WO2018228464A1

WO2018228464A1 - Tumour-targeted nanocapsule, preparation method therefor and use thereof

Info

Publication number: WO2018228464A1
Application number: PCT/CN2018/091218
Authority: WO
Inventors: 原续波; 康春生; 卢云峰
Original assignee: 天津纳诺生物科技有限公司
Priority date: 2017-06-14
Filing date: 2018-06-14
Publication date: 2018-12-20
Also published as: CN109078009A

Abstract

Disclosed is a tumour-targeted nanocapsule comprising a polymer sensitive to a tumour microenvironment, a substance having an anti-tumour activity and encapsulated by the polymer, and a tumour diagnostic reagent or a contrast agent; wherein the polymer sensitive to the tumour microenvironment comprises a cross-linking agent sensitive to the tumour microenvironment and 2-methacryloyloxyethyl phosphorylcholine, and the cross-linking agent sensitive to the tumour microenvironment at least comprises a polypeptide cross-linking agent capable of being degraded by an enzyme overexpressed in the tumour microenvironment. The polypeptide cross-linking agent capable of being degraded by an enzyme overexpressed in the tumour microenvironment is preferably one or more of a polypeptide cross-linking agent capable of being degraded by matrix metalloproteinases and a polypeptide cross-linking agent capable of being degraded by hyaluronidases, and is more preferably one or more of a polypeptide cross-linking agent capable of being degraded by matrix metalloproteinases.

Description

Tumor-targeted nano microcapsule and preparation method and application thereof

Cross-reference to related applications

The present application claims the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the disclosure.

Technical field

The invention belongs to the fields of biomedicine, preparation and polymer materials, and particularly relates to a tumor targeting nano microcapsule based on tumor microenvironment characteristics and preparation method and application thereof

Background technique

The ultimate goal of malignant tumor treatment is to increase the survival time of patients and improve the quality of life by reducing the systemic toxicity of the drug. The targeted delivery and release of drugs is a key link to improve efficacy and reduce toxicity. One of the important branches is nanocapsules containing anti-tumor active substances.

In the blood vessels of normal tissues, the distance between the two endothelial cells is about 2 nm, and in the tumor blood vessels, the distance between the two endothelial cells is 100-150 nm. This structural feature leads to enhanced permeability of the tumor tissue. Enhanced permeability and retention effect (EPR effect). The nanocapsules generally have a particle size of 10 to 150 nm and have a strong penetrating power, which can penetrate into tumor tissues and accumulate. This nano-targeted drug delivery system based on the EPR effect of tumors can only be called "passive targeting." With the deepening of research on tumors and tumor microenvironment, active targeting of nanocapsules has become a research hotspot.

Early cancer research focuses on tumor cells, focusing on gene mutations, proliferation and apoptosis of tumor cells. With the deepening of research, it is recognized that tumors are organisms composed of tumor cells and various stromal cells and non-cellular components. The core of them is tumor cells, and the surrounding stromal cells and non-cellular components constitute the growth of tumor cells. Environment, the "tumor microenvironment". When stromal cells in the tumor microenvironment are transformed by tumor cells, a large number of growth factors, cell chemokines and matrix degrading enzymes are produced around them, which promote tumor development by inducing neovascularization, inhibiting immune response and infecting cancer stem cells. To regulate a variety of biological behaviors of tumors (Du Gangjun, et al. A new approach to cancer treatment: targeting tumor microenvironment [J]. International Journal of Pharmaceutical Research, 2011, 38 (5): 336-341).

Yan Ming et al reviewed the microenvironment and tumor malignant transformation, pointing out that microenvironment hypoxia, high pressure, large growth factors and hydrolyzed protease production and its immunoinflammatory reaction are the main biological characteristics of tumor tissue metabolic environment, which is for tumor growth. Invasion, metastasis, and angiogenesis have very important effects (Ming Ming, et al. Tumor microenvironment and malignant transformation of tumors [J]. Cancer mutation distortion, 2008, 20 (5): 412-417). The characteristics of the above tumor blood vessels are also the result of the action of a large number of vascular growth factors in the tumor microenvironment.

Ma et al. showed that in the process of breast cancer from normal tissue to precancerous lesions to invasive ductal carcinoma, the extracellular matrix (ECM) of the tumor matrix includes collagen, laminin and proteoglycan complexes. Expression of matrix metalloprotease (MMP) and cell cycle related genes is up-regulated; precancerous lesions to invasive changes are characterized by several matrix metalloproteinases (MMP-2, MMP-11 and MMP-14). High expression (MaXJ, etal. Gene expression profiling of the tumor micro-environment during breast cancer progression [J]. Breast Cancer Res, 2009, 11(1): R7.). In prostate cancer, MMP-1 and MMP-13 have a role in promoting tumor cell invasion; among numerous family members, MMP-2 and MMP-9, which are the only two gelatinases in the MMP family, are known. Important members closely related to cancer metastasis and invasion (Hua Dan, et al. Progress in metalloproteinase anti-cancer targets and targeted peptide drugs] [J]. Chinese Journal of New Drugs, 2014, 23(19): 2231-2237 ).

Wang Yu et al. pointed out in the review that due to the rapid proliferation of tumors, the nutrition and oxygen provided by the vasculature can not meet their needs, and the lack of oxygen leads to insufficient energy, resulting in the production of lactic acid and ATP hydrolysate, resulting in increased extracellular acidity of the tumor, pH. It is lower than normal tissue (Wang Yu, et al. Overview of pH-sensitive nanoformers [J]. Chinese Journal of Pharmaceutics, 2009, 7(2): 72-76).

In summary, the tumor microenvironment is characterized by hypoxia, low pH, growth factors, and protease aggregation. In response to these characteristics, in recent years, researchers have conducted a lot of research on the active targeted modification of tumor nanoparticles. Ge et al. describe in their review: "Different units of enzyme degradable crosslinkers linked to block copolymers and polymers thereof can be used to load chemotherapeutic drugs or developers and release the loaded drug or developer by enzymatic degradation. The review further indicates that the MMP-2 degradable polypeptide sequence (Gly-Pro-Val-Gly-Leu-Ile-Gly-Lys) crosslinks temperature-sensitive multi-block copolymers and poloxamer triblock copolymers. The temperature-sensitive gel condenses in the body temperature range and encounters the over-expressed MMP-2 in the tumor tissue and degrades (Ge, ZSet al. Functional block copolymer assemblies responsive to tumor and intracellular microenvironments for site specific drug delivery and enhanced imaging performance [ J]. Chemical Society Reviews. 2013, 42(17): 7289-7325).

Daniel et al. used open-loop metathesis polymerization (ROMP) to synthesize amphiphilic block copolymers using highly reactive functional groups that are resistant to sulfhydryl initiators, and designed and prepared matrix metalloproteinases (MMPs) and reactive oxygen species. (ROS) dual-responsive nanoparticle for the delivery of inflammatory diseases (Daniel KB, et al. Dual-responsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species [J], Chemical Communications, 2016, 52(10): 2162-2128).

Prior art studies have shown that tumor microenvironment-specifically expressed enzymes, such as MMPs, as well as special biochemical conditions, such as hypoxia and low pH, can be used as targets for drug delivery systems. However, based on the complexity of the tumor, there are still many difficulties to overcome in developing nano-microcapsules with clinical value.

Summary of the invention

In order to meet the clinical therapeutic and diagnostic needs, the present invention provides a tumor-targeted nanocapsule, and the nanocapsule of the present invention can be compared to the conventional passively targeted nanocapsule based only on the EPR effect. Respond to multiple specific factors in the tumor microenvironment, rapid release at the tumor site, and achieve active targeting, thereby improving the efficacy of tumor treatment drugs, reducing systemic toxicity, or more conducive to early diagnosis of tumors.

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

A tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or a developer; wherein the tumor is microenvironment sensitive The polymer comprises a tumor microenvironment sensitive crosslinker and 2-methacryloyloxyethylphosphocholine, the tumor microenvironment sensitive crosslinker comprising at least a polypeptide crosslinkable by an enzyme that is overexpressed in the tumor microenvironment Agent.

Preferably, the polypeptide crosslinking agent capable of being degraded by an enzyme overexpressed in the tumor microenvironment is selected from one of a matrix metalloproteinase degradable polypeptide crosslinking agent and a hyaluronidase degradable polypeptide crosslinking agent or A variety.

More preferably, the polypeptide crosslinking agent capable of being degraded by an enzyme overexpressed in the tumor microenvironment is selected from one or more of the matrix metalloproteinase degradable polypeptide crosslinking agents.

Preferably, the matrix metalloproteinase degradable polypeptide crosslinking agent is selected from the group consisting of a matrix metalloproteinase-2 degradable polypeptide crosslinking agent, a matrix metalloproteinase-7 degradable polypeptide crosslinking agent, and matrix metalloproteinase-9. One or more of the polypeptide crosslinkers; more preferably one or both of the matrix metalloproteinase-2 degradable polypeptide crosslinker and the matrix metalloproteinase-9 degradable polypeptide crosslinker.

Preferably, the tumor microenvironment sensitive crosslinking agent may also be selected from one or more of a pH sensitive crosslinking agent and a redox sensitive crosslinking agent.

Preferably, the pH sensitive crosslinking agent is selected from the group consisting of an ester crosslinking agent and/or a Schiff base crosslinking agent; more preferably ethylene glycol dimethacrylate.

Preferably, the oxidation-reduction sensitive crosslinking agent is selected from the group consisting of disulfide-containing crosslinking agents; more preferably diallyl disulfide.

Preferably, the tumor microenvironment sensitive polymer further comprises a hydrophobic polymerizable monomer.

As a preferred embodiment, the present invention provides a tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or a developer Wherein the tumor microenvironment sensitive polymer comprises the above tumor microenvironment sensitive crosslinker, 2-methacryloyloxyethylphosphocholine and a hydrophobic polymerizable monomer; preferably, the above tumor microenvironment is sensitive The molar ratio of the crosslinking agent, 2-methacryloyloxyethylphosphocholine and the hydrophobic polymerizable monomer is:

Tumor microenvironment sensitive crosslinker: 2-methacryloyloxyethylphosphocholine: hydrophobic polymerizable monomer = 1:10:1 to 1:100:1.

As a preferred embodiment, the present invention provides a tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or a developer Wherein the tumor microenvironment sensitive polymer is polymerized by 2-methacryloyloxyethylphosphocholine, a hydrophobic polymerizable monomer, and one or more matrix metalloproteinase degradable polypeptide crosslinkers. The matrix metalloproteinase degradable polypeptide crosslinking agent is selected from the group consisting of a matrix metalloproteinase-2 degradable polypeptide crosslinking agent, a matrix metalloproteinase-7 degradable polypeptide crosslinking agent, and a matrix metalloproteinase-9 degradable Polypeptide crosslinker.

As another preferred embodiment, the present invention provides a tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or development The tumor microenvironment sensitive polymer is polymerized by 2-methacryloyloxyethylphosphocholine, a hydrophobic polymerizable monomer, and a polypeptide crosslinker degradable by two or more matrix metalloproteinases. The matrix metalloproteinase degradable polypeptide crosslinking agent is selected from the group consisting of a matrix metalloproteinase-2 degradable polypeptide crosslinking agent, a matrix metalloproteinase-7 degradable polypeptide crosslinking agent, and matrix metalloproteinase-9. Polypeptide crosslinker.

In still another preferred embodiment, the present invention provides a tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or development The tumor microenvironment sensitive polymer is polymerized by 2-methacryloyloxyethylphosphocholine, a hydrophobic polymerizable monomer, and two or more tumor microenvironment sensitive crosslinkers. The tumor microenvironment sensitive cross-linking agent comprises a matrix metalloproteinase-2 degradable polypeptide cross-linking agent, a matrix metalloproteinase-7 degradable polypeptide cross-linking agent and a matrix metalloproteinase-9 degradable polypeptide cross-linking agent. One or more of them, and one or more selected from the group consisting of the pH-sensitive crosslinking agent or the oxidation-reduction crosslinking agent.

Preferably, the hydrophobic polymerizable monomer is selected from the group consisting of acrylic compounds, more preferably from acrylic acid and its salts, esters, amide derivatives, or alkyl-substituted acrylic acids and salts, esters, and amides thereof. derivative.

Further preferably, the hydrophobic monomer is selected from the group consisting of acrylic acid, methacrylic acid, acrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, ethylene glycol dimethacrylate, methylene bis acrylamide, hydroxy acrylate One of propyl ester, N-methylol acrylamide, N-2-hydroxypropyl-methacrylamide, and N-(3-aminopropyl)methacrylic acid hydrochloride, or a plurality of ratios.

Preferably, the substance having antitumor activity is selected from the group consisting of an anti-tumor monoclonal antibody, an anti-tumor monoclonal antibody-small molecule compound conjugate, an anti-tumor bispecific antibody or an anti-tumor small molecule compound.

The anti-tumor monoclonal antibody may be selected from the group consisting of Nimotuzumab, Cetuximab, Trastuzumab, Bevacizumab, Panitumumab (Panitumumab), Denosumab, Iprimimumab (or Ipilimumab), Pertuzumab, Ramucirumab, Pamuzumab (or called pembrolizumab), Nivolumab, Rituximab, Alemtuzumab, Tositumomab, and Alpha Resistance (Ofatumumab), Atolzuzumab (Obinutuzumab), Dinutuximab, and the like.

Anti-tumor monoclonal antibody-small molecule compound conjugate selected from

(ado-trastuzumab emtansine),

(ziv-aflibercept),

(Gemtuzumab-ozogamicin),

(brentuximab-vedotin), etc.

The anti-tumor bispecific antibody can be selected from

(Catumaxomab),

(blinatumomab) and so on.

The anti-tumor small molecule compound may be selected from the group consisting of a vinca alkaloid (eg, vinblastine, vincristine #, vinflunine §, vindesine, vinorelbine), doxorubicin and its derivatives, yew Alkanes (such as cabazitaxel, docetaxel, lalottan, ortaxel, paclitaxel, tesitastat, etc.), dihydrofolate reductase inhibitors (such as aminopterin, methotrexate #, Pemetrexed, prasic acid, etc.), thymidylate synthase inhibitors (such as raltitrexed, pemetrexed, etc.), adenosine deaminase inhibitors (such as pentastatin, etc.), halogenated /ribonucleotide reductase inhibitors (such as cladribine, clorabin, fludarabine, nairabin, etc.), thiopurines (such as thioguanine, guanidine, etc.), thymidylate synthase inhibition Agents (5-fluorouracil #, capecitabine, tegafur, carmofur, fluorouridine), polymerase chain reaction inhibitors (such as cytarabine, etc.), ribonucleotide reductase inhibitors ( Such as gemcitabine, etc.), DNA methylation inhibitors (such as azacitidine, decitabine, etc.), ribonucleotide reductase inhibitors (such as hydroxyurea), camptothecin (such as camptothecin, topology Kang, irinotecan, rubiconcan, beloxicon, etc.), podophyllin alkaloids (such as etoposide, teniposide, etc.), anthracyclines (such as arubicin, daunorubicin) , doxorubicin, epirubicin, idarubicin, amrubicin

Pirarubicin, valrubicin, zorubicin, steroids (such as mitoxantrone, pilocarpine, etc.), nitrogen mustard, cyclophosphamide (such as cyclophosphamide, ifosfamide) , tromethamine, etc., chlorambucil (such as melphalan, prednistatin, bendamustine, uraramustine, estramustine, etc.), nitrosourea (such as Camo Statin, lomustine, formoterol, nimustine, ramustine, streptozotocin, etc.), alkyl sulfonates (such as busulfan, etc.), nitrogen propylene ring ( Such as carbopol, thiotepa, triammine, tritamine, etc.), organoplatinum compounds (such as carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatinum tetrachloride, satraplatin, etc.), hydrazine Derivatives (such as procarbazine, etc.), triazenes (such as dacarbazine, temozolomide, etc.), hexamethylene melamine, dibromomannitol, piperacin, streptomycin (such as actinomycin, Bo L-mycin, mitomycin, pucamycin, etc.), 5-aminolevulinic acid/aminolevulinic acid methyl ester, ethylene propionate, porphyrin derivatives (such as sodium porphin, talaporfin , temoprofen, verteporfin, etc.), tyrosine kinase inhibitors (such as Imazine mesylate) Niger, gefitinib, lapatinib, nilotinib, telatinib, sunitinib malate, erlotinib hydrochloride, neratinib, dasatinib, bosutinib, Imatinib, axitinib, erlotinib, vandetanib, sectinib, carnitinib dihydrochloride, carnitinib, sunitinib, dasatinib, tan Dentinib, mulletinib, lapatinib di-p-toluenesulfonate, tenidap, dovetinib, butatinib, N-desmethylimatinib, bafluinib, Latini, bafilinib, tenidololol, pirinidine, tennic acid, tenidacetin, afatinib, molitinib, nilotinib, carnitinib, metformin Sunitinib, tenilapine, antipyrine, tenipazone, imatinib, ernoteinib, soratinib toluene, tinidopap sodium, etc., Fani Base transferase inhibitors (such as tififene and the like), cyclin-dependent kinase inhibitors (such as flurazepam, celesil, etc.), proteasome inhibitors (such as bortezomib, etc.), phosphodiesterase inhibition Agent (such as anagrelide, etc.), inosine dehydrogenase inhibition Formulations (such as thiazolidine, etc.), lipoxygenase inhibitors (such as masobolol), poly ADP-ribose polymerase inhibitors (such as olaparib), tissue protein deacetylase inhibitors (such as Vorinostat, romidepsin, etc.), endothelin receptor antagonists (such as atrasentan, etc.), retinoid X receptor agonists (such as bismuthine, etc.), sex steroids (such as testis Lactones, etc., ampicillin, trobeidine, retinoids (such as 9-cis retinoic acid, retinoic acid, etc.), arsenic trioxide, asparagine (such as asparaginase / angkasip) ), celecoxib, colchicine, Ili Simo, elsamitrucin, etodogr, chlordamine, thioxanthone, mitoxantrone, mitoxantrone, olimex, high serrata Ester base, rapamycin target protein inhibitors (such as everolimus, sirolimus, etc.) and the like.

Another object of the present invention is to provide a method for preparing the above-mentioned tumor-targeted nanocapsules, which comprises the steps of: in situ polymerization, first: 2-methacryloyloxyethylphosphocholine and the hydrophobicity The polymerizable monomer is added to the solution of the antitumor activity substance, the tumor diagnostic reagent or the developer, and then the tumor micro-environment sensitive cross-linking agent is added, and finally the initiator is added, and the reaction is carried out at 0 to 30 ° C for 0.1 to 24 hours. .

Preferably, the molar ratio of the antitumor activity substance, the tumor diagnostic reagent or the developer to the tumor microenvironment sensitive crosslinker is 1:100 to 1:10000.

Preferably, the molar ratio of the substance having antitumor activity, the tumor diagnostic reagent or the developer to 2-methacryloyloxyethylphosphocholine is 1:500 to 1:10000.

Preferably, the molar ratio of the substance having antitumor activity, the tumor diagnostic reagent or the developer to the hydrophobic polymerizable monomer is from 1:100 to 1:10000.

Preferably, the initiator consists of a persulfate and one selected from the group consisting of tetramethylethylenediamine, sodium sulfite and sodium hydrogen sulfite, and the molar ratio of the two is 1:100 to 100:1;

Still preferably, the persulphate is selected from the group consisting of ammonium persulfate or potassium persulfate.

In the above preparation method, the reaction temperature after the addition of the initiator is preferably such that the substance having antitumor activity, the tumor diagnostic reagent or the developer is not deactivated. For monoclonal antibodies or bispecific antibodies, the reaction temperature does not exceed 4 ° C. For small molecule compounds or developers, in situ polymerization can be carried out at higher temperatures, such as room temperature.

The invention also provides the use of the above tumor-targeted nanocapsules for preparing a tumor therapeutic drug or a tumor diagnostic drug.

The metal matrix protease-2 degradable polypeptide cross-linking agent, the metal matrix protease-7 degradable polypeptide cross-linking agent, the metal matrix protease-9 degradable polypeptide cross-linking agent, etc. of the invention are all commercially available, and Buy through open channels. The metal matrix protease-2 degradable polypeptide cross-linking agent as used in Example 1 is produced by Shanghai Qiangyao Biotechnology Co., Ltd. and contains an amino acid series of "VPLGVRTK".

The hydrophobic polymerizable monomer of the present invention has an unsaturated double bond in a molecular structure, and can be bonded to 2-methacryloyloxyethylphosphocholine or a crosslinking agent by an addition reaction.

The tumor-targeted nanocapsules provided by the invention have a shell-core structure. Taking a cross-linking agent as a polypeptide degradable by MMPs and a tumor-targeting nano-microcapsule carrying an anti-tumor monoclonal antibody, as shown in FIG. 1, the "shell" is linked at both ends of the polypeptide degradable by the MMPs. a biocompatible hydrophilic 2-methacryloyloxyethylphosphocholine and the hydrophobic polymerizable monomer, or linked by 2-methacryloyloxyethylphosphocholine and said The polymerized segments of the hydrophobic units containing double bonds or the block copolymers of the two, thereby forming a shell structure similar to a "mesh". The "nucleus" encapsulated in the shell is an anti-tumor monoclonal antibody. The tumor-targeted nanocapsules are intravenously injected into the blood circulation, firstly enriched in the tumor tissue based on the EPR effect of the tumor tissue, and the MMPs degradable polypeptide present in the nanocapsule shell is highly expressed by the matrix metal in the tumor microenvironment. The degradation of proteases (MMPs) causes the nanocapsules to disintegrate, and the encapsulated monoclonal antibodies are rapidly released, acting on tumor cells and inhibiting their proliferation.

The tumor-targeted nanocapsules of the present invention have small and uniform particle size (within 50 nm), and have the function of long-circulating blood in addition to targeting tumors, site-delivering and releasing the entrapped drugs and reagents. Taking the nanocapsules prepared in Example 1 as an example, the half-life time in the blood reached 48 h. Since the 2-methacryloyloxyethylphosphocholine is biocompatible and can pass through a "delivery barrier" in the blood brain barrier or the like, the nanocapsules provided by the present invention are particularly useful for the treatment or diagnosis of brain tumors. Advantageous means and methods are provided.

DRAWINGS

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

1 is a three-dimensional schematic diagram of the in situ polymerization process of the matrix metalloproteinase (MMPs)-sensitive tumor-targeted nanocapsules of the present invention and its degradation process in tumor tissues;

Indicates 2-methacryloyloxyethylphosphocholine,

Represents a hydrophobic polymerizable monomer,

a polypeptide that degrades MMPs (with double bonds at both ends),

Representing anti-tumor monoclonal antibodies,

Represents matrix metalloproteinases (MMPs).

2 is a transmission electron micrograph of the nanocapsules prepared in Example 1.

Figure 3 is a graph showing the particle size distribution of the nanocapsules prepared in Example 1.

Figure 4 is a graph showing the Zeta charge distribution of the nanocapsules prepared in Example 1.

5 is a diagram showing the detection of brain targeting function of the in situ model of U87 glioma nude mice prepared by the nanocapsules prepared in Example 1, wherein 1 is a control group, 2 is a pure antibody treatment group, and 3 is a nanocapsule treatment group.

Figure 6 is a graph showing the evaluation of the effect of the nanocapsules prepared in Example 1 on the in situ model of U87 glioma nude mice.

Fig. 7 is a graph showing the evaluation of the therapeutic effect of the nanocapsules prepared in Example 1 on the subcutaneous model of MGC803 gastric cancer nude mice.

Figure 8 shows a transmission electron micrograph of the nanocapsules prepared in Example 2.

Figure 9 shows a transmission electron micrograph of the nanocapsules prepared in Example 3.

The best way to implement the invention

The invention is further illustrated by the following examples, without however limiting the scope of the invention. Unless otherwise stated, the reagents or starting materials referred to in the following examples are all available through publicly available commercial sources. among them:

2-methacryloyloxyethylphosphocholine: Sigma

N-(3-aminopropyl)methacrylic acid hydrochloride: Sigma

Matrix metalloproteinase-2 degradable peptide crosslinker: Shanghai Qiang Yao Biotechnology Co., Ltd.

Nimotuzumab: Baitai Biopharmaceutical Co., Ltd., batch number 0120120207

Cetuximab: German Merck Company, Imported Drug Registration No.: S20130004

Example 1 Preparation and Characterization of Nimotuzumab Nanocapsules Containing MMP-2 Degradable Crosslinker

Take 1 mg of nimotuzumab solution (200 μL), add N-(3-aminopropyl)methacrylate, molar of nimotuzumab and N-(3-aminopropyl)methacrylate The ratio is 1:300, then add 2-methacryloyloxyethylphosphocholine, the molar ratio of nimotuzumab to 2-methacryloyloxyethylphosphocholine is 1:4000; then press The matrix metalloproteinase-2 degradable polypeptide crosslinker was added at a molar ratio of nimotuzumab to crosslinker of 1:500, allowed to stand for 10 min, and enriched around the mAb by electrostatic and hydrogen bonding. The reaction monomer and the enzyme degrade the polypeptide crosslinking agent; then, ammonium persulfate and tetramethylethylenediamine are added, and the molar ratio of nimotuzumab to ammonium persulfate and tetramethylethylenediamine is 1:500:1000. The reaction was carried out at 4 ° C for 2 h to prepare a nitruzumab nanocapsule encapsulated by poly-2-methacryloyloxyethylphosphocholine.

The transmission electron micrograph of the nimotuzumab nanocapsules coated with poly-2-methacryloyloxyethylphosphocholine was observed. As shown in Fig. 2, the surface of the nanocapsules was smooth and uniform in particle size; further use The particle size analyzer (BI-90Plus, Brookhaven Instruments, USA) tested the particle size distribution and surface charge of the obtained product. The surface of the nanocapsules was smooth and uniform in particle size, and the particle size (nm) was 30±5. (shown in Figure 3); surface charge (mV) is 2.3 (as shown in Figure 4).

Test Example 1 Evaluation of the effect of nanocapsules prepared in Example 1 on the in situ model of U87 glioma nude mice

On the day before virus infection, U87 cells (ATCC, USA, HTB-14) were plated in 24-well plates at a density of 1×105-1×106, and cultured at 5% CO2 at 37°C. The medium was DMEM medium. (GBICO, USA, 11965-092), serum was selected from imported calf serum (HyClone, SH30071.03). After 24h, add 10-500μl of viral solution encoding luciferase (Shanghai Jima Pharmaceutical Technology Co., Ltd.), and add 10-50μl of polybrene to increase the infection efficiency; after 12h, replace it with DMEM medium; after 48h The screening drug 10-100 μl of Puromycin was added, and after 2 weeks of press screening, positive clones were obtained, and the culture was expanded to prepare an in situ model for transplantation into the brain of nude mice.

After the anesthesia of the nude mice was stabilized, a surgical incision was made at the top of the head and punched on the skull. Placed on a stereotactic instrument (Stoelting, lab standard ^TM ); the two cells were digested and resuspended in an appropriate amount of medium. In the middle, the syringe was slowly injected into the rat brain, and then the suture was sutured to the skin. After tumor inoculation for 10 days, the tumor formation rate was observed by in vivo imaging. The animals were randomly divided into groups of 10, and the naprozumab and the nanocapsules prepared in Example 1 were injected into the tail vein at a dose of 5 mg per kg of body weight. , a total of 10 injections. The intracranial tumor size of the animals was monitored every 10 days using a living imager (Xenogen, Waltham, MA, USA, 200), and the survival time of the animals was counted. The statistical results were: control vs pure antibody treatment group, p= 0.1745; control vs nanocapsule treatment group, p=0.0385; pure antibody treatment group vs nanocapsule treatment group, p=0.0061.

As shown in Figures 5 and 6, the growth rate of brain tumors in the nanocapsule treatment group prepared in Example 1 was significantly slower than that in the pure antibody-treated group, and 5 times after the start of treatment. The results of quantitative detection of tumor size at time observation points (10, 20, 30, 40, 50 days) further confirmed the above results.

Test Example 2 Evaluation of the therapeutic effect of the nanocapsules prepared in Example 1 on the subcutaneous model of MGC803 gastric cancer in nude mice

MGC803 gastric cancer cells stably expressing luciferase were inoculated subcutaneously into 4 week old nude mice at a dose of 5×105 cells per injection point using a 100 μl microinjector to establish a tumor source. When the subcutaneous tumor reached a long diameter of 3 cm, the tumor mass was removed, evenly chopped, and implanted into the experimental group (n=6) under the skin of the nude mice, and the feeding was continued. When the long diameter reached about 5 mm, the experimental groups began treatment. The treatment method is tail vein injection, the therapeutic dose is 5 mg per kilogram of body weight, and the number of treatments is once. At the same time, the therapeutic effect was observed. The luciferase activity of the tumor cells was collected by a living imager every two days for 30 days, and then the nude mice of each treatment group were sacrificed and the tumor pieces were peeled off. As shown in FIG. 7, the tumor growth rate of the nanocapsule treatment group prepared in Example 1 was significantly reduced as compared with the pure nimotuzumab treatment group.

Example 2 Encapsulation-specific double antibody containing a pH-sensitive crosslinker

Preparation and characterization of nanocapsules

Take a solution containing 1 mg of double-antibody (200 μL) and add acrylamide monomer.

The molar ratio to acrylamide is 1:300, followed by the addition of 2-methacryloyloxyethylphosphocholine, the molar ratio of the double antibody to 2-methacryloyloxyethylphosphocholine is 1:4000; The ethylene glycol dimethacrylate crosslinker was added at a molar ratio of the double antibody to the crosslinker of 1:500, and allowed to stand for 10 min, and the reaction monomer was enriched around the double antibody by hydrogen bonding. pH-sensitive cross-linking agent; then adding ammonium persulfate and tetramethylethylenediamine, the molar ratio of the double antibody to ammonium persulfate and tetramethylethylenediamine is 1:500:1000, and reacting at 4 ° C for 2 h, Nano-microcapsules containing a specific sensitive double-antibody containing a pH-sensitive crosslinker were prepared.

The nanocapsules of the above-prepared specific double-antibody were observed by transmission electron microscopy. As shown in Fig. 8, the surface of the nanocapsules was smooth and uniform in particle size.

Example 3 Preparation and Characterization of Cetuximab-Containing Nanocapsules Containing Redox-Sensitive Crosslinking Agent

Take 1 mg of cetuximab solution (200 μL), add acrylamide monomer, the molar ratio of cetuximab to acrylamide is 1:300, then add 2-methacryloyloxyethylphosphocholine The molar ratio of cetuximab to 2-methacryloyloxyethylphosphocholine is 1:4000; then the molar ratio of cetuximab to crosslinker is 1:500. Allyl disulfide cross-linking agent, standing for 10 min, using hydrogen bonding, enriching the reaction monomer and redox-sensitive cross-linking agent around cetuximab; then adding ammonium persulfate and tetramethylethylene Amine, cetuximab with ammonium persulfate, tetramethylethylenediamine in a molar ratio of 1:500:1000, reacted at 4 ° C for 2 h, prepared containing redox sensitive cross-linking agent contained cetuximab Resistance to nanocapsules.

The nanocapsules coated with cetuximab prepared above were observed by transmission electron microscopy. As shown in Fig. 9, the surface of the nanocapsules was smooth and uniform in particle size.

Example 4 Encapsulation-specific double antibody containing MMP-2 and MMP-9 crosslinkers

Preparation and characterization of nanocapsules

The molar ratio to acrylamide is 1:300, followed by the addition of 2-methacryloyloxyethylphosphocholine, the molar ratio of the double antibody to 2-methacryloyloxyethylphosphocholine is 1:4000; The ethylene glycol dimethacrylate crosslinker was added at a molar ratio of the double antibody to the crosslinker of 1:500, and allowed to stand for 10 min, and the reaction monomer was enriched around the double antibody by hydrogen bonding. MMP-2 and MMP-9 crosslinker; then add ammonium persulfate and tetramethylethylenediamine, the molar ratio of the double antibody to ammonium persulfate and tetramethylethylenediamine is 1:500:1000, at 4 ° C Under the conditions of 2 h, nano-microcapsules containing specific double antibodies containing MMP-2 and MMP-9 crosslinkers were prepared.

The nanocapsules of the above-prepared specific double-antibody were observed by transmission electron microscopy, and the surface of the nanocapsules was smooth and uniform in particle size (transmission electron micrograph, slightly omitted).

Example 5 Preparation and Characterization of Cetuximab-Containing Nanocapsules Containing MMP-9 Crosslinker

Take 1 mg of cetuximab solution (200 μL), add acrylamide monomer, the molar ratio of cetuximab to acrylamide is 1:300, then add 2-methacryloyloxyethylphosphocholine The molar ratio of cetuximab to 2-methacryloyloxyethylphosphocholine is 1:4000; then the molar ratio of cetuximab to crosslinker is 1:500. Allyl disulfide crosslinker, allowed to stand for 10 min, enriched with reactive monomer and MMP-9 sensitive crosslinker around cetuximab by hydrogen bonding; then added ammonium persulfate and tetramethyl b The diamine, the molar ratio of cetuximab to ammonium persulfate and tetramethylethylenediamine is 1:500:1000, and reacted at 4 ° C for 2 h to prepare a mixture containing MMP-9 cross-linking agent. Nanocapsules of monoclonal antibodies.

The nanocapsules coated with cetuximab prepared above were observed by transmission electron microscopy. The surface of the nanocapsules was smooth and uniform in particle size (transmission electron micrograph, slightly omitted).

Comparative example 1

Take a solution containing 1 mg of double-antibody (200 μL), add acrylamide monomer, the molar ratio of double antibody to acrylamide is 1:300, then add 2-methacryloyloxyethylphosphocholine, double antibody and 2- The molar ratio of methacryloyloxyethylphosphocholine is 1:400; then the ethylene glycol dimethacrylate crosslinker is added in a ratio of the molar ratio of the double antibody to the crosslinker of 1:500. After 10 min; then adding ammonium persulfate and tetramethylethylenediamine, the molar ratio of the double antibody to ammonium persulfate and tetramethylethylenediamine is 1:500:1000, and reacting at 4 ° C for 2 h, the result is in transmission The nanocapsules were not observed under electron microscope, indicating that the nanocapsules carrying the double-antibody could not be prepared.

Comparative example 2

Take 1 mg of cetuximab solution (200 μL), add acrylamide monomer, the molar ratio of cetuximab to acrylamide is 1:30, then add 2-methacryloyloxyethylphosphocholine The molar ratio of cetuximab to 2-methacryloyloxyethylphosphocholine is 1:4000; then the ratio of cetuximab to crosslinker is 1:50. The allyl disulfide cross-linking agent was allowed to stand for 10 min; then ammonium persulfate and tetramethylethylenediamine were added, and the molar ratio of cetuximab to ammonium persulfate and tetramethylethylenediamine was 1:500: 1000, reacted at 4 ° C for 2 h, the results were not observed under transmission electron microscopy nanocapsules, indicating that the nanocapsules encapsulating cetuximab could not be prepared.

Claims

A tumor-targeted nanocapsule comprising a tumor microenvironment sensitive polymer and a substance having antitumor activity encapsulated by the polymer, a tumor diagnostic reagent or a developer; wherein the tumor microenvironment sensitive polymerization The invention comprises a tumor microenvironment sensitive crosslinker and 2-methacryloyloxyethylphosphocholine, the tumor microenvironment sensitive crosslinker comprising at least a polypeptide crosslinker capable of being degraded by an enzyme overexpressed in the tumor microenvironment. .
The nanocapsule according to claim 1, wherein the polypeptide crosslinking agent capable of being degraded by an enzyme overexpressed in the tumor microenvironment is selected from the group consisting of a matrix metalloproteinase degradable polypeptide crosslinking agent and hyaluronidase One or more of the degradable polypeptide crosslinkers;

Preferably, the polypeptide crosslinking agent capable of being degraded by an enzyme overexpressed in the tumor microenvironment is selected from one or more of matrix metalloproteinase degradable polypeptide crosslinking agents.
The nanocapsule according to claim 2, wherein the matrix metalloproteinase degradable polypeptide crosslinking agent is selected from the group consisting of a matrix metalloproteinase-2 degradable polypeptide crosslinking agent and a matrix metalloproteinase-7 degradable One or more of a polypeptide crosslinking agent and a matrix metalloproteinase-9 degradable polypeptide crosslinking agent;

Preferably, the matrix metalloproteinase degradable polypeptide crosslinking agent is selected from one or both of a matrix metalloproteinase-2 degradable polypeptide crosslinking agent and a matrix metalloproteinase-9 degradable polypeptide crosslinking agent.
The nanocapsule according to any one of claims 1 to 3, wherein the tumor microenvironment-sensitive crosslinking agent is further selected from one of a pH-sensitive crosslinking agent and a redox-sensitive crosslinking agent. Multiple

Preferably, the pH-sensitive crosslinking agent is selected from the group consisting of an ester crosslinking agent and/or a Schiff base crosslinking agent, more preferably ethylene glycol dimethacrylate;

Preferably, the oxidation-reduction sensitive crosslinking agent is selected from the group consisting of disulfide-containing crosslinking agents, more preferably diallyl disulfide.
The nanocapsule according to any one of claims 1 to 4, wherein the tumor microenvironment sensitive polymer further comprises a hydrophobic polymerizable monomer;

Preferably, the molar ratio of the tumor microenvironment sensitive crosslinking agent, 2-methacryloyloxyethylphosphocholine and the hydrophobic polymerizable monomer is:

The tumor microenvironment sensitive crosslinker: 2-methacryloyloxyethylphosphocholine: the hydrophobic polymerizable monomer = 1:10:1 to 1:100:1.
The nanocapsule according to claim 5, wherein the hydrophobic polymerizable monomer is selected from the group consisting of acrylic compounds, preferably acrylic acid and salts thereof, esters, amide derivatives, or alkyl-substituted acrylic acids. And its salts, esters, amide derivatives;

More preferably, the hydrophobic polymerizable monomer is selected from the group consisting of acrylic acid, methacrylic acid, acrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, ethylene glycol dimethacrylate, methylene bis acrylamide, One or any ratio of hydroxypropyl acrylate, N-methylol acrylamide, N-2-hydroxypropyl-methacrylamide, and N-(3-aminopropyl) methacrylate hydrochloride Kind.
The nanocapsule according to any one of claims 1 to 6, wherein the substance having antitumor activity is selected from the group consisting of an antitumor monoclonal antibody, an antitumor monoclonal antibody-small molecule compound conjugate, An anti-tumor bispecific antibody or an anti-tumor small molecule compound.
The method for producing a nanocapsule according to any one of claims 1 to 7, wherein the step of in-situ polymerization comprises the steps of: firstly, 2-methacryloyloxyethylphosphocholine and the hydrophobic polymerizable The monomer is added to the solution of the antitumor activity substance, the tumor diagnostic reagent or the developer, and then the tumor microenvironment sensitive crosslinker is added, and finally the initiator is added, and the reaction is carried out at 0 to 30 ° C for 0.1 to 24 hours.
The preparation method according to claim 8, wherein the molar ratio of the substance having antitumor activity, the tumor diagnostic reagent or the developer to the tumor microenvironment sensitive crosslinking agent is 1:100 to 1:10000 ;

Preferably, the molar ratio of the substance having antitumor activity, the tumor diagnostic reagent or the developer to 2-methacryloyloxyethylphosphocholine is 1:500 to 1:10000;

Preferably, the molar ratio of the substance having antitumor activity, the tumor diagnostic reagent or the developer to the hydrophobic polymerizable monomer is 1:100 to 1:10000;

Preferably, the initiator consists of a persulfate and one selected from the group consisting of tetramethylethylenediamine, sodium sulfite and sodium hydrogen sulfite, and the molar ratio of the two is 1:100 to 100:1;

Still preferably, the persulphate is selected from the group consisting of ammonium persulfate or potassium persulfate.
The nanocapsule according to any one of claims 1 to 7, or the nanocapsule prepared by the preparation method according to claim 8 or 9, for use in preparing a tumor therapeutic drug or a tumor diagnostic drug.