WO2021098621A1

WO2021098621A1 - Immunoadjuvant-encapsulated nanoparticle and use thereof

Info

Publication number: WO2021098621A1
Application number: PCT/CN2020/128862
Authority: WO
Inventors: 蔡林涛; 周海梅; 刘兰兰; 何华美; 梁锐晶
Original assignee: 深圳先进技术研究院
Priority date: 2019-11-20
Filing date: 2020-11-13
Publication date: 2021-05-27
Also published as: CN110755387A; CN110755387B

Abstract

Provided is a nanoparticle having a simple preparation process, stable properties and good biocompatibility. An immunoadjuvant can be efficiently encapsulated in the nanoparticle, so as to form a stable drug-loaded nanoparticle having a high encapsulation rate and a high loading capacity. The nanoparticle can be rapidly degraded under acidic (pH<7. 4) conditions, and can achieve tumor immunotherapy by means of the immunomodulatory function of the immunoadjuvant per se and the tumor-killing effect of the loaded related drugs, improving the anti-tumor efficacy and having important use prospects in the field of nanomedicine.

Description

Nanoparticles containing immune adjuvant and application

Technical field

The invention belongs to the field of nanomedicine, and specifically relates to a nanoparticle containing an immune adjuvant, a preparation method thereof, and an application in tumor immunotherapy.

Background technique

Tumor treatment is a complicated process. The three traditional methods of cancer treatment mainly include surgical resection, chemotherapy, and radiotherapy. These methods can effectively treat some tumors and control tumor metastasis. However, long-term clinical practice has found that there are still some shortcomings, such as It is highly traumatic, low-targeting, and easy to produce drug resistance. In recent years, studies have reported that the use of tumor immunotherapy can achieve more effective anti-tumor effects by taking advantage of its wide trial range, fewer side effects, significant curative effects and high sustained effects. Tumor immunotherapy has developed rapidly and has achieved a series of major breakthroughs. In 2013, it was named the top ten scientific and technological breakthroughs by Science magazine. Tumor immunotherapy is an emerging tumor treatment after traditional surgery, chemotherapy, and radiotherapy. It has attracted the attention of scholars because of its high specificity and remarkable curative effect. Tumor immunotherapy mainly achieves the purpose of treating tumors by activating or improving the immune function of the human body. It has little side effects on the human body, and at the same time, immunotherapy has long-term anti-tumor immune function, which can permanently and specifically recognize tumor cells, thereby effectively inhibiting tumor metastasis and recurrence. With the in-depth understanding of the tumor microenvironment and tumor escape mechanism, mobilizing the body's immune system to resist tumors has gradually become a new research direction. As an emerging tumor treatment method, tumor immunotherapy effectively compensates for some shortcomings of traditional treatment methods and provides new ideas and directions for the treatment of certain malignant tumors.

Poly(lactic-co-glycolic acid), PLGA) is formed by the random polymerization of two monomers-lactic acid and glycolic acid. It is a degradable functional polymer organic compound with good biocompatibility, non-toxicity, and good encapsulation and synthesis. The performance of the membrane is widely used in pharmaceuticals, medical engineering materials and modern industrial fields. In the United States, PLGA passed FDA certification and was officially included in the United States Pharmacopoeia as a pharmaceutical excipient.

Poly I:C alias polyinosinic acid, polyinosinic acid, polycytidylic acid, is an analog of double-stranded RNA, one chain is poly(I), the other chain is poly(C), Poly I:C It is an interferon inducer, which produces interferon under the induction of cells in the body. It has a wide range of antiviral and immunoregulatory functions and is used for adjuvant treatment of viral infectious diseases and tumors. Poly I:C is a ligand for type III Toll-like receptors in animals. After activating TLR-3, it can mediate a series of immune responses in the body, such as inducing the secretion of interferon, interleukin, tumor necrosis factor and other cytokines, and promoting cells Proliferation and maturation of, monocytes, macrophages, lymphocytes and dendritic cells, etc., promote the production of antibodies in the body, so Poly I:C has a good promotion effect on the body's specific and non-specific immunity . Because Poly I:C has an immune adjuvant effect, it can stimulate the reticuloendothelial system, enhance the phagocytic function of phagocytes, enhance the formation of antibodies, stimulate allograft reactions and delayed allergic reactions, etc., so it has a certain anti-tumor effect .

technical problem

The present invention provides a poly I:C nanoparticle encapsulating immune adjuvant and a preparation method and application thereof, and aims to provide a nanoparticle with simple preparation process, stable properties and good biocompatibility, and adopts Poly I: The immunomodulatory function and tumor killing effect of C itself can achieve tumor immunotherapy, which has important application prospects in the field of nanomedicine.

Technical solutions

An object of the present invention is to provide a nanoparticle encapsulating an immune adjuvant, the nanoparticle is composed of liposomes, an immune adjuvant, maleimide, and a copolymer of about 5000 to 200,000 daltons. Said liposome is selected from natural lecithin, synthetic lecithin, distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phospholipid (HSPC), soybean lecithin, egg yolk egg Any one or more of phospholipids, the immune adjuvant is selected from any one of monophosphoryl lipid (MPLA), imiquimod (Imiquimod), and polyinosinic acid (Poly(I:C)) Or more, the copolymer is selected from polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid (PLGA), polylactic acid-polyethylene glycol (PLA-PEG), polyglycolic acid- Any one or more of polylactic acid-polyethylene glycol (PLGA-PEG) or polycaprolactone-polyethylene glycol (PCL-PEG).

Preferably, the particle size of the nanoparticles is 10-150, preferably 30-100 nm, more preferably 60-90 nm.

Preferably, the liposome is lecithin, preferably soy lecithin.

Preferably, the immune adjuvant is Poly(I:C).

Preferably, the copolymer is PLGA.

Preferably, the maleimide is phospholipid polyethylene glycol maleimide (DSPE-PEG-Mal).

Preferably, the nanoparticles have the performance of loading, delivery and/or slow release, and the nanoparticles are easier to release in an environment with pH<7.4; preferably, the nanoparticles are released in an environment with pH=5. The best results.

Another object of the present invention is to provide a composition including the nanoparticle, and the composition also includes an anti-tumor drug.

Preferably, the anti-tumor drugs include anti-tumor broad-spectrum drugs and/or anti-tumor targeted drugs.

Preferably, the anti-tumor broad-spectrum drug is selected from any one or more of camptothecin drugs, doxorubicin drugs, paclitaxel drugs or platinum drugs.

Preferably, the anti-tumor targeted drug is selected from the group consisting of zebutinib, nilotinib, imatinib, vermoderil, verofenib, temsirolimus, sunitinib, and ceritin Ni, regorafenib, afatinib, trametinib, pranatinib, bortezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib , Levatinib, Dalafenib, Crizotinib, Carfilzomib, Ostinib, Cabotinib, Carbitinib, Gefitinib, Vorinostat, Vandetanib , Alectinib, denosumab, sondeji, sorafenib, bosutinib, belisstat, olaparib, aflibercept, lapatinib, dasatinib, Any one or more of Pabocinib, Pabisstat or Erlotinib.

Preferably, the composition further includes a polypeptide substance, and the polypeptide includes an antigen or an antibody.

Preferably, the antibody is selected from the group consisting of adalimumab, cetuximab, ibrituximab, trastuzumab, nivolumab, darrilimumab ramucirumab, and navastin Razizumab, pembrolizumab, pembrolizumab, ofatumumab, Bonatumumab, bevacizumab, panitumumab, obiniutuzumab, bentuximab , Denutuximab, Tositumomab, Errotuzumab, Trastuzumab or Rituximab.

Preferably, the antigen is a tumor antigen.

Preferably, the tumor is selected from basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.

Preferably, the anti-tumor drug or the polypeptide substance is embedded in the nanoparticle or adsorbed on the surface of the nanoparticle.

Another object of the present invention is to provide a medicine containing the nanoparticle or the composition.

Preferably, the drug is an anti-tumor drug.

Another object of the present invention is to provide a nanoparticle adjuvant containing the nanoparticle or the composition.

Preferably, the drug or the nanoparticle adjuvant is administered by injection and/or oral administration.

Preferably, the injection administration includes any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumor injection or subfoot injection.

Preferably, the drug or the nanoparticle adjuvant further includes medically or pharmaceutically acceptable auxiliary substances and/or excipients.

Another object of the present invention is to provide an application of the nanoparticle or the composition in the preparation of antitumor drugs or nanoparticle adjuvants.

Another object of the present invention is to provide a method for preparing the nanoparticles, which includes the following steps:

(1) Prepare an acetone solution of the copolymer to obtain a copolymer solution; prepare an ethanol solution of the liposome to obtain a liposome solution; prepare an ethanol solution of maleimide to obtain a maleimide solution ; Prepare an aqueous solution of immune adjuvant to obtain an aqueous solution of immune adjuvant;

(2) Mix the liposome solution and the maleimide solution together; the mass ratio of the liposome solution to the maleimide solution is (1-5): (2) -10), the total mass of the liposome solution and the maleimide solution is 5-30% of the added copolymer solution;

(3) Add the immune adjuvant aqueous solution obtained in step (1) to the solution obtained in step (3);

(4) Ultrasound the mixed solution obtained in step (3) for 1-15 min with an ultrasonic disruptor, during which time the copolymer solution is added dropwise with a syringe;

(5) Transfer the nanoparticle solution containing the immune adjuvant obtained in step (4) to a dialysis bag and dialyze to obtain purified nanoparticles containing the immune adjuvant.

Preferably, the concentration of the copolymer solution in the step (1) is 0.2-10 mg/mL, preferably 2 mg/mL.

Preferably, the concentration of the liposome solution in the step (1) is 0.2-50 mg/mL, preferably 10 mg/mL.

Preferably, the concentration of the maleimide solution in the step (1) is 0.2-50 mg/mL, preferably 10 mg/mL.

Preferably, the concentration of the immune adjuvant solution in the step (1) is 0.2-10 mg/mL, preferably 2.5 mg/mL.

Preferably, the mass ratio of the liposome solution to the maleimide solution in the step (2) is 2:3.

Preferably, the total mass of the liposome solution and the maleimide solution in the step (2) is 15% of the added copolymer solution.

Preferably, the ultrasound in the step (4) is ultrasound with a power of 20HZ and 130W for 5 minutes.

Beneficial effect

The characteristics of the encapsulated immune adjuvant nanoparticles of the present invention are: ①The immune adjuvant can be efficiently encapsulated in the nanoparticles to form stable drug-loaded nanoparticles with high encapsulation efficiency and high loading; ②Polylactic acid- Glycolic acid copolymer has good biocompatibility and biodegradability, which can reduce the toxicity of drugs to the body; ③The self-assembly of polylactic acid-glycolic acid copolymer, lecithin, immune adjuvant and maleimide Nanoparticles can be rapidly degraded under acidic conditions (pH<7.4) to achieve controlled drug release; ④After the nanoparticles containing immune adjuvant reach the tumor site, the nanoparticles can release the immune adjuvant in situ to stimulate the body Produce a series of immune effects and improve anti-tumor efficacy.

Description of the drawings

Figure 1 shows the particle size distribution diagram of Poly I:C nanoparticles encapsulating immune adjuvant.

Figure 2 shows the potential diagrams of Poly I:C nanoparticles without immune adjuvant and Poly I:C nanoparticles with immune adjuvant.

Figure 3 shows the release diagram of Poly I:C in Poly I:C nanoparticles encapsulated with immune adjuvant at different pH.

Figure 4 shows Poly without encapsulated immune adjuvant Ultraviolet-visible absorption spectra of I:C nanoparticles and Poly I:C nanoparticles containing immune adjuvant.

Embodiments of the present invention

Hereinafter, the present invention will be further described in detail through specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

Example 1 Preparation of Poly I:C Nanoparticles Encapsulated Immunity Adjuvant

①Prepare the acetone solution (2 mg/mL) of polylactic acid-glycolic acid copolymer (PLGA); prepare the ethanol solution of soybean lecithin (10 mg/mL); prepare the aqueous solution of Poly I:C (2.5 mg/mL); Prepare maleimide (DSPE-PEG-Mal) ethanol solution (10 mg/mL);

②Mix 120μL of soybean lecithin solution and 180μL of maleimide solution; the mass ratio of soybean lecithin solution to maleimide solution is 2:3, and the total weight of soybean lecithin solution and maleimide solution is 2:3. The mass is 15% of the added PLGA solution;

③Add 200μL Poly I:C aqueous solution (2.5 mg/mL) to the above solution;

④Using an ultrasonic breaker to sonicate the above-mentioned mixed solution with a power of 20HZ and 130W for 5 minutes, during which time 10 mL of PLGA solution (2 mg/mL) was added dropwise;

⑤Transfer the poly I:C nanoparticle solution containing the immune adjuvant to a dialysis bag with a molecular weight of 3500, and dialyze it with pure water for 24 h to obtain the purified poly I:C nanoparticle containing the immune adjuvant.

Example 2 Performance and Characterization of Poly I:C Nanoparticles Encapsulated Immunological Adjuvant

1. Particle size distribution of Poly I:C nanoparticles containing immune adjuvant

A Malvern particle size analyzer was used to determine the particle size distribution of the encapsulated immunoadjuvant Poly I:C nanoparticles. It was found that the size of PIP nanoparticles was about 70 nm, and the particle size distribution diagram is shown in Figure 1.

2. Potential detection of Poly I:C nanoparticles containing immune adjuvant

A Malvern particle size analyzer was used to measure the potentials of Poly I:C nanoparticles without immune adjuvant and Poly I:C nanoparticles with immune adjuvant. We found that after encapsulating the immune adjuvant Poly I:C, the potential of the particles decreased from -18mV to -24mV, indicating that the immune adjuvant Poly I:C was successfully encapsulated. The potential diagram is shown in Figure 2.

3. Detection of the release of Poly I:C in Poly I:C nanoparticles encapsulated in immune adjuvant Poly I:C at different pH

Through testing, it was found that both pH5.0 and pH7.4 maintained the release capacity of Poly I:C, but the release volume of pH5.0 was about twice that of pH7.4. Obviously, Poly I:C is easier to release in an acidic environment. As shown in Figure 3.

4. UV-Vis Absorption Spectroscopy Detection of Poly I:C Nanoparticles with Immunity Adjuvant

Ultraviolet-visible spectrophotometer was used to measure the UV-visible absorption spectra of Poly I:C nanoparticles without immune adjuvant and Poly I:C nanoparticles with immune adjuvant. It can be seen from the UV-visible absorption spectrum results in Fig. 4 that the poly I:C nanoparticle encapsulated immune adjuvant has the characteristic absorption peak of Poly I:C, indicating that the immune adjuvant Poly I:C is successfully encapsulated.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. Range of.

Claims

A nanoparticle encapsulating an immune adjuvant, the nanoparticle is composed of liposomes, an immune adjuvant, maleimide, and a copolymer of about 5000 to 200,000 daltons, and the liposomes are selected from natural Any one or more of lecithin, synthetic lecithin, distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean lecithin (HSPC), soybean lecithin, egg yolk lecithin Species, the immune adjuvant is selected from any one or more of monophosphoryl lipid (MPLA), imiquimod (Imiquimod), polyinosinic acid (Poly(I:C)), and the copolymerization The material is selected from polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid (PLGA), polylactic acid-polyethylene glycol (PLA-PEG), polyglycolic acid-polylactic acid-polyethylene glycol (PLGA-PEG) or any one or more of polycaprolactone-polyethylene glycol (PCL-PEG); the particle size of the nanoparticles is 10-150, preferably 30-100nm, more preferably It is 60-90nm.
The nanoparticle according to claim 1, which has the performance of loading, delivery and/or slow release, and the nanoparticle is more easily released in an environment of pH<7.4; preferably, the nanoparticle is in an environment of pH=5 The slow-release effect is the best.
The nanoparticle according to claim 1, wherein the liposome is lecithin, preferably soy lecithin; the immune adjuvant is Poly(I:C); the copolymer is PLGA; the horse Leimide is phospholipid polyethylene glycol maleimide (DSPE-PEG-Mal).
A composition comprising the nanoparticle according to any one of claims 1 to 3, the composition further comprising an anti-tumor drug and/or a polypeptide substance; the anti-tumor drug includes an anti-tumor broad-spectrum drug and/or an anti-tumor drug Tumor targeting drugs; the polypeptides include antigens or antibodies.
The composition according to claim 4, wherein the anti-tumor broad-spectrum drug is selected from any one or more of camptothecin drugs, doxorubicin drugs, paclitaxel drugs or platinum drugs.
The composition according to claim 4, wherein the anti-tumor targeted drug is selected from the group consisting of zebutinib, nilotinib, imatinib, vermodil, verofenib, temsirolimus, Sunitinib, Ceritinib, Regorafenib, Afatinib, Trametinib, Pranatinib, Bortezomib, Pazopanib, Axitinib, Romidepsin, Ive Moss, Ibrutinib, Levatinib, Dabrafenib, Crizotinib, Carfilzomib, Ostinib, Cabotinib, Carbitinib, Gefitinib, Vorib Nota, vandetanib, alectinib, denosumab, sondeji, sorafenib, bosutinib, belisstat, olaparib, aflibercept, lapatinib Any one or more of Ni, Dasatinib, Pabocinib, Pabistat or Erlotinib;.
The composition according to claim 4, wherein the antibody is selected from the group consisting of adalimumab, cetuximab, ibrituzumab, trastuzumab, nivolumab, darelimumab, ramo Lutuzumab, Nexituzumab, Pembrolizumab, Pembrolizumab, Ofatumumab, Bonatumumab, Bevacizumab, Panitumumab, Obinutuzumab Any one or more of anti-, Bentuximab, Denutuximab, Tositumomab, Erotuzumab, Trastuzumab, or Rituximab.
The composition according to claim 4, wherein the anti-tumor drug or the polypeptide substance is embedded in the nanoparticle or adsorbed on the surface of the nanoparticle.
A drug or nanoparticle adjuvant containing the nanoparticle according to any one of claims 1-3 or the composition according to any one of claims 4-8.
The drug or nanoparticle adjuvant according to claim 9, which is administered by injection and/or oral administration.
The drug or nanoparticle adjuvant according to claim 10, wherein the injection administration includes any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumoral injection or subfoot injection .
The drug or nanoparticle adjuvant according to any one of claims 9-11, further comprising medically or pharmaceutically acceptable auxiliary substances and/or excipients.
An application of the nanoparticle according to any one of claims 1 to 3 or the composition according to any one of claims 4-8 in the preparation of anti-tumor drugs or nanoparticle adjuvants.
The composition according to any one of claims 4-8 or the application of claim 13, wherein the tumor is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, One or more of breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.
A method for preparing the nanoparticles of any one of claims 1-14, comprising the following steps:

(1) Prepare an acetone solution of the copolymer to obtain a copolymer solution; prepare an ethanol solution of the liposome to obtain a liposome solution; prepare an ethanol solution of maleimide to obtain a maleimide solution ; Prepare an aqueous solution of immune adjuvant to obtain an aqueous solution of immune adjuvant;

(2) Mix the liposome solution and the maleimide solution together; the mass ratio of the liposome solution to the maleimide solution is (1-5): (2) -10), the total mass of the liposome solution and the maleimide solution is 5-30% of the added copolymer solution;

(3) Add the immune adjuvant aqueous solution obtained in step (1) to the solution obtained in step (3);

(4) Ultrasound the mixed solution obtained in step (3) for 1-15 min with an ultrasonic disruptor, during which time the copolymer solution is added dropwise with a syringe;

(5) Transfer the nanoparticle solution containing the immune adjuvant obtained in step (4) to a dialysis bag, and dialyze to obtain purified nanoparticles containing the immune adjuvant.
The method according to claim 15, wherein the concentration of the copolymer solution in the step (1) is 0.2-10 mg/mL, preferably 2 mg/mL; the concentration of the liposome solution is 0.2-50 mg/mL, preferably 10 mg/mL; the concentration of the maleimide solution is 0.2-50 mg/mL, preferably 10 mg/mL; the concentration of the immune adjuvant solution is 0.2-10 mg/mL, preferably 2.5 mg/mL.
The method according to claim 15, wherein the mass ratio of the liposome solution to the maleimide solution in the step (2) is 2:3; the liposome solution and the maleimide solution The total mass of the imide solution is 15% of the added copolymer solution.
The method according to any one of claims 15-17, wherein the ultrasound in the step (4) is ultrasound with a power of 20HZ and 130W for 5 minutes.