CN115501334B

CN115501334B - PD-1 nano vesicle loaded with Iguratimod-rhodium nano particles, and preparation method and application thereof

Info

Publication number: CN115501334B
Application number: CN202210973221.1A
Authority: CN
Inventors: 黄曦; 穆罕默德·尤尼斯
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2023-09-19
Anticipated expiration: 2042-08-15
Also published as: CN115501334A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an Iguratimod-rhodium nanoparticle-loaded PD-1 nanovesicle, and a preparation method and application thereof. The invention provides a PD-1 nano vesicle loaded with Iguratimod-rhodium nano particles, which takes the PD-1 nano vesicle as a drug carrier to wrap the Iguratimod and the rhodium nano particles. The nano vesicle has the functions of resisting tumor growth and tumor metastasis. First, the nanovesicles express the PD-1 receptor, enhancing cancer immunotherapy by disrupting the PD-1/PD-L1 immunosuppressive axis; second, IGU provides chemotherapeutic function by inhibiting the AKT/mTOR pathway; finally, rh inhibited tumor growth by photothermal treatment via the JNK/ERK pathway. Therefore, the combined therapy can provide a new idea for tumor treatment, and has good clinical application prospect.

Description

PD-1 nano vesicle loaded with Iguratimod-rhodium nano particles, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an Iguratimod-rhodium nanoparticle-loaded PD-1 nanovesicle, and a preparation method and application thereof.

Background

Lung cancer is the most common cause of cancer-related death worldwide. 180 ten thousand people are diagnosed with lung cancer each year, 160 ten thousand of which die from the disease. Despite the differences in different times and different regions, the overall 5-year survival rate of lung cancer is only 4-17%. Combination therapy is an important treatment regimen for lung cancer that overcomes multiple drug resistance of tumors by modulating multiple cell signaling pathways simultaneously, has synergistic therapeutic effects, and reduces systemic toxicity.

Cancer immunotherapy aims at destroying cancer cells using the human immune system. Among them, immune checkpoint blockade (immune checkpoint blockade, ICB) methods directed against programmed death-1 (pd-1) and programmed death ligand 1 (pd-L1) pathways exert significant clinical effects in partial types of cancers such as melanoma, renal cell carcinoma, non-small cell lung carcinoma, bladder carcinoma, etc. PD-L1 is a ligand for PD-1, and is up-regulated in expression in cancer cells, inhibiting effector T cells. Thus, by blocking the interaction between PD-1 and PD-L1 by antibodies, the immune response of T cells to cancer cells can be enhanced.

However, anti-PD-1 treatment is not effective against all types of cancer, and only a few patients benefit from ICB therapy. Meanwhile, most of the existing humanized antibodies are derived from mice, and require complicated design and separation processes, so that the manufacturing cost is increased, and the cost of immune checkpoint blocking antibody treatment is not yet affordable for many patients. Accordingly, there is a need to develop a method to replace or improve PD-1/PD-L1 inhibitors.

Photodynamic therapy (photodynamic therapy, PDT) is promising to replace traditional methods for the treatment of different types of cancer because of its high selectivity, which prevents damage to healthy tissue. The use of nanomaterials in photodynamic therapy has several advantages over conventional photosensitizers due to their unique structural and functional characteristics. Interestingly, in the use of metal nanoparticles it was found that it is capable of absorbing electromagnetic radiation and transferring this energy into oxygen molecules to generate Reactive Oxygen Species (ROS), or to emit it as heat. Although previous reports have demonstrated the ability of Rh derivatives as antitumor agents, to our knowledge, no study has been made on the potential use of Rh nanoparticles as photosensitizers in photodynamic therapy.

In addition, iguratimod is a novel antirheumatic drug, which is currently widely used in China and Japan. Previous studies have shown that Iguratimod has anti-inflammatory effects. In vitro experiments, iguratimod was able to reduce the expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-8 (IL-8) in monocytes, synovial fibroblasts and alveolar macrophages.

Disclosure of Invention

Aiming at the defect and the defect of poor monotherapy effect in the prior art, the invention provides an engineering cell membrane source expression PD-1 nano vesicle (PD-1 NVs) which can enhance cancer immunotherapy by inhibiting a PD-1/PD-L1 signal axis, and the invention takes the PD-1 bionic nano vesicle as a drug carrier and loads Iguratimod (IGU) as a chemotherapeutic agent and rhodium nano particles (Rh-NPs) as a photothermal agent and a photosensitizer respectively.

The first object of the invention is to provide a PD-1 nano vesicle loaded with Iguratimod-rhodium nano particles, wherein the PD-1 nano vesicle is taken as a drug carrier to wrap Iguratimod (IGU) and rhodium nano particles (Rh-NPs).

The second aim of the invention is to provide a preparation method of the PD-1 nano vesicle loaded with the Iguratimod-rhodium nano-particles.

In order to achieve the above object, the present invention adopts the following technical scheme:

the preparation method of the PD-1 nano vesicle loaded with the Iguratimod-rhodium nano particles comprises the following steps of:

s1, constructing cells stably expressing PD-1, collecting the cells and suspending the cells in a separation buffer solution, centrifuging the cells after homogenizing and grinding to collect supernatant, centrifuging the supernatant again, discarding the supernatant to obtain plasma membrane precipitate, washing and purifying the plasma membrane precipitate, and extruding the plasma membrane precipitate by a polycarbonate membrane extruder after ultrasonic treatment to obtain nano vesicles;

s2, introducing rhodium nano-particles and Iguratimod into the nano-vesicles obtained in the step S1 by adopting an electroporation technology.

Preferably, the cells stably expressing PD-1 in step S1 are any one of HEK293T, hepG, huh-7, hepa1-6, SK-Hep-1, lymphocytes, and the like.

Preferably, the nanovesicles of step S2: iguratimod: rhodium nanoparticles were used in a 1:1:2 ratio.

The third object of the invention is to provide an application of the PD-1 nanovesicle loaded with the Iguratimod-rhodium nanoparticles in preparing a tumor therapeutic drug.

Preferably, the tumor includes liver cancer, lung cancer, esophageal cancer, gastric cancer, melanoma, colorectal cancer, bladder cancer, pancreatic cancer, ovarian cancer, breast cancer and neuroblastoma.

More preferably, the tumor is lung cancer.

Preferably, the medicament treats a tumor by inhibiting proliferation of tumor cells.

Preferably, the medicament treats a tumor by inhibiting metastasis of tumor cells.

Compared with the prior art, the invention has the beneficial effects that:

the synthesized cell membrane-derived nano vesicle loaded IGU and Rh-NPs (IGU-Rh-PD-1 NVs) have the effects of resisting tumor growth and tumor metastasis. First, the drug expresses the PD-1 receptor, enhancing cancer immunotherapy by disrupting the PD-1/PD-L1 immunosuppressive axis; second, IGU provides chemotherapeutic function by inhibiting the AKT/mTOR pathway; finally, rh inhibited tumor growth by photothermal treatment via the JNK/ERK pathway. Therefore, the combination therapy shows good clinical application prospect.

Drawings

FIG. 1 is a schematic representation of IGU-Rh-PD-1NVs synthesis, with reference numerals illustrating: 1 engineering a HEK293T cell line expressing a mouse PD-1 receptor on a cell membrane; 2 is harvesting the cell membrane expressing the PD-1 receptor; 3 is the preparation of vesicles expressing PD-1 by extrusion; 4 is loading of IGU and Rh-NPs into PD-1 vesicles.

FIG. 2 is a representation of HEK-293T stably transformed cell lines expressing PD-1 receptors, wherein FIG. 2A is a representation of HEK293 cell membranes stably expressing mouse PD-1 receptors on cell membranes, showing expression of DsRed-PD-1NVs by fluorescence on confocal images; scale bar: 10 μm; FIG. 2B is a Western blot used to detect expression of mouse PD-1 (mPD-1) receptor on vesicles, whole cell lysates, 293T cells and 293T vesicles, actin protein as loading control; the TEM image of FIG. 2C shows the shape and size of the expressed PD-1 vesicles, scale bar, 100nm; FIG. 2D Dynamic Light Scattering (DLS) was used for size and zeta potential analysis of 293T vesicles, expression PD-1 vesicles and IGU-Rh-PD-1 vesicles.

FIG. 3 is an in vitro targeting validation of IGU-Rh-PD-1NVs, wherein FIG. 3A is the binding of Dil-labeled PD-1 vesicles to PDL-1 receptors on LLC cell membranes compared to 293T vesicles, marking LLC cell membranes with Dio dye (scale: 10 μm); FIG. 3B is a plot of PD-1 receptor orientation on vesicles as confirmed by immunoprecipitation assay, PD-1 vesicles were incubated with aPD-1 antibodies for 4 hours, followed by immunoprecipitation and Western blot analysis; FIG. 3C is CO-IP for detecting interactions between PD-1 (on the vesicle surface) and PD-L1 (on LLC cell membrane).

FIG. 4 shows the therapeutic effect of IGU-Rh-PD-1NVs on lung cancer cells in vitro, wherein FIGS. 4A-4D are CCK-8 analysis of LLC cell and L929 (as normal cell controls) cell viability at different concentrations of IGU and Rh-NPs (near infrared 808 nm); fig. 4E, fig. 4F are graphs of LLC cell viability after analysis of the following conditions: without any treatment, IGU, rh-NPs, PD-1NVs or IGU-Rh-PD 1NVs, and with CD8 cells; FIG. 4G is an analysis of cytotoxicity after NC, IGU, rh NPs, PD-1NVs and IGU-Rh-PD 1NVs treatment at different time points.

FIG. 5 is an IGU-Rh-PD-1NVs effect on apoptosis and improving ROS production, wherein FIG. 5A is an analysis of apoptosis after treatment with an Annexin-V/P1 kit for flow cytometry analysis; FIG. 5B is an analysis of ROS levels following NC, IGU, rh NPs, PD-1NVs and IGU-Rh-PD 1NVs treatment; FIGS. 5C,5D are statistical graphs representing the results of FIGS. 5A and 5B, respectively; FIG. 5E is an evaluation of protein expression by Western blot analysis of p-ERK, ERK, p-JNK and JNK.

FIG. 6 shows the therapeutic effect of IGU-Rh-PD-1NVs on in vivo lung cancer mice models, wherein FIG. 6A is an in vivo xenograft mouse model established by inoculation of LLC cells in C57/L6 mice. Then, PBS, 293T NVs, IGU, rh NPs, PD-1NVs and IGU-Rh-PD 1NVs treatments were provided, mice were sacrificed after 21 days and tumors were collected for further analysis; fig. 6B-6D) are graphs showing tumor weight, tumor size, and mouse body weight.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The test methods used in the embodiment of the invention are all conventional methods unless specified otherwise; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1

A method for preparing a PD-1 nanovesicle (IGU-Rh-PD-1 NVs) loaded with an Iguratimod-rhodium nanoparticle, comprising the steps of:

1. preparation of plasmid and stable cell line

(1) Mouse PD-1 was cloned into pCMV6 mammalian expression vector.

(2) The PD-1 plasmid was confirmed by DNA sequencing.

(3) Lipofectamine 2000 plasmid was transiently transfected into HEK293T cells.

(4) To establish stable cells, we transfected HEK293T cells with pCMV6-OFR-PD-1 and further screened with hygromycin B.

2. Preparation of nanovesicles expressing PD-1 cell membrane source:

(1) HEK293T cells stably expressing DsRed-PD-1 were cultured in DMEM medium containing 10% fetal bovine serum. Cells were collected after digestion with trypsin.

(2) Cells were washed 3 times with pre-chilled PBS and centrifuged at 1000 rpm.

(3) Then with 1mM NaHCO ₃ Cells were suspended in isolation buffer composed of 0.2mM EDTA, 1mM PMSF, and overnight at 4 ℃.

(4) After this, the cells were milled at least 20 times in an ice bath by a dounce homogenizer.

(5) The whole solution was centrifuged at 1000 Xg for 5 minutes, the supernatant collected and centrifuged at 10,000Xg for 25 minutes.

(6) Discarding the precipitate; the supernatant was centrifuged at 150,000Xg for 35 minutes, the supernatant was discarded, and the bottom plasma membrane, which was an off-white precipitate, was collected.

(7) The plasma membrane pellet was washed 1 with 1mM EDTA and then sonicated gently for subsequent experiments.

(8) Cell membrane fragments were dispersed in PBS at ph=7.4, sonicated for 30min, and then extruded with polycarbonate membrane extruders at 800nm, 400nm, 200nm, 100nm, respectively, and repeated 20 times to obtain nanovesicles.

(9) The membrane protein content was determined using coomassie brilliant blue.

3. Preparation of rhodium nanoparticles

First we synthesized silver triangular nanoplates by the following method

1) Synthesis of Ag nano-seeds by reduction of Ag (I) in the presence of shape control additives

2) mu.L of 10mM AgNO was added ₃ The solution, 300. Mu.L of 30mM sodium citrate dihydrate solution, 1.5mL, 3.5mM polyvinylpyrrolidone solution, and 24.75mL deionized water were sequentially added to a 50mL round bottom flask. Fully stirring;

3) Adding 60 mu L of 30% hydrogen peroxide into the solution in the step 1) under the magnetic stirring condition, and oxidizing and etching for 4 minutes;

4) To the prepared Ag nano seeds were sequentially added 333 μl of a 75mM trisodium citrate dihydrate solution and 1ml of 100mM L-ascorbic acid under magnetic stirring.

5) Ag growth solution consists of 20mL of 1mM AgNO ₃ 0.125mL 100mM citric acid and 10. Mu.L 75mM trisodium citrate dihydrate.

6) 13mL of the prepared Ag growth solution was added to the seed solution at a rate of 0.2 mL/s. During the growth reaction, the solution color changed to a deep blue.

7) After an additional 10min of reaction at room temperature, the Rh nanoplatelets were prepared by reverse direction electrodisplacement without any purification.

8) 20mL of 1mM AgNO ₃ Sequentially adding the solution, 0.125mL 100mM citric acid solution and 10. Mu.L 75mM sodium citrate dihydrate solution into a 25mL round bottom flask;

9) 13mL of the growth solution of 4) was slowly added to the solution of 3), and after 10 minutes of reaction, the solution color eventually turned dark blue.

10 The synthesized silver triangular nanoplate solution was centrifuged at 11000rpm for 10 minutes, the supernatant was discarded, the precipitate was resuspended in distilled water, centrifuged at 11000rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected.

11 The nanoparticles were weighed and resuspended in distilled water and stored in a refrigerator at 4 ℃ for subsequent experiments.

Next, we reverse-direction electro-displacement of Ag nanoplates with Rh (III).

1) To a clear 25mL glass vial was added 4mL of the synthesized Ag nanoplates followed by 10mL EG and pipetting multiple times to achieve uniform mixing.

2) To an Ag nanoplate in deionized water/EG mixed solvent was added an appropriate volume of 4.2mM aqueous Rh (III) stock solution.

3) To successfully synthesize nanoshells, nanoshells and porous nanoplates, 500, 600 and 900 μl of 4.2mM Rh (III) should be added to each vial, respectively.

4) The IGR reaction was completed by heating at 190 ℃ for 4 hours without capping to evaporate the deionized water content.

5) During the reaction, the colors of the nanoshell, and porous nanoshell became indigo blue, blue brown, and dark brown, respectively.

6) After heating for 4 hours, the heat source was removed and 10mL DI water was added directly to each vial to quench the reaction.

7) The synthesized Rh nanostructures were purified by centrifugation at 9000rpm for 10min and washed 3 times with deionized water.

8) The final product was redispersed in 4mL of deionized water and expressed as 1equiv concentration.

9) The Rh nanostructures produced can be used without any colloidal stability problems, even if stored at room temperature for more than 3 months.

4. IGU and Rh-NP are loaded in the nano vesicles, and the specific steps are as follows:

1) mu.g/mL IGU and 2mg/mL Rh-NPs were loaded into PD-1NVs, and 1mg (protein mass) of purified vesicles (100 mg/mL diluted in PBS) were gently mixed in 1mL of 4℃electroporation buffer (1.15 mM potassium phosphate pH7.2, 25mM potassium chloride, 21% Optiprep).

2) Samples were electroporated at 300V and 150. Mu.F in 0.4cm electroporation cuvettes using a MicroPulser Electro-corporation (Bio-Rad, USA).

3) Thereafter, the electroporation cuvette with the sample was incubated on ice for 30 minutes to restore membrane integrity.

4) The NV is then washed 3 times with pre-chilled PBS by ultracentrifugation at 100,000Xg.

5) For another method of loading IGU to nanovesicles, 1mg of purified vesicles and 500 μg IGU were gently mixed in 1mL PBS and incubated for 2 hours at 37 ℃. NV was then washed 3 times with PBS.

Example 2

Characterization of HEK-293T stably transformed cell lines expressing PD-1 receptors

HEK293T cells expressing PD-1 receptor on cell membranes were used for PD-1NV production. The PD-1 receptor protein contains a DsRed protein tag in the C-terminal region. PD-1NV was isolated by squeezing vesicles from engineered HEK-293T cells through a polycarbonate membrane filter. Density gradient ultracentrifugation was used to further isolate and purify NV. Furthermore, confocal imaging and western blotting were used to assess the expression of PD-1 receptors on stable cells and NV (see fig. 2A, 2B). Confocal images showed red bright spots, indicating the presence of DsRed-PD-1 receptor. The morphology of NV after negative staining was examined using Transmission Electron Microscopy (TEM) and confirmed to be closed vesicles (see fig. 2C). Dynamic Light Scattering (DLS) techniques were used to determine the size distribution and zeta potential of free NV, PD-1NV and IGU-Rh-PD-1 NV. DLS analysis showed a slight increase in average diameter (142 nm) at loaded IGU and Rh-NPs compared to PD-1NVs (130 nm). The Zeta potential of NV was also determined to be-5 mV and-7 mV before and after loading, respectively (FIG. 2D).

Example 3

In vitro targeting of IGU-Rh-PD-1NVs

Antigen-specific cd8+ T cells are depleted due to over-expression of PDL-1 binding to the PD-1 receptor on cancer cells. To examine whether PD-1NVs bind to lung cancer cells, PD-1NVs were co-cultured in vitro with LLC. Free NV and PD-1NV were labeled with Dio dye, which was used to label the cell membrane of LLC. We observed that PD-1NV bound efficiently to the cell membrane surface of LLC cells after 4 hours of incubation (fig. 3A). To verify whether the PD-1 receptor remained externally oriented on the NVs surface, we performed an immunoprecipitation assay (IP). Most of the PD-1NV was pulled down by the PD-1 antibody, indicating the correct orientation of the PD-1 receptor outside of most of the PD-1NV (FIG. 3B). In addition, CO-IP results also indicate that PD-1NVs successfully bind to PDL-1 receptors on cancer cells (FIG. 3C).

Example 4

IGU-Rh-PD-1NVs therapeutic effects on lung cancer cells in vitro

We used the CCK8 kit to detect in vitro cytotoxic effects of IGU, rh-NPs and IGU-Rh-PD-1NVs on LLC cells. First, we used different concentrations of IGU and Rh-NPs to evaluate IC50 against cancer cells and cytotoxic effects against normal cell lines (L929) (fig. 4A-D). Second, we studied and compared the effects of IGU, rh-NPs and PD-1NVs and IGU-Rh-PD-1NVs in the presence and absence of CD8T cells (fig. 4e, f). Time-dependent cytotoxicity experiments also showed similar effects (fig. 4G). Taken together, the results indicated that IGU, rh-NPs have anticancer capabilities, while no significant effect was observed on normal cell lines. Furthermore, IGU-Rh-PD-1NVs significantly inhibited LLC growth compared to NC, indicating that combination treatment has greater anti-tumor potential than monotherapy.

Example 5

Effect of IGU-Rh-PD-1NVs on apoptosis and improving ROS production

We examined whether IGU-Rh-PD 1NVs treatment affected apoptosis of LLC cells. The proportion of apoptotic cells was determined by staining cells with PI and Annexin V-FITC using flow cytometry. A significant increase in apoptosis induced by IGU-Rh-PD 1NVs was observed compared to the control and other groups alone (FIGS. 5A, 5C).

Elevated ROS levels activate cell signaling pathways such as mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF- κb), and Wnt, which are involved in apoptosis. Based on the above results, we hypothesize that our combination therapy may improve apoptosis in a ROS-dependent manner. To confirm our hypothesis, we analyzed the effect of IGU-Rh-PD 1NVs treatment on ROS production, we found that IGU-Rh-PD 1NVs promoted ROS production (fig. 5b, d). Next, we assessed the expression level of ROS-dependent apoptosis pathway proteins by using western blot analysis. As shown in FIG. 5E, LLC cells treated with IGU-Rh-PD 1NVs demonstrated increased p-ERK and p-JNK expression levels in IGU, rh-NPs and IGU-Rh-PD 1NVs treated groups.

Example 6

Therapeutic effects of IGU-Rh-PD-1NVs on in vivo lung cancer mouse models

The antitumor effect of IGU-Rh-PD 1NVs was also demonstrated in xenograft tumor mouse models. The C57/L6 mice were divided into six groups: PBS, free NVs, IGU, rh-NPs, PD 1NVs and IGU-Rh-PD1 NVs. Treatment was injected into tumor-bearing mice every three days later. Tumors received a laser beam at 808nm (200 mW cm-2) for 5 minutes after injection for 3 hours in Rh-NPs and IGU-Rh-PD-1NVs treatment groups. Tumor growth was monitored by using vernier calipers. Macroscopic, the growth rate of xenograft tumors treated with IGU-Rh-PD 1NV was significantly slower compared to PBS and IGU, rh-NPs and PD1-NV treated groups (FIG. 6A). The tumor growth curves of mice treated with IGU-Rh-PD 1NVs showed a relatively slow trend compared to the control group (fig. 6b, c). The body weights of the mice in the treatment group and the control group did not change significantly during all the measured times (FIG. 6D).

It should be understood that the foregoing description of the specific embodiments is merely illustrative of the invention, and is not intended to limit the invention, and that any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. The PD-1 nano vesicle loaded with the Iguratimod-rhodium nano particles is characterized in that the nano vesicle of which the engineering cell membrane source expresses the PD-1 is taken as a drug carrier to wrap the Iguratimod and the rhodium nano particles.

2. A method for preparing the Iguratimod-rhodium nanoparticle-loaded PD-1 nanovesicle according to claim 1, comprising the steps of:

s1, preparation of nano vesicles from PD-1 cell membrane expression: constructing cells stably expressing PD-1, collecting the cells and suspending the cells in a separation buffer solution, centrifuging the cells after homogenizing and grinding to collect supernatant, centrifuging the supernatant again, discarding the supernatant to obtain plasma membrane precipitate, washing and purifying the plasma membrane precipitate, and extruding the plasma membrane precipitate by a polycarbonate membrane extruder after ultrasonic treatment to obtain nano vesicles;

3. The method according to claim 2, wherein the PD-1 stably expressing cells in step S1 are any one of HEK293T, hepG2, huh-7, hepa1-6, SK-Hep-1 and lymphocytes.

4. Use of a PD-1 nanovesicle loaded with an Iguratimod-rhodium nanoparticle according to claim 1 for the preparation of a tumor therapeutic drug.

5. The use according to claim 4, wherein the tumor comprises liver cancer, lung cancer, esophageal cancer, gastric cancer, melanoma, colorectal cancer, bladder cancer, pancreatic cancer, ovarian cancer, breast cancer, neuroblastoma.

6. The use according to claim 5, wherein the tumour is lung cancer.

7. The use according to any one of claims 4 to 6, wherein the medicament is for inhibiting tumor cell proliferation.

8. The use according to any one of claims 4 to 6, wherein the medicament is for inhibiting tumor cell metastasis.