Water-soluble protein-encapsulating boron nano-carrier and boron nano-carrier complex

Abstract

To provide a boron nano-carrier that enables treatment of a primary tumor site by boron neutron capture therapy and additionally enables treatment of metastatic sites through immunoactivation.SOLUTION: A boron nano-carrier for encapsulating a water-soluble protein, which is capable of encapsulating a water-soluble protein and to which boron neutron capture therapy (BNCT) is applicable, wherein a water-soluble polyphenol having a catechol structure is modified with a boronic acid compound at the catechol structure, the boron nano-carrier being represented by the following formula (I). In formula (I), R1 represents a structure derived from a boronic acid compound, and R2 and R3 represent structures derived from the water-soluble polyphenol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to boron nanocarriers and boron nanocarrier complexes for encapsulating water-soluble proteins. [Background technology]

[0002] With the advancement of molecular biology, it has long been possible to design and synthesize any protein in a test tube, and the development of protein drugs using this technology is accelerating. In particular, antibody drugs have been shown to be effective in treating diseases such as cancer, and the competition to develop them is fierce. To improve the efficacy of these protein drugs, it is important to develop a delivery system that improves their delivery to the target site.

[0003] Conventionally, antibody drugs have shown longer blood retention compared to general protein drugs, making them excellent for delivery to tumor tissue, but it has been difficult to ensure that their function is expressed only at the target site. Some antibodies have been designed to induce conformational changes in the antibody by binding to ATP in the tumor environment, thereby showing affinity for the target substance, but the technology is still in development.

[0004] Recently, boron compounds have attracted attention as carriers for transporting proteins into cells. For example, Non-Patent Document 1 discloses a boron delivery system using a complex composed of a fructose-modified poly(ethylene glycol)-poly(L-lysine) block copolymer and p-phenylboronic acid. This complex is used in boron neutron capture therapy (BNCT). Non-Patent Document 2 describes a boron compound that mainly uses a dendrimer as a protein delivery system.

[0005] Boron neutron capture therapy is a treatment in which boron compounds are incorporated into cancer cells and then neutron beams are used to destroy the cancer cells locally. In this treatment, it is important to selectively accumulate the boron compounds within the tumor. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] T. Nomoto et al., J. Control. Release, 332 (2021) 184-193 [Non-patent document 2] Q. Ren et al., Adv. Healthcare Mater. 2023, 12, 2202049 Summary of the Invention [Problem to be solved by the invention]

[0007] The boron compound described in Non-Patent Document 1 forms micelle-type nanoparticles in which polymers are highly accumulated. This makes it difficult to stably retain proteins inside the nanoparticles and control their release. Furthermore, although the boron compound described in Non-Patent Document 2 has high structural stability, it is difficult to complex proteins in a stable state and in a state in which they are coated with polymers to impart resistance to degrading enzymes.

[0008] Furthermore, while the boron agents currently used in clinical BNCT demonstrate excellent therapeutic efficacy against primary tumors, their effectiveness in treating metastatic cancer is insufficient. In recent years, it has been discovered that in BNCT, immune activation, triggered by damage to cancer cells due to neutron irradiation, plays an important role in treating metastatic cancer. However, the immunosuppressive environment of cancer cells prevents this immune activation, making it difficult to treat metastatic cancer.

[0009] The technology disclosed here has been developed in light of these issues, and its purpose is to provide a boron nanocarrier that can treat not only primary lesions by boron neutron capture therapy but also metastatic lesions by immunostimulation. It also aims to improve the delivery of water-soluble proteins to target tissues, reduce side effects as a drug, and release water-soluble proteins in response to external stimuli. [Means for solving the problem]

[0010] The inventors of the present application have developed a boron nanocarrier that can stably complex water-soluble proteins, such as antigens and antibodies, inside it and release them in response to external stimuli.

[0011] The present disclosure provides a boron nanocarrier for encapsulating water-soluble proteins, which is capable of encapsulating water-soluble proteins and is applicable to boron neutron capture therapy (BNCT), and which comprises a water-soluble polyphenol having a catechol structure, the catechol structure of which is modified with a boronic acid compound and is represented by the following formula (I):

[0012] [ka]

[0013] In formula (I), R 1 indicates a structure derived from a boronic acid compound, and R 2 and R 3 indicates a structure derived from a water-soluble polyphenol.

[0014] As a result of intensive research by the present inventors, the findings have shown that the boron nanocarrier for encapsulating water-soluble proteins of the present disclosure can easily and stably complex proteins through weak supramolecular chemical interactions, efficiently deliver the proteins to the target site, and release the complexed protein in response to an external stimulus. Furthermore, the boron nanocarrier for encapsulating water-soluble proteins of the present disclosure can easily encapsulate proteins and form complexes by simply mixing with the proteins, and the complexes can exhibit behavior sufficient to protect the proteins from degrading enzymes.

[0015] Furthermore, according to the present disclosure, it is possible to provide a boron nanocarrier complex in which a water-soluble protein acting as an immune checkpoint inhibitor is encapsulated in the boron nanocarrier. By encapsulating an immune checkpoint inhibitor, combined therapy with boron neutron capture therapy (BNCT), a type of radiation therapy, and an immune checkpoint inhibitor not only improves the response rate of primary lesions to boron neutron capture therapy, but also enables the treatment of metastatic lesions through immune activation. The boron nanocarrier complex of the present disclosure preferably has a core-shell structure in which the boron nanocarrier serves as the shell and the water-soluble protein serves as the core.

[0016] The water-soluble polyphenol is preferably at least one selected from the group consisting of tannic acid, gallic acid, catechins, and derivatives thereof.

[0017] The boronic acid compound is preferably at least one selected from the group consisting of phenylboronic acid, bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, and borofarane, and more preferably phenylboronic acid. [Effects of the Invention]

[0018] As described above, the present disclosure makes it possible to provide boron nanocarriers that can not only treat primary lesions through boron neutron capture therapy, but also treat metastatic lesions through immune activation. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is an IR spectrum of a boron nanocarrier according to this embodiment. [Figure 2] 1 is a microscopic image of a boron nanocarrier complex according to this embodiment. [Figure 3] 1 is a microscopic image of a boron nanocarrier complex according to this embodiment. [Figure 4] 1 shows the results of single particle analysis showing the release capacity of the boron nanocarrier according to this embodiment. [Figure 5] 1 shows the results of single particle analysis showing the release capacity of the boron nanocarrier according to this embodiment. [Figure 6] 1 shows the results of single particle analysis showing the release capacity of the boron nanocarrier according to this embodiment. [Figure 7] 1 is a graph showing the antitumor activity of a boron nanocarrier according to this embodiment. [Figure 8] 1 is an image showing the IgG tumor delivery of a boron nanocarrier according to this embodiment. [Figure 9] 1 is a graph of a pharmacokinetic evaluation showing the accumulation of boron. [Figure 10] 1 is a graph of primary tumor growth. [Figure 11] 1 is a graph of tumor growth in metastatic lesions. DETAILED DESCRIPTION OF THE INVENTION

[0020] The boron nanocarriers of the present disclosure are described in detail below.

[0021] The boron nanocarriers disclosed herein are designed to stably encapsulate water-soluble proteins such as antigens and antibodies and deliver the water-soluble proteins to target tissues. They can be used as carriers for immune checkpoint inhibitors and can be used in boron neutron capture therapy (BNCT).

[0022] The boron nanocarrier of the present disclosure is represented by the following formula (I), in which a water-soluble polyphenol having a catechol structure is modified with a boronic acid compound at the catechol structure.

[0023] [ka]

[0024] In formula (I), R 1 is a structure derived from a boronic acid compound, and R 2 and R 3 indicates a polysaccharide-derived structure.

[0025] The water-soluble polyphenols used in the boron nanocarriers of the present disclosure are not limited as long as they have a catechol structure, but may be at least one selected from tannic acid, gallic acid, catechins, and their derivatives. Examples of tannic acid derivatives include compounds in which at least a portion of the hydroxyl groups of tannic acid are substituted with alkyl ethers or alkyl esters. Examples of gallic acid derivatives include methyl gallate, ethyl gallate, butyl gallate, pentyl gallate, and propyl gallate. Examples of catechins and their derivatives include epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, theasinensins, theaflavins, proanthocyanidins, and thearubigins. All of these water-soluble polyphenols can be bound to boronic acid via the catechol structure. Furthermore, these water-soluble polyphenols can encapsulate water-soluble proteins in a stable state. The combination of interactable polyphenols and proteins is not limited, but may be, for example, those disclosed in Polyphenol-Mediated Assembly of Proteins for Engineering Functional Materials (Y. Han et al., Angew. Chem. Int. Ed. 2020, 59, 15618-15625).

[0026] The boronic acid compounds used in the boron nanocarriers of the present disclosure are not limited as long as they can be introduced into the catechol structure of water-soluble polyphenols and are usable for boron neutron capture therapy. Examples of boronic acid compounds that can be used include phenylboronic acid (BPA), known for its selective uptake by melanoma cells and various cancer cells, and anticancer drugs such as bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, and borofarane. The electronic interaction between the boron atom and water-soluble proteins enables boron nanocarriers to transport water-soluble proteins in a stable state. Accumulating structures derived from such boronic acid compounds at primary tumors not only enables effective boron neutron capture therapy, but also enables the treatment of metastatic lesions through immune activation by the action of the water-soluble proteins loaded as complexes.

[0027] The boron nanocarrier of the present disclosure can easily form a boron nanocarrier complex encapsulating the water-soluble protein simply by mixing it with a water-soluble protein. The water-soluble protein that can be encapsulated in the boron nanocarrier can be, for example, one that acts as an immune checkpoint inhibitor and can be any known protein in the art, including antigens and antibodies. The boron nanocarrier of the present disclosure has a core-shell structure with the boron nanocarrier as the shell and the water-soluble protein as the core.

[0028] The water-soluble protein that forms the boron nanocarrier complex may be any protein that can interact with the boron nanocarrier of the present disclosure and form a core-shell structure. Examples of water-soluble proteins include horseradish peroxidase (HRP), which is used as a labeling enzyme for secondary antibodies, and antibodies such as IgG, PD-L1 antibody, PD-1 antibody, and CTLA-4 antibody. [Example]

[0029] The present invention will be described in more detail below with reference to examples. These examples are merely illustrative examples for suitably explaining the present invention, and are not intended to limit the present invention.

[0030] [Synthesis of boron nanocarriers] As an example of a boron nanocarrier, L-BPA-modified polyphenol was synthesized by modifying tannic acid with L-BPA, as shown in the following formula (II).

[0031] [ka]

[0032] Specifically, tannic acid (100 mg, 0.059 mmol), boronophenylalanine (L-BPA) (35.8 mg, 0.294 mmol), and 5 mL of DMF were placed in a recovery flask, followed by the addition of DBU (313 mg). The mixture was stirred at 45°C for 24 hours and allowed to react. The mixture was then reprecipitated with acetone (stirred for 1 hour), centrifuged twice (10,000 rpm, 10°C, 15 min), and the supernatant was removed. The mixture was then dried in a desiccator to obtain the target product. The synthesis of the target product was confirmed by quantification of boron concentration using ICP emission spectroscopy and measurement of B-O bonds using infrared absorption spectroscopy (IR). As shown in Figure 1, IR measurements detected vibrations associated with B-O bonds. Furthermore, ICP emission spectroscopy confirmed the incorporation of boron atoms.

[0033] Next, we attempted to conjugate the synthesized L-BPA-modified polyphenol (TA-BPA) as a boron nanocarrier with proteins of different properties, such as molecular weight and isoelectric point, using horseradish peroxidase (HRP) and antibody (IgG).

[0034] [Combination of boron nanocarriers and proteins] (HRP conjugation) HRP dissolved in PBS was mixed with TA-BPA. Conjugation was performed using 400 μg of HRP and varying amounts of TA-BPA (4, 40, 80, 100, and 400 μg, respectively, (TA-BPA solution volume: 0, 1, 10, 20, 50, and 100 μL). Twenty-four hours after mixing, dynamic light scattering measurements, zeta potential measurements, and HRP activity were evaluated. HRP activity was measured using TMB as a substrate and expressed as a specific activity relative to the free enzyme activity. The results of these experiments are shown in Table 1. The TA-BPA / HRP complex (TA-BPA / HRP) had a diameter of approximately 100 nm, demonstrating that the enzyme retained its activity in the complexed state.

[0035] [Table 1]

[0036] (IgG conjugation) IgG dissolved in PBS was mixed with TA-BPA. Conjugation was performed using 200 μg of IgG and varying amounts of TA-BPA (1, 20, and 100 μL of TA-BPA solution). Dynamic light scattering, zeta potential, and HRP activity were evaluated 24 hours after mixing. The results are shown in Table 2. Figure 2 shows an electron microscopic image of the complex. Figure 3 shows the electron microscopic results of TA-BPA conjugated to gold nanoparticles and TA-BPA conjugated to IgG. The TA-BPA / IgG complex (TA-BPA / IgG) was stable with a particle size of less than 100 nm and low dispersibility. It also possessed a core-shell structure, as shown in Figure 2. Furthermore, in the TA-BPA / IgG complex with gold nanoparticles, gold nanoparticles were scattered within the aggregates, as shown in Figure 3, confirming that the protein was indeed encased in tannic acid for conjugation.

[0037] [Table 2]

[0038] [Release test based on ATP response of TA-BPA / IgG] To evaluate the antibody's ability to selectively release ATP in the tumor environment, single-particle analysis was performed using imaging flow cytometry. Here, fluorescently labeled IgG was conjugated and evaluated. Gating was performed using non-fluorescent IgG as a control. The results are shown in Figures 4 to 6. Figure 4 shows the single-particle analysis results after the addition of ATP when non-fluorescent IgG was used, Figure 5 shows the results when fluorescent IgG was used, and Figure 6 shows the results when fluorescent IgG was used. Fluorescent IgG conjugates in Figure 4 were considered positive when fluorescence intensity was higher than the right end of the histogram for the non-fluorescent IgG conjugate. The fluorescent IgG conjugate in Figure 5 showed a tendency for over 88% of the particles to be derived from IgG. Furthermore, when 1 mM ATP was added to the fluorescent IgG conjugate, the histogram shifted to the left, as shown in Figure 6, confirming the release of IgG. These results suggest that the boron nanocarriers disclosed herein can stably conjugate water-soluble proteins, transport them to target tissues (tumors), and release the water-soluble proteins in response to ATP, which is abundant in tumors.

[0039] [BNCT activity of TA-BPA / IgG (in vitro)] To compare the antitumor activity of L-BPA-modified polyphenols (TA-BPA / IgG, TA-PB / IgG) with that of the clinical drug L-BPA / fructose complex, the following procedure was used to evaluate the activity. TA-PB / IgG is a complex of IgG and a boron nanocarrier in which tannic acid is modified with phenylboronic acid (PB). (Day 1) 1×10 5 EG7-OVA cells were seeded into a 12-well plate for cell culture. (Day 2) The prepared TA-BPA / IgG, TA-PB / IgG, and L-BPA / fructose complexes were added to 1 mL of medium so that the final boron concentrations were 0, 0.05, 0.1, 0.5, and 1 ppm, respectively, and the cells were allowed to incorporate the boron for 24 hours. On day 3, the cells were collected in cryotubes and neutron-irradiated (1 MW, 15 min). 100 μL of the irradiated cell suspension was seeded onto a 96-well plate and incubated for 24 and 48 hours. On day 4, WST-8 reagent was added to the medium and incubated for 2 hours to allow color development. The absorbance (450 nm) of this solution was measured to calculate the cell viability 24 hours after neutron irradiation. (Day 5) The same procedure as on Day 4 was repeated to calculate the cell viability 48 hours after neutron irradiation. The cell viability was calculated by normalizing the absorbance of EG7-OVA cells without the addition of boron drugs, which means there is no dose contribution from nuclear reactions due to boron capture.

[0040] The results 24 hours after neutron irradiation are shown in Figure 7. The clinical drugs BPA, TA-PB, and TA-BPA each had a calculated IC50 cell growth inhibition concentration. 50 The values ​​were 0.177, 0.441, and 0.072 ppm. TA-BPA, the boron nanocarrier of the present disclosure, has therapeutic properties that surpass those of clinical drugs such as BPA, and it is believed that efficient treatment was achieved by granulating it. TA-PB was also confirmed to have sufficient therapeutic properties.

[0041] [Pharmacokinetic evaluation of TA-BPA / IgG] The accumulation of antibodies and boron in tumors was evaluated using the following method, and the delivery ability was clarified, while the time of neutron irradiation was determined. Tumor-bearing mice were generated by transplanting EG7-OVA into C57BL6 mice.

[0042] A 100μL sample containing 50ppm boron was administered to each mouse. To examine the distribution of fluorescence within the body, the mouse was imaged using an in vivo imaging system at 0, 1, 3, 6, and 24 hours after administration. After 24 hours, the mice were dissected, and the collected organs (liver, lung, heart, tumor, spleen, kidney, and skin) were analyzed for accumulation. The collected organs were then subjected to an oxidation treatment and analyzed using an ICP analyzer. To examine blood retention, blood was collected at each time point, centrifuged, and the serum was then subjected to an oxidation treatment and analyzed using an ICP analyzer.

[0043] The results of tumor accumulation are shown in Figure 8. IgG accumulation in the tumor was confirmed over time. This is thought to be due to tumor accumulation achieved through the general EPR effect. As for boron accumulation, as shown in Figure 9, tumor accumulation reached its peak after 24 hours, so it was decided to irradiate with neutrons at this time.

[0044] [BNCT activity of TA-BPA / ICI (in vivo)] The therapeutic activity of TA-BPA / ICI (TA-BPA complex) was evaluated for metastatic cancer using the following method. The PDL-1 antibody was used as the ICI. Tumor-bearing mice were generated by implanting EG7-OVA into C57BL6 mice.

[0045] 100 μL of a sample prepared to a boron concentration of 100 ppm was administered to each mouse, followed by neutron irradiation (5 MW, 30 min) 24 hours later. The tumor volume and weight change of the mice after irradiation were measured to evaluate the antitumor effect. For comparison, mice administered with an L-BPA / fructose complex were also neutron irradiated in the same way, and the antitumor effect was evaluated.

[0046] The therapeutic effect of primary lesions is shown in Figure 10, and the therapeutic effect of metastatic lesions is shown in Figure 11. The tumor volume in the control group not irradiated with neutrons continued to increase over time. The same was true for the group irradiated with neutrons alone. However, tumor growth was significantly suppressed in the group irradiated with neutrons and administered in combination with clinical drugs L-BPA and ICI (BPA+ICI hot in Figures 10 and 11). The therapeutic performance of the newly prepared TA-BPA / ICI (TA-BPA / ICI hot in Figures 10 and 11) surpassed that of the former. On the other hand, TA-BPA / ICI also showed the highest therapeutic performance in metastatic lesions, demonstrating the usefulness of the L-BPA-modified polyphenols of the present disclosure.

Claims

1. A boron nanocarrier for encapsulating a water-soluble protein, which can encapsulate a water-soluble protein and can be used for boron neutron capture therapy (BNCT), A boron nanocarrier for encapsulating water-soluble proteins, represented by the following formula (I), in which a water-soluble polyphenol having a catechol structure is modified with a boronic acid compound at the catechol structure. 【Chemistry 1】 [In formula (I), R １ represents a structure derived from a boronic acid compound, and R ２ and R ３ represents a structure derived from a water-soluble polyphenol.]

2. The boron nanocarrier for encapsulating water-soluble proteins according to claim 1, wherein the water-soluble polyphenol is at least one selected from the group consisting of tannic acid, gallic acid, catechins, and derivatives thereof.

3. The boron nanocarrier for encapsulating a water-soluble protein according to claim 2, wherein the boronic acid compound is at least one selected from the group consisting of phenylboronic acid, bortezomib, ixazomib, tavaborole, crisaborole, vaborbactam, and borofarane.

4. The water-soluble protein is an immune checkpoint inhibitor, and the boron nanocarrier complex comprises the boron nanocarrier described in any one of claims 1 to 3 encapsulating the immune checkpoint inhibitor.

5. The water-soluble protein is an immune checkpoint inhibitor, and the boron nanocarrier complex has a core-shell structure in which the boron nanocarrier described in any one of claims 1 to 3 serves as a shell and the immune checkpoint inhibitor serves as a core.

Images