Diarylboronic acid-catechin drug-loaded aggregate with high stability and antioxidant activity and application of diarylboronic acid-catechin drug-loaded aggregate

Abstract

The invention discloses a diarylboronic acid-catechin drug-loaded aggregate with high stability and antioxidant activity and application, diarylboronic acid molecules exist in a positive charge participated interaction aggregate form, and boric acid groups are exposed on the outer surface of the aggregate. Catechin reacts with boric acid groups on the surface of the aggregate to generate dynamic covalent bond boric acid ester, the form of the aggregate is not changed, and finally the diarylboronic acid-catechin drug-loaded aggregate with high stability and antioxidant activity is obtained. The natural antioxidant catechin in the diarylboronic acid-catechin drug-loaded aggregate can be used as a health care and therapeutic drug or an auxiliary therapeutic drug to be conveyed in vivo, remove oxygen free radicals harmful to health and release antioxidant activity at tumor sites with overexpressed H2O2 or sialoglycoprotein.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier design, research and development and application technology, specifically relating to a diaromatic boric acid-catechin drug-loaded aggregate with high stability and antioxidant activity and its application. Background Technology

[0002] Catechins are a class of natural antioxidant molecules, abundant in plants such as tea. Tea is a major source of catechins for many people, offering certain health benefits. However, the ionization of phenolic hydroxyl groups in a weakly alkaline environment reduces the stability of catechins, causing them to lose their antioxidant properties. Increasing research indicates that various oxygen free radicals produced by oxygen metabolism are closely related to the pathogenesis of many chronic diseases, such as neurodegenerative diseases, aging, and cancer. Therefore, intravenous injection of catechins to remove various oxygen free radicals and other reactive oxygen species is of significant value for the health care and drug treatment or adjuvant drug therapy of chronic patients. However, intravenous injection carries the risk of catechin inactivation and triggering the body's clearance mechanisms. Constructing a biocompatible drug delivery system is a practically feasible application. Encapsulation-targeted release is currently the delivery mechanism for many drugs, but such carrier preparation techniques limit the antioxidant function of scavenging free radicals during blood circulation. Therefore, chemically binding catechins to the carrier surface becomes a necessary choice, and the selection of modified chemical bonds and the development of the carrier are key to achieving this goal. This invention considers several basic requirements that must be met in the development of such a carrier: (1) the modified material must be biocompatible and biodegradable; (2) it must have strong chemical bonding ability; (3) the modified catechin must have sufficient stability in an environment close to physiological pH 7.4, i.e., it must be able to effectively inhibit the auto-oxidation of catechin; (4) it must retain the antioxidant function of catechin in scavenging free radicals; and (5) it must be able to rapidly release catechin at the site of malignant tumors (overexpression of H2O2 or overexpression of sialic acid, etc.). Aromatic boric acid can form dynamic covalent bonds with ortho-dihydroxy molecules and has become a specific receptor for polyhydroxy sugar molecules. Although there have been some studies on binding with catechin, there are currently no reports on carriers that can simultaneously meet the above requirements. Summary of the Invention

[0003] The purpose of this invention is to provide a highly stable and antioxidant drug-loaded aggregate of diarylboronic acid-catechin and its application. In this diarylboronic acid-catechin drug-loaded aggregate, the natural antioxidant catechin is delivered in vivo as a health care and therapeutic drug or adjuvant therapy drug, scavenges harmful healthy oxygen free radicals, and releases antioxidant activity at tumor sites that overexpress H2O2 or sialic acid glycoprotein.

[0004] To achieve the above objectives, this invention employs the following technical solution: a highly stable and antioxidantly active diarylboronic acid-catechin drug-loaded aggregate, wherein the diarylboronic acid molecules exist in the form of aggregates with positive charges participating in weak interactions, and the boronic acid groups are exposed on the outer surface of the aggregates. Catechins react with the boronic acid groups on the surface of the aggregates to form dynamically covalently bonded boronic esters, maintaining the aggregate morphology unchanged. This ultimately yields a highly stable and antioxidantly active diarylboronic acid-catechin drug-loaded aggregate. The structural formula of the diarylboronic acid is:

[0005]

[0006] Furthermore, the diarylboronic acid exists in the form of weakly interacting aggregates with positive charge in the concentration range in which it is used, wherein the concentration range in which it is used is higher than the critical aggregation concentration of the corresponding diarylboronic acid.

[0007] Furthermore, all of the diaromatic boronic acids have symmetrical aromatic boronic acid groups, wherein the aromatic group of BTEAB is m-benzyl and the spacer group is ethyl diammonium; the aromatic group of PyBBA is m-pyridinium and the spacer group is butadiyl; and the aromatic group of PyPBA is m-pyridinium and the spacer group is 1,4-phenylenebis(methylene).

[0008] Furthermore, each of the diaromatic boric acids contains two symmetrical quaternary ammonium cations, whose electron-withdrawing effect gives the boric acid group of BTEAB an acid dissociation constant pK close to the physiological pH of 7.4. a 7.2–7.8, while PyBBA and PyPBA have pK a These pH values ​​are much higher than the physiological pH of 4.24 and 4.01, respectively, which allows them to have sufficiently large binding constants with catechin esterification at physiological pH.

[0009] Furthermore, the main force driving the formation of BTEAB aggregates in the diaromatic boric acid is the cation-π interaction, the main force driving the formation of PyBBA aggregates is the anion-involved pyridine ring π-π stacking interaction, and the main force driving the formation of PyPBA aggregates is the same π-π stacking interaction as PyBBA aggregates, as well as the π-π interaction between the coexisting spacer benzene rings. The common feature of these interactions is that the cations in the diaromatic boric acid are embedded in the aggregates, thereby effectively avoiding the influence of positive charge on the diaromatic boric acid-catechin drug-loaded aggregates during in vivo delivery.

[0010] Furthermore, the diaromatic boric acid-catechin drug-loaded aggregate exhibits anti-oxidative stability in an aqueous environment with a pH of 6.5–8.5 and retains the anti-free radical oxidation activity of catechins. This enables it to remove harmful oxygen free radicals during human blood circulation at physiological pH, and can be used to prepare drug-loaded and delivered systems with anti-free radical oxidation activity for health care and drug therapy.

[0011] Furthermore, under normal low-concentration H2O2 conditions in vivo, the catechins bound in the diaromatic boric acid-catechin drug-load aggregates are not oxidized. When overexpressed H2O2 is present, such as at the site of malignant tumor cells, the BTEAB-catechin drug-load aggregates can directly remove the overexpressed H2O2. When overexpressed sialic acid-terminated glycoproteins are present, such as at the site of malignant tumor cells, both PyBBA-catechin and PyPBA-catechin drug-load aggregates can respond to the overexpressed sialic acid-terminated glycoproteins and release catechins to remove H2O2.

[0012] The application of the diaromatic boric acid-catechin drug-loaded aggregate described in this invention as a targeted anticancer drug or a targeted adjuvant anticancer drug.

[0013] This invention offers the following advantages and benefits: Compared to drug encapsulation delivery systems, the catechin system bound to the aggregate surface in this invention can scavenge harmful oxygen free radicals during in vivo delivery, thus better leveraging the health benefits of catechins; compared to monomeric catechins, the high proportion of binding to the aggregate surface enhances their antioxidant efficiency; it simultaneously satisfies the requirements of catechins' stability against auto-oxidation and the retention of high antioxidant capacity and efficiency; it can responsively release antioxidant functions at malignant tumor sites with H2O2 and sialic acid telomere glycoprotein overexpression; the formation of self-assembled aggregates and in-situ dynamic covalent bonding offer advantages such as low formulation cost and the ability to be readily prepared. This invention holds potential application prospects for catechins in health care and drug therapy or adjuvant drug therapy. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the structure and antioxidant properties of three diaromatic boric acids and epicatechin and their drug-loaded aggregates.

[0015] Figure 2 (a) Light scattering intensity versus concentration curves of PyBBA and PyPBA; (b) Light scattering intensity versus C EC / C DiarylBA Curve, where C DiarylBA =3.00mM, incident light wavelength is 384nm, initial scattered light intensity is zero, and solution pH is 7.4.

[0016] Figure 3 The absorbance of equimolar concentration DiarylBA and EC aggregate solutions as a function of time at a wavelength of 434 nm is shown. The concentrations of DiarylBA and EC were both 0.15 mM. The maximum incubation time in the dark was 24 h, and the incubation temperature was 25 °C.

[0017] Figure 4 For ABTS ·+ Scavenging activity (a r % as a function of EC concentration. ABTS ·+ The initial concentration was 0.10 mM, and the pH values ​​of the solutions were (a) pH 6.5, (b) pH 7.4 and (c) pH 8.5.

[0018] Figure 5 The absorbance difference ΔAbs between EC and DiarylBA-EC aggregates 434nm The curves showing the change with H2O2 concentration. The pH values ​​of the solutions were (a) pH 6.5, (b) pH 7.4 and (c) pH 8.5, respectively. The concentrations of EC and each DiarylBA were 0.15 mM, and the solutions were incubated at 25°C in the dark for 24 h. Detailed Implementation

[0019] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0020] Example 1

[0021] Using epicatechin (EC) as a model molecule, and employing positively charged diarylboronic acids (BTEAB, PyBBA, and PyPBA, abbreviated as DiarylBA) as key groups of the carrier molecule, a compound with 10... 3 The DiarylBA-EC aggregate drug delivery system, characterized by its order-of-magnitude binding constant, ability to highly inhibit catechin apoptosis in pH < 8.5 environments, strong free radical antioxidant activity, and ability to scavenge reactive oxygen species (ROS) at malignant tumor sites, is illustrated in the following diagram, which shows the overall structure and antioxidant properties of three diaromatic boric acids and epicatechin, along with their drug-loaded aggregates. Figure 1 As shown. This drug delivery carrier has antioxidant functions that target malignant tumors in vivo. The BTEAB-EC system can directly scavenge overexpressed H2O2, while PyBBA-EC and PyPBA-EC can release the antioxidant activity of ECs to exert the drug's effect.

[0022] The formation of self-assembled aggregates of BTEAB in this invention has been reported in the literature, with a critical aggregation concentration of 0.30 mM. The aggregation behavior of PyBBA and PyPBA was characterized by the variation of light scattering intensity with concentration, and the results are shown in... Figure 2 In (a), the critical aggregation concentration of PyBBA was found to be 0.30 mM, while that of PyPBA was less than 0.3 mM. The aggregate size of PyPBA abruptly changed at concentrations greater than 8.0 mM. The aggregate sizes of BTEAB, PyBBA, and PyPBA remained unchanged at smaller EC / DiarylBA concentration ratios. Figure 2 The aggregation morphology changes at the critical concentration ratio shown in (b). This invention limits the preparation of DiarylBA-EC drug-loaded aggregates to a concentration ratio range lower than this.

[0023] This invention measured the binding constants of three types of DiarylBA and EC at pH 6.5, pH 7.4, and pH 8.5 using isothermal titration calorimetry. For the BTEAB-EC system, the binding constant decreased from a minimum of 10 to an absolute value for each pH unit increase. 3 Increasing the binding constant by an order of magnitude, for both the PyBBA-EC and PyPBA-EC systems, the binding constant remains at 10. 3 With the same order of magnitude, all three systems possess sufficiently large integration capabilities.

[0024] This invention uses isothermal titration calorimetry to measure the stoichiometric coefficients of three types of DiarylBA and EC binding. The stoichiometric coefficients of all systems were greater than 1 at each pH, ​​indicating that the boric acid groups of the DiarylBA aggregates are distributed on the outer surface of the aggregates, resulting in a higher surface concentration of EC and thus improving the antioxidant efficiency.

[0025] This invention assesses the stability of EC by examining the UV-vis absorption spectra of EC and its auto-oxidation product δ-type DhC2 and its absorbance at 434 nm. The structural formulas of EC(a) and δ-type DhC2(b) are shown below, and the absorbance versus time curves are shown. Figure 3 At pH 6.5, both EC and the drug-loaded system covalently linked to DiarylBA aggregates showed a very small increase in absorbance, indicating a very weak auto-oxidation ability. However, at pH 7.4 and pH 8.5, BTEAB could almost completely inhibit the strong auto-oxidation of EC, while PyBBA and PyPBA could also inhibit about 70% of the auto-oxidation of EC, which could meet the need to improve the stability of EC.

[0026]

[0027] This invention is achieved through ABTS ·+The free radical scavenging assay characterized the anti-free radical oxidation activity of the DiarylBA-EC drug-loaded aggregates using ABTS. ·+ Free radical scavenging percentage (a) r The curve illustrating the change in % with EC concentration is shown below. Figure 4 The results in the graph clearly show that removing 50% of ABTS... ·+ EC concentration (IC50) corresponding to free radicals 50 The presence of DiarylBA is irrelevant; that is, the DiarylBA-EC drug-loaded aggregate retains the same antioxidant activity as pure EC, and can completely scavenge 0.10 mM ABTS at an EC concentration of 0.01 mM. ·+ Free radicals, which can scavenge more than ten times the concentration of ABTS. ·+ Free radicals.

[0028] This invention characterizes the oxidative activity of DiarylBA-EC drug-loaded aggregates against non-radical reactive oxygen species (ROS) through H2O2 scavenging experiments. The results are represented by the increase in absorbance of the EC oxidation products at 434 nm, as shown in the figure. Figure 5 The increase in absorbance represents the relative content of H2O2 oxidation products after deducting EC auto-oxidation. Because auto-oxidation is faster at higher pH, the actual EC concentration participating in the H2O2 oxidation reaction decreases. Figure 5 The results still showed that increasing pH enhanced the oxidative capacity of H2O2 on BTEAB-EC aggregates or increased its H2O2 scavenging ability, while the PyBBA-EC and PyPBA-EC systems exhibited weaker H2O2 scavenging capabilities. These results indicate that BTEAB-EC aggregates can release antioxidant activity in response to overexpressed H2O2, while PyBBA-EC and PyPBA-EC require the prior release of ECs bound to the aggregates before they can exert their antioxidant function, such as at malignant tumor sites where sialic acid telomeres are overexpressed.

[0029] in conclusion:

[0030] 1. The DiarylBA synthesized in this invention was synthesized in our laboratory. The molecular structure and purity of the product were characterized by NMR and mass spectrometry. The acid dissociation constant of the borate group, the self-assembly of the molecule, and the structural stability in the presence of dissolved oxygen, oxygen free radicals, or H2O2 were studied. The results showed that the properties and structural characteristics of BTEAB, PyBBA, and PyPBA are suitable for the modification of epicatechin.

[0031] 2. The benzylammonium boric acid of BTEAB and the pyridinium boric acid of PyBBA or PyPBA are the key groups of the three DiarylBAs. They are the key groups that have a large binding constant in their esterification reaction with catechins. Simply changing the structure of the spacer group has no significant impact on the application involved in this invention.

[0032] 3. All three types of DiarylBA used in this invention have aggregation mechanisms involving cationic charges, including the cation-π interaction of BTEAB and the π-π stacking interaction of PyBBA or PyPBA, which embed positive charges within the aggregates, thereby avoiding the influence of cations on the drug carrier during in vivo delivery.

[0033] 4. The drug formulation of the diaromatic boric acid-catechin drug-loaded aggregate of the present invention, which has high stability and antioxidant activity, can be prepared at room temperature using solid mixtures and can quickly reach an equilibrium aggregation state without the need for special conditions and instruments.

[0034] In summary, in this invention, epicatechin (EC) is used as the representative catechin molecule, and diarylboronic acid is an N-type catechin synthesized in our laboratory. 1 N 2 -bis(3-boronic acid benzyl)-N 1 N 1 N 2 N 2The three diarylboronic acid molecules are tetramethylethane-1,2-diammonium bromide (BTEAB), 1,1′-(1,4-butadiyl)-bis(3-boronyl-1-onyl)bromopyridine (PyBBA), and 1,1′-(1,4-phenylenebis(methylene))-bis(3-boronyl-1-onyl)bromopyridine (PyPBA). All three exist as aggregates with positively charged interactions (cation-π or pyridinium ring π-π stacking) within the concentration ranges used, with the boric acid groups exposed on the outer surface of the aggregates. Epicatechin reacts with the boric acid groups on the surface of the aggregates to form dynamically covalently bonded borate esters, maintaining the aggregate morphology and yielding catechin drug-loaded aggregates. Experiments show that this covalently bound epicatechin aggregate exhibits excellent anti-oxidative stability in aqueous solutions with pH 6.5–8.5 while retaining the free radical oxidizing activity of pure epicatechin. It can scavenge harmful oxygen free radicals during blood circulation at physiological pH, thus possessing health-promoting and therapeutic functions. Under normal low-concentration H2O2 conditions in vivo, the bound epicatechin is not oxidized. However, when overexpressed H2O2 is present, such as in malignant tumor cells, BTEAB-EC can directly scavenge reactive oxygen species (ROS) such as H2O2. In contrast, PyBBA-EC and PyPBA-EC require the release of EC in response to co-expressed sialic acid telomerase glycoproteins to scavenge ROS. This targeted antioxidant function of EC has significant application value for the treatment or adjuvant therapy of malignant tumors.

[0035] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A diaromatic boric acid-catechin drug-loaded aggregate with high stability and antioxidant activity, characterized in that: Diarylboronic acid molecules exist as aggregates with positive charges participating in weak intermolecular interactions. Boric acid groups are exposed on the outer surface of the aggregates. Catechins react with the boric acid groups on the surface of the aggregates to form dynamically covalently bonded boronic acid esters. The aggregate morphology remains unchanged, ultimately yielding a highly stable and antioxidant drug-loaded diarylboronic acid-catechin aggregate. The structural formula of diarylboronic acid is as follows:

2. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: The diaromatic boric acids are used in the concentration range in which they exist as aggregates with positive charges participating in weak intermolecular interactions, and the concentration range used is higher than the critical aggregation concentration of the corresponding diaromatic boric acid.

3. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: All of the diaromatic boric acids have symmetrical aromatic boric acid groups, wherein the aromatic group of BTEAB is m-benzyl and the spacer group is ethyl diammonium; the aromatic group of PyBBA is m-pyridinium and the spacer group is butadiyl; and the aromatic group of PyPBA is m-pyridinium and the spacer group is 1,4-phenylenebis(methylene).

4. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: Each of the diaromatic boric acids contains two symmetrical quaternary ammonium cations, giving the boric acid group of BTEAB an acid dissociation constant pK close to the physiological pH of 7.

4. a Sections 7.2-7.8: PyBBA vs. PyPBA a These values ​​are 4.24 and 4.01, respectively, which are much smaller than physiological pH. This allows them to have sufficiently large binding constants for the esterification of catechins at physiological pH.

5. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: The main force driving the formation of BTEAB aggregates in the diaromatic boric acid is the cation-π interaction, the main force driving the formation of PyBBA aggregates is the anion-involved pyridine ring π-π stacking interaction, and the main force driving the formation of PyPBA aggregates is the same π-π stacking interaction as PyBBA aggregates, as well as the π-π interaction between the coexisting spacer benzene rings. The common feature of these interactions is that the cations in the diaromatic boric acid are embedded in the aggregates, which can effectively avoid the influence of positive charge on the diaromatic boric acid-catechin drug-loaded aggregates during in vivo delivery.

6. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: The diaromatic boric acid-catechin drug-loaded aggregate exhibits anti-oxidation stability in an aqueous environment with a pH of 6.5–8.5 and retains the anti-free radical oxidation activity of catechins. This enables it to remove harmful oxygen free radicals during human blood circulation at physiological pH, and can be used to prepare drug-loaded and delivered systems with anti-free radical oxidation activity for health care and drug therapy.

7. The diaromatic boric acid-catechin drug-loaded aggregate according to claim 1, characterized in that: Under normal low-concentration H2O2 conditions in vivo, the catechins bound in the diaromatic boric acid-catechin drug-load aggregates are not oxidized. When overexpressed H2O2 is present, such as at the site of malignant tumor cells, the BTEAB-catechin drug-load aggregates can directly remove the overexpressed H2O2. When overexpressed sialic acid-terminated glycoproteins are present, such as at the site of malignant tumor cells, both PyBBA-catechin and PyPBA-catechin drug-load aggregates can respond to the overexpressed sialic acid-terminated glycoproteins and release catechins to remove H2O2.

8. The use of the diaromatic boric acid-catechin drug-loaded aggregates according to claims 1 to 8 as targeted anticancer drugs or targeted adjuvant anticancer drugs.

Images