancer gradually tends to be younger, and the health of women is seriously harmed. Cervical cancer is mainly caused by Human Papillomavirus (HPV) infection, and the occurrence and development of cervical cancer are promoted by the continuous infection of high-risk HPV, the reduction of the immune function of an organism and the interaction between local microenvironments of cervical cancer. Current treatment methods include surgical resection, radiation therapy, chemotherapy. Surgery is one of the main methods for treating cervical cancer, and although the surgery can temporarily remove a lesion, tumor cells are very invasive and metastatic, and the lesion cannot be completely removed. The adjuvant chemotherapy is widely used for preoperative and postoperative treatment in clinic, can effectively reduce the tumor stage and reduce parauterine infiltration, provides good conditions for operation, but has poor targeting property of chemotherapy, is easy to generate drug resistance and has obvious toxic and side effects on the whole body. Cervical cancer is a radiation-sensitive tumor, and the effect of radiotherapy on patients with middle and advanced stages and patients with relapse is positive, but serious complications thereof bring great difficulty to treatment. Therefore, the method explores the pathogenesis of the cervical cancer, searches a new target point for treating the cervical cancer and has important clinical significance for early diagnosis, treatment and prognosis evaluation of the cervical cancer.

Disclosure of Invention

Aiming at the technical problems, the invention provides a targeted PD-L1 microbubble, and the targeted PD-L1 microbubble is applied to the preparation of a medicine for inhibiting cervical cancer transplantable tumors. In a further preferred scheme, the application is applied to the preparation of the medicine for inhibiting the U14 cervical cancer cells.

The targeted PD-L1 microbubble is a biotinylated lipid microbubble prepared by a thin film hydration method and a mechanical oscillation method, and comprises the following specific steps:

(1) mixing distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), distearoyl phosphatidylethanolamine-polyethylene glycol 2000-Biotin (DSPE-PEG2000-Biotin) and stearic acid-polyethyleneimine (PEI-600) in a glass test tube, preheating in a water bath kettle at 60 ℃, and filling nitrogen in a vortex state to enable the mixed solution to form a film on the wall of the test tube; (distearoylphosphatidyl choline (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG2000), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000-Biotin (DSPE-PEG2000-Biotin), stearic acid-polyethyleneimine (PEI-600), all available from Avanti corporation, USA).

Distearoyl phosphatidylcholine, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-biotin, stearic acid-polyacetyl imine in a molar ratio of 75-90:3-6:3-6: 2-6.

In a preferred scheme, the molar ratio of distearoyl phosphatidylcholine to distearoyl phosphatidylethanolamine-polyethylene glycol 2000-biotin to stearic acid-polyacetimide is 85:5:5: 5.

(2) Vacuumizing the glass tube formed in the step (1) (vacuumizing for more than 3 hours to fully volatilize liquid), adding Tris buffer solution, performing water bath ultrasonic treatment (fully dissolving the film on the wall of the glass tube in the Tris buffer solution, stopping the water bath ultrasonic treatment after the liquid is clarified), sealing in a penicillin bottle, and filling C₃F₈Gas, refrigerating and storing;

(3) oscillating the refrigerated penicillin bottle in the step (2) to obtain white emulsion (generally oscillating for 25-30 s); centrifuging the white milky liquid at 1000rpm for 3min, and discarding the lower layer liquid as biotinylated lipid microbubble (the lower layer liquid is free lipid and impurities);

(4) dropwise adding streptavidin (purchased from Avanti company of America) to the biotinylated lipid microbubble obtained in the step (3), and carrying out constant-temperature oscillation incubation at 2-4 ℃ in the dark to obtain microbubble suspension;

(5) centrifuging the microbubble suspension obtained in the step (4) (centrifuging for 3min at a centrifugal speed of 1000 rpm), discarding the supernatant (to remove free streptavidin), rinsing with PBS buffer, centrifuging, and discarding the supernatant (preferably repeating the steps of rinsing with PBS buffer, centrifuging, and discarding the supernatant 3 times) to obtain microbubbles;

(6) biotinylated PD-L1 antibody (purchased from eBioscience, USA) is added into the microvesicle obtained in step (5), incubated overnight at 2-4 ℃ under constant temperature shaking in the dark, centrifuged (at a centrifugation speed of 1000rpm for 3min), the supernatant is discarded (to remove free biotinylated PD-L1 antibody), PBS buffer is added for rinsing, centrifugation is carried out, and the supernatant is discarded (preferably, the steps of PBS buffer rinsing, centrifugation and supernatant discarding are repeated for 3 times), thus obtaining the targeted PD-L1 microvesicle.

Counting the obtained target microbubbles by a blood counting plate, measuring the particle size by a particle size analyzer, and observing the form of the microbubbles under a microscope; observed by laser scanning confocal fluorescence microscope.

The streptavidin is added in excess relative to the biotinylated lipid microbubbles; biotinylated PD-L1 antibody was added in excess relative to the microbubbles.

The invention discloses application of the prepared targeted PD-L1 microvesicle in preparing a medicament for inhibiting cervical cancer transplantable tumor.

The targeted PD-L1 microvesicle is applied to the preparation of a medicament for inhibiting U14 cervical cancer cells.

The concentration of the targeting PD-L1 microbubble is 1 x 10⁷-1.5×10⁹One per ml.

And (3) incubating the obtained targeted microvesicle with Hela cervical carcinoma cells highly expressed by PD-L1, repeatedly washing by PBS, and observing the targeted aggregative property of the targeted PD-L1 microvesicle on a cell level under an optical microscope. The prepared targeted microvesicle and non-targeted microvesicle tail are injected into a tumor-bearing mouse intravenously, and the ultrasonic imaging effect of the microvesicle in a mouse transplantation tumor is observed by an ultrasonic diagnostic apparatus.

The conditions of the mice and cell lines used to implement the protocol of the present application are as follows:

SPF-grade BALB/c mice, female, about 18g, were provided by the university of Sanxia animal laboratories, housed in the university of Sanxia animal laboratories in a pathogen-free barrier environment, under strict SPF-grade laboratory animal protocols. The U14 cervical cancer cell tumor strain is provided by the tumor microenvironment of the university of the three gorges and an important laboratory of immunotherapy Hubei province.

Establishment and grouping treatment of mouse cervical cancer transplantable tumor

Ascites is extracted from the abdominal cavity of a mouse with U14 cervical carcinoma cells, and the concentration of the U14 cervical carcinoma cells is adjusted to be about 2 multiplied by 10⁷Perml, 0.2ml was subcutaneously inoculated in the right anterior axilla of mice, and approximately 0.5cm subcutaneous tumor nodules were formed after one week. The successfully established mouse U14 cervical carcinoma subcutaneous transplantation tumor model is randomly divided into 5 groups, and each group comprises 10 mice: control group (tumor inoculation), empty microvesicle group (tumor inoculation, pure microvesicle without targeted PD-L1), and targeted PD-L1 microvesicle group obtained in the present application. The injection of different kinds of microbubble preparations is given according to groups, the tumor body is irradiated once by tail vein injection (0.2 ml/tube) and ultrasonic every two days, and the irradiation conditions are as follows: frequency 1MHZ, power 1W/cm²Duty ratio of 50%, irradiation time of 90 s/tube, wherein the control group was treated with the same dose of physiological saline, and the tumor body major axis (a) and minor axis (b) were measured before treatment, according to formula V of 1/2ab²And calculating the tumor volume and drawing a tumor growth curve. After 5 times of treatment, some mice were sacrificed, tumor bodies were stripped, and tumor volume and mass inhibition rate were calculated. The remaining mice were treated further, one mouse per group was sacrificed every other day, spleens were removed, and splenocytes were isolated.

Detection of NK cell immune killing activity by lactate dehydrogenase release method

Adjusting the spleen lymphocyte concentration to 10 with the isolated mouse spleen lymphocytes as effector cells⁶each/mL of the cells was added to a 96-well culture plate at 100. mu.L/well, and cocultured with spleen lymphocytes at an effective target ratio of 25:1, 50:1, and 100:1 using U14 cervical cancer cells as target cells, and tumor antigen-stimulated, and cell culture supernatants were collected after 96 hours. A blank set, maximum release hole, is also provided. The detailed steps of LDH detection are shown in the operation steps of a lactate dehydrogenase cytotoxicity detection kit. The killing rate calculation formula is as follows:

the killing rate is (killing effect group OD value-blank group OD value)/(maximum release group OD value-blank group OD value) × 100%.

Tumor tissue immunity and apoptosis related gene and protein expression analysis and detection

The stripped tumor tissue is preserved at the temperature of minus 80 ℃, total RNA of the tumor tissue is extracted, the RNA is reversely transcribed into cDNA, and the expression conditions of bcl-2, bax, CD80 and CD86 in the tumor tissue are detected by real-time fluorescence quantitative PCR (RT-qPCR). Fixing the rest tumor tissue by 4% paraformaldehyde, dehydrating, embedding paraffin, slicing by 4 μm, detecting Bax and Bcl-2 protein expression by immunohistochemistry, observing the result under microscope, and shooting.

The test data of the invention is statistically analyzed by SPSS18.0 software, the result is expressed by (x +/-s), and the difference is P <0.05, which has statistical significance.

Drawings

Fig. 1 is a graph of targeted PD-L1 microbubble morphology and particle size, wherein, a. microbubble microscopic morphology; B. the microbubble particle size.

FIG. 2 is a confocal laser image of targeted PD-L1 microbubbles, wherein A is a red fluorescence image emitted by a PE marker (streptavidin), B is an image of a FITC marker (anti-mouse IgG), which reflects the presence of PD-L1 antibody on the microbubbles, and C is an overlay of A and B.

Figure 3 is targeting of PD-L1 targeted microvesicles in vitro, wherein a. B. The group of microbubbles is targeted.

FIG. 4 is a tumor growth curve of mice in the control group, the empty microvesicle group and the targeted PD-L1 microvesicle group.

FIG. 5 shows Bax and Bcl-2 protein expression targeting the antitumor effect of PD-L1 microvesicles, wherein a is a control group, b is an empty microvesicle group, and c is a targeted PD-L1 microvesicle group.

FIG. 6 is a diagram of the quantitative analysis of Bax/Bcl-2 protein expression in a group of targeted PD-L1 microvesicles, compared with a control group^*P<0.05。

Figure 7 is control, empty microvesicle, targeted PD-L1 microvesicle anti-tumor immune activity, wherein a.ldh detects immunolethality; comparing the expression of CD80 genes; CD86 Gene expression comparison with control^*P<0.01。

Detailed Description

Example 1

Preparation and characteristics of targeted PD-L1 microvesicles

1) Mixing distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), distearoyl phosphatidylethanolamine-polyethylene glycol 2000-Biotin (DSPE-PEG2000-Biotin) and stearic acid-polyethyleneimine (PEI-600) in a molar ratio of 85:5:5:5 in a glass test tube, slightly shaking, mixing uniformly, preheating in a water bath at 60 ℃, placing the glass test tube on a vortex oscillator, and using N to stir the glass test tube with the N to stir the glass test tube₂The liquid in the glass test tube is blown, and the vortex is carried out while the blowing, so that a layer of homogeneous film is formed at the bottom of the wall of the glass tube.

2) And connecting the glass tube after film formation with a vacuum air extractor, and vacuumizing for 3h to fully volatilize the liquid.

3) And taking out the glass test tube, adding 5ml of Tris buffer solution into the glass test tube, putting the glass test tube into an ultrasonic cleaning machine for water bath for 5min to fully dissolve the film on the wall of the glass tube into the Tris buffer solution, and stopping water bath ultrasonic treatment after the liquid is clarified. Using a pipette to divide the solution into 5 penicillin bottles and sealing the bottles by caps.

4) Respectively filling C into each penicillin bottle₃F₈And (5) putting the gas into a refrigerator at 4 ℃ for storage.

5) Fixing a penicillin bottle on a high-speed oscillator, adjusting the oscillation time to be 30s, and enabling the liquid to be white emulsion after oscillation; transferring the liquid into a centrifuge tube, centrifuging for 3min at 1000rpm of a desk-top low-speed centrifuge, and discarding the lower layer liquid which is the common biotinylated lipid microbubble, wherein the lower layer liquid is free lipid and impurities.

6) Adding 1ml of biotinylated lipid microbubble into a test tube, slowly dropwise adding excessive PE-labeled streptavidin, and incubating for 30min at 4 ℃ in a constant temperature oscillator protected from light.

7) Centrifuging the microbubble suspension at 1000rpm for 3min by using a desk-top low-speed centrifuge, and removing the clear liquid to remove free streptavidin; add PBS buffer for rinsing, centrifugation, discard the supernatant, repeat 3 times.

8) Adding biotinylated PD-L1 antibody into the above microvesicle, and incubating overnight in a constant temperature oscillator at 4 deg.C in the absence of light; the incubated microbubble suspension was centrifuged at 1000rpm for 3min using a tabletop low speed centrifuge and the supernatant was discarded to remove free biotinylated PD-L1 antibody; adding PBS buffer solution for rinsing, centrifuging, removing the supernatant, repeating the process for 3 times, and preparing the targeted PD-L1 microvesicle.

The prepared target PD-L1 microbubble is milky white suspension, is spherical under microscope (figure 1.A), has uniform dispersion, average particle diameter of (1.09 + -0.21) μm (figure 1.B), and average concentration of 6.4 × 10⁹The concentration of the extract is adjusted to 1 × 10⁹One/ml was used for the experiment. Whether the targeted PD-L1 microbubble is successfully connected with the anti-PD-L1 antibody is observed through a laser scanning confocal fluorescence microscope, and the prepared PD-L1 targeted microbubble can emit red fluorescence (PE-labeled streptavidin) and green fluorescence (FITC-labeled anti-PD-L1 monoclonal antibody) under the laser confocal microscope, and emits yellow fluorescence after synthesis, which indicates that the antibody is successfully connected to the surface of the microbubble (figure 2).

Targeting microvesicles targeting in vitro and in vivo

Then, whether the prepared targeted PD-L1 microvesicle has good targeting property or not is verified through a cell level targeting experiment, and the targeted PD-L1 microvesicle annularly surrounds tumor cells under an optical microscope, so that the targeted microvesicle has better cell targeted aggregation property compared with a non-targeted microvesicle (figure 3). The targeted and non-targeted microbubbles are respectively injected into a tumor-bearing mouse through the tail vein, and after the same time period (59-60s), microbubble contrast agent residues can be seen in the targeted microbubble group, the non-targeted microbubble group does not have the contrast agent residues, and the retention time of the targeted microbubbles in tumor tissues is longer than that of the non-targeted microbubbles (figure 4), which shows that the prepared targeted PD-L1 microbubbles have certain targeting specificity in vivo and have the effect of targeted development.

Example 2

Application of targeted PD-L1 microvesicles prepared in example 1 to preparation of drugs for treating cervical cancer

Targeting PD-L1 microvesicles to inhibit tumor growth

The tumors of the control group and the empty microvesicle group are continuously increased in different degrees, part of the affected limbs of the mice are in dysfunction, the tumor growth of the targeted microvesicle group is slower than that of the other two groups, the tumor tissue is stripped from the mice, the nodular lumps are seen to be slightly hard, the surface of the tumor is pseudo-enveloped, the tumor is easily stripped from the surrounding tissues, the tumor volume inhibition rate and the tumor mass inhibition rate are reduced (table 1), and the result shows that the tumor growth of the targeted microvesicle group is slower than that of the control group, the volume and mass growth are inhibited, the tumor volume inhibition rate is 39.21%, and the tumor mass inhibition rate is 44.44% (the results show that the tumor growth of the targeted microvesicle group is slow, the tumor volume and mass growth are inhibited, and the tumor mass inhibition rate is 39.21%^#*P<0.05)。

TABLE 1 comparison of tumor inhibition rates of mice of each experimental group

Note: comparing with control group^*P<0.01, compared to the empty microbubble group^#P<0.05

Induction of tumor cell apoptosis by targeting PD-L1 microvesicles

Immunohistochemistry method for detecting bcl-2 and bax gene and protein expression in individual tumor tissue shows that the targeted microvesicle group has reduced bcl-2 expression and bax expression is increased relative to the control group ()^*P<0.05, FIGS. 5 and 6)

Targeting PD-L1 microvesicles enhances the immune activity of the body

With splenic lymphocytes of miceAfter co-culture with U14 cervical cancer, LDH measures the in vitro immune killing effect of mouse spleen lymphocytes on U14 cervical cancer cells, and the result shows that the mouse spleen lymphocytes of the targeted microvesicle group can kill the U14 cervical cancer cells in vitro with high efficiency, and the difference has significant meaning compared with a control group (the difference is shown in the specification)^*P<0.01, fig. 5. a). Real-time fluorescent quantitative PCR (RT-qPCR) data showed up-regulation of CD80 and CD86 in targeted microvesicle group expression: (RT-qPCR)^*P<0.01, FIG. 7. B/C).

Claims

1. The application of the targeted PD-L1 microvesicle in preparing the medicine for inhibiting the cervical cancer transplantable tumor is characterized in that the preparation method of the targeted PD-L1 microvesicle comprises the following steps:

(1) the preparation method comprises the following steps of mixing distearoyl phosphatidylcholine, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-biotin and stearic acid-polyacetyl imine in a glass test tube, preheating a water bath kettle at 50-60 ℃, and filling nitrogen in a vortex state to enable the mixed solution to form a film on the wall of the test tube;

(2) vacuumizing the glass tube formed in the step (1), adding Tris buffer solution, performing water bath ultrasonic treatment, sealing in a penicillin bottle, and filling with C₃F₈Gas, refrigerating and storing;

(3) oscillating the refrigerated penicillin bottle in the step (2) to obtain white emulsion; centrifuging the white milky liquid, and removing the lower layer liquid to obtain biotinylated lipid microbubble;

(4) dropwise adding streptavidin into the biotinylated lipid microbubble obtained in the step (3), and carrying out constant-temperature oscillation incubation at 2-4 ℃ in the dark to obtain microbubble suspension;

(5) centrifuging the microbubble suspension obtained in the step (4), discarding the supernatant, rinsing with PBS buffer solution, centrifuging, and discarding the supernatant to obtain microbubbles;

(6) adding biotinylated PD-L1 antibody into the microvesicle obtained in the step (5), carrying out constant-temperature oscillation incubation overnight at 2-4 ℃ in a dark place, centrifuging, removing the clear liquid, adding PBS buffer solution for rinsing, centrifuging, and removing the clear liquid to obtain the targeted PD-L1 microvesicle.

2. The use of claim 1, wherein the molar ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000-biotin, stearic acid-polyacetylimine is 75-90:3-6:3-6: 2-6.

3. The use of claim 2, wherein the molar ratio of distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000-biotin, stearic acid-polyacetylimine is 85:5:5: 5.

4. Use according to claim 1, characterized in that the streptavidin is added in excess with respect to the biotinylated lipid microvesicles; the amount of biotinylated PD-L1 antibody added was in excess of the amount of microbubbles added.

5. The use of claim 1, wherein the targeted PD-L1 microvesicle is used for preparing a medicament for inhibiting U14 cervical cancer cells.

6. The use of claim 1, wherein the targeted PD-L1 microvesicles are present at a concentration of 1 x 10⁷-1.5×10⁹One per ml.