Schiff base-mediated peptide self-assembly and preparation method and application thereof

Abstract

The application belongs to the field of bioactive peptides, and relates to a Schiff base-mediated peptide self-assembly and a preparation method and application thereof, in particular to a supermolecular self-assembly of an egg white-derived active peptide with an amino acid sequence of Gln-Ile-Gly-Leu-Phe (QIGLF) and application thereof. The self-assembly is assembled by a group of mixed construction modules (mainly 2QIGLF-3GA, molecular formula: C 71 H 104 N 12 O 16 ) generated by Schiff base reaction of QIGLF and glutaraldehyde (GA). The peptide self-assembly provided by the application can effectively combine with hydrophobic material curcumin, has excellent encapsulation capacity (>22%), can significantly improve the antioxidant capacity of curcumin and the transport level of curcumin on a Caco-2 cell monolayer membrane, and has the characteristics of safety and non-toxic side effects, can be used as a high-loading rate delivery carrier of hydrophobic food and medicine ingredients, and has important significance in the field of peptide-based supermolecular biomaterials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioactive peptides, and particularly relates to an egg white-derived active peptide supermolecular self-assembly with an amino acid sequence of Gln-Ile-Gly-Leu-Phe (QIGLF) as well as a preparation method and applications thereof. BACKGROUND

[0002] Inspired by the harmony and beauty of natural protein and peptide structures and functions, researchers pursue the supreme wisdom of nature, simulate the manufacture of complex and highly ordered superstructures, and thus generate new biological materials with multiple functions. Peptides released after the degradation of food proteins are not only basic materials for the construction and repair of organisms, but also undertake the tasks of human growth and repair and renewal of damaged cells. They also provide an important treasure trove for obtaining self-assembling peptide building blocks to organize high-order structures. Although some food-derived peptides with clear structural characteristics can self-assemble into diverse fine structures based on non-covalent interactions, self-assembly with the aid of covalent chemistry is an important way to help food-derived peptides lacking secondary structures (such as β-sheets), intrinsic disorder or amphiphilic properties to develop superstructures with subtle architecture, dynamic interactions and multifunctionality to perform biological functions. Currently, a series of dynamic reactions with reversible covalent bonding characteristics have been successfully used to design functional materials, bottom-up assembly processes and molecular machines.

[0003] Schiff base bonds (imine bonds -N=CH-) are dynamic covalent bonds formed between aldehyde (or ketone) groups and amino groups, which can synergistically regulate the structure and function of assemblies with non-covalent interactions. This bond endows the assembly with spontaneous fluorescence properties, which helps to track biological processes in vivo without external fluorescent dyes. More importantly, the regulation of dynamic covalent interactions enables the assembly to have unique adaptability, dynamic characteristics and relatively stable networks in biological systems. This Schiff base-induced dynamic covalent assembly has become a powerful and effective strategy for constructing peptide-based nanocarriers to encapsulate and deliver food and medicinal ingredients. It also provides a new idea for solving the problem of limited bioavailability caused by the confinement of hydrophobic components in extremely low water solubility.

[0004] However, the practical application of supramolecular nanocarriers still faces many challenges, especially in the encapsulation of hydrophobic compounds. One significant problem is the difficulty in effectively improving the encapsulation capacity, which is usually less than 10%. In addition, the efficiency of assembly penetrating the intestinal epithelial cells into the blood circulation is extremely low, which significantly limits the amount of loaded active ingredients reaching the target site and their subsequent ability to exert physiological regulation functions. Therefore, it is urgent to construct high-quality nanocarriers with excellent encapsulation capacity and effective intestinal absorption barrier penetration. It is of great significance and prospect to design a hydrophobic delivery carrier with high drug encapsulation capacity, high biocompatibility, and effective penetration of the intestinal epithelial absorption barrier based on the dynamic covalent bond of Schiff base to endow food-derived bioactive peptides with supramolecular structure.

[0005] The present application aims to provide a Schiff base dynamic covalent bond mediated peptide-based supramolecular self-assembly that can be used to efficiently encapsulate hydrophobic curcumin and improve its bioavailability. SUMMARY

[0006] The present application discloses a peptide-based supramolecular self-assembly (QIGLF-GA SA) assembled by a group of mixed construction modules mediated by Schiff base dynamic covalent bond based on the amino acid sequence of Gln-Ile-Gly-Leu-Phe. The self-assembly can be used for efficient encapsulation, antioxidant activity and bioavailability gain of hydrophobic component curcumin (Cur).

[0007] The technical solution of the present application is:

[0008] Step one, peptide self-assembly construction and identification

[0009] 1.0 ~ 4.0 mg / mL of QIGLF was dissolved in ultrapure water, and 1.0 ~ 3.0% (v / v) glutaraldehyde (GA, 50%) solution was added under continuous magnetic stirring. The pH of the system was adjusted to 5.0 ~ 7.0, and the reaction was carried out in a water bath at 30 ~ 45 ℃ for 18 ~ 36 h. After the reaction was completed, the system was dialyzed in a 3.5 kDa dialysis bag for 24 ~ 36 h, and the dialysis fluid was water, which was replaced twice during the dialysis process to remove the unreacted GA. Thereafter, the reaction system was freeze-dried to obtain the peptide self-assembly reported in the present application. Thereafter, the self-assembly was measured by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) with water as the solvent and a-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. The test molecular weight range was 1000-10000 Da, and the positive ion detection was carried out.

[0010] The reagent for adjusting the pH of the solution is preferably NaOH, more preferably 1.0 M NaOH; the water is preferably deionized water; the reaction temperature is preferably 37 °C, and the reaction time is preferably 24 h. The mixed building blocks are identified by MALDI-TOF-MS method, including 2QIGLF-3GA (molecular formula: C 71 H 104 N 12 O 16 ), 2QIGLF-2GA, 2QIGLF-4GA, 2QIGLF-5GA, and the content ratio of the four is about 11:3:6:2.

[0011] Step two: determination of the encapsulation capacity of self-assembly for hydrophobic curcumin under pH-driven by centrifugation method

[0012] The aqueous solution of self-assembly (0.25 ~ 1.0 mg / mL) is mixed with an equal volume of curcumin solution (0.1 mg / mL, previously dissolved in water at pH = 12.0), then the pH of the mixture is adjusted to 12.0 with NaOH solution, and continuously stirred. Then, the pH of the system is quickly adjusted to 7.0 with HC1 solution to obtain the curcumin-loaded supramolecular co-assembly. After the sample is centrifuged to remove the unencapsulated curcumin, the concentration of curcumin in the supernatant is quantified by the method of establishing a standard curve (R² > 0.999) at 425 nm by ultraviolet spectrophotometer, and the encapsulation capacity (EC) of self-assembly for curcumin is calculated according to formula (1).

[0013] (1)

[0014] The NaOH for adjusting the pH is preferably 6 M, and the HC1 is preferably 1 M; the centrifugation conditions of the sample are 8000 ~ 12000 rpm / min, 10 min, preferably 10000 rpm / min, 10 min.

[0015] Step three: determination of the gain of antioxidant activity by DPPH, ABTS

[0016] (1) DPPH free radical scavenging activity: first, 0.2 mM DPPH solution was prepared with 75% ethanol, and appropriately diluted to make its ultraviolet absorption at 517 nm 0.8 ± 0.02. After the sample to be tested was prepared, it was reacted in the dark for 1.5 ~ 2 h, and after the reaction was completed, the sample was centrifuged to take the supernatant, and then the sample (curcumin concentration 10 ug / ml) was mixed with DPPH solution in a volume ratio of 1:2 and incubated in the dark for 0.5 h. DPPH was mixed with pure water as a control, and after the reaction was completed, the absorbance value of the sample at 517 nm was measured by ultraviolet spectrophotometry. The DPPH free radical scavenging activity calculation formula is as follows:

[0017] (2)

[0018] In the formula, A C and A S are the absorbance values of the control group and the experimental group, respectively.

[0019] The centrifugation conditions are 4 ℃, 9000 ~ 12000 r / min, 5 ~ 15 min, preferably 10000 r / min, 10 min.

[0020] (2) ABTS free radical scavenging activity: 7 mM ABTS reagent and 2.45 mM potassium persulfate were mixed in a volume ratio of 1:1, incubated at room temperature for 12 ~ 16 h in the dark, and diluted with PBS buffer before use to make its absorbance value at 734 nm 0.70 ± 0.02, obtaining ABTS working solution. In a 96-well plate, 20 μl of sample (curcumin concentration 10 μg / mL) was added to each well, then 180 μl of ABTS working solution was added, shaken for 10 s, and then incubated at room temperature for 5 ~ 10 min. The absorbance value of the reaction system at 734 nm was measured by an enzyme marker. The ABTS free radical scavenging activity calculation formula is:

[0021] (3)

[0022] In the formula, A C , A S and A B are the absorbance values of the control group, the experimental group and the blank group, respectively.

[0023] Step four: MTS method for determining cell toxicity and hemolysis experiment for evaluating biocompatibility

[0024] (1) MTS method for determining cytotoxicity: Caco-2 or L02 cells are seeded in 96-well plates (90 μL), incubated for 12 ~ 16 h, and then 10 μL of sample is added to each well for further incubation for 24 ~ 36 h. 20 μL of MTS is added and treated for 2 ~ 4 h. The absorbance value at 490 nm is recorded using a microplate reader. The blank group uses 90 μL of DMEM instead of the cell suspension, and the control group uses 10 μL of DMEM instead of the sample. Cell viability is calculated according to formula (4):

[0025] (4)

[0026] wherein A S , A B and A C represent the absorbance values of the sample group, the blank group and the control group, respectively.

[0027] (2) Hemolysis experiment: Fresh sterile defibrinated rabbit blood is washed with physiological saline several times to obtain a 4% red blood cell suspension. An appropriate amount of sample is mixed with an equal volume of red blood cell suspension and shaken at 37 °C for 3 ~ 6 h. Ultra-pure water and physiological saline are used as positive and negative control groups, respectively. After incubation, the sample is centrifuged. The absorbance value of the supernatant at 540 nm is detected using a microplate reader, and the hemolysis rate is calculated using formula (5):

[0028] (5)

[0029] wherein A S , A B and A C represent the absorbance values of the sample, the negative control group and the positive control group, respectively.

[0030] The sample centrifugation conditions are 2000 ~ 4000 r / min for 10 min, preferably 3000 r / min for 10 min.

[0031] Step five: Caco-2 cell monolayer membrane transport evaluation experiment for bioavailability gain

[0032] Caco-2 cells are seeded in 12-well Transwell plates and continuously cultured for more than 21 days until the transmembrane electrical resistance (TEER) value reaches 400 ~ 600 Ω / cm². Different samples are added to the top of the monolayer membrane at the same curcumin content as the standard, incubated for 2 ~ 3 h, and then sampled from the other side. The curcumin content in the sample is quantified using a microplate reader, and the apparent permeability coefficient (P app ) is calculated according to formula (6).

[0033] (6)

[0034] Wherein, Q is the total amount of permeation sample (μg), A is the membrane area (cm²), C is the initial concentration of sample in the donor zone (μg / mL), and t is the total time of the experiment (s).

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] (1) The present application is based on the self-assembly of food-derived peptides mediated by Schiff base dynamic covalent bonds, and the reported self-assembly has high biocompatibility.

[0037] (2) The present application significantly improves the encapsulation capacity of curcumin (>22%), which is much higher than the current reality level of less than 10% of the loading rate of nano-carriers,

[0038] (3) The present application significantly improves the antioxidant activity of curcumin, and the DPPH and ABTS free radical scavenging activities of the self-assembly encapsulating curcumin are more than 35.4% and 19.4%, respectively, which are about 11.8 times and 2.6 times, respectively, of the free curcumin under the same experimental conditions.

[0039] (4) After the self-assembly encapsulates 100 μg / mL of curcumin (corresponding to a self-assembly concentration of 250 μg / mL), the results of the cytotoxicity experiment are as follows: the survival rate of Caco-2 cells is 83.4% ~ 88.2%, the survival rate of L02 cells is 89.2% ~ 91.5%, and the hemolysis rate is 0.414% ~ 0.307%, which demonstrates the high biocompatibility of the self-assembly.

[0040] (5) The self-assembly can significantly improve the bioavailability of curcumin, and the apparent permeability coefficient P aap of curcumin on the Caco-2 cell monolayer membrane can be improved to 3.996 × 10 -7 ~ 4.223 × 10 -7 cm / s, which is 11.23 ~ 12.68 times of the free curcumin under the same experimental conditions.

[0041] The supramolecular self-assembly reported in the present application has important practical significance and broad application prospects in the encapsulation and delivery of hydrophobic components. BRIEF DESCRIPTION OF DRAWINGS

[0042] The present application is accompanied by Figure 5 a drawing, wherein:

[0043] Figure 1 Chemical composition identification of self-assembly (MALDI-TOF-MS);

[0044] Figure 2 Encapsulation capacity of self-assembly for curcumin;

[0045] Figure 3 Gain of self-assemblies on curcumin DPPH, ABTS radical scavenging activity;

[0046] Figure 4 Evaluation of self-assemblies biocompatibility;

[0047] Figure 5 Gain of self-assemblies on curcumin bioavailability. DETAILED DESCRIPTION

[0048] The application will be further described in the following specific examples.

[0049] Example 1. Peptide self-assemblies construction and identification

[0050] QIGLF was dissolved in ultrapure water at a concentration of 2.0 mg / mL, and 2.5% (v / v) GA solution (50%) was added. The pH of the system was adjusted to 7.0 with 1 M NaOH, and the reaction was carried out in water at 37°C for 24 h. After the reaction was completed, the system was dialyzed in a 3.5 kDa dialysis bag for 36 h, with water as the dialysate, and the dialysate was replaced twice during the dialysis. Thereafter, the reaction system was freeze-dried to obtain the peptide self-assemblies reported in the present application. Thereafter, the self-assemblies were measured by MALDI-TOF-MS with water as the solvent and CHCA as the matrix, with a molecular weight range of 1000-10000 Da and positive ion detection. The results, as shown in Figure 1 Figure 1, demonstrate that the reported self-assemblies are assembled from a group of mixed basic building blocks mainly composed of 2QIGLF-3GA.

[0051] Example 2. Determination of self-assemblies' encapsulation capacity for hydrophobic curcumin

[0052] The prepared self-assemblies aqueous solution (0.25 mg / mL) was mixed with an equal volume of curcumin solution (0.1 mg / mL, previously dissolved in water at pH = 12.0), and then the pH of the mixture was adjusted to 12.0 with 6 M NaOH, and the stirring was continued. Subsequently, the pH of the solution was rapidly reduced to 7.0 with 1 M HC1 to obtain the curcumin-loaded supramolecular co-assemblies. The encapsulation capacity of self-assemblies for curcumin was quantitatively determined by UV-visible spectrophotometer (UV-2250, Shimadzu, Japan) at 425 nm with a standard curve (R² > 0.999) established. Specifically, the freshly prepared sample was centrifuged at 10000 rpm / min for 10 min at 4°C to remove any unreacted or unencapsulated curcumin. Then, 0.5 mL of supernatant was diluted and uniformly mixed with 3.5 mL of 75% ethanol for determination. The encapsulation capacity of self-assemblies for curcumin was calculated according to the following formula.

[0053]

[0054] Figure 2 The results shown indicate that the self-assembly formed at physiological pH has an encapsulation capacity for curcumin of more than 22%, which is about 23 times that of the original peptide chain QIGLF, and the self-assembly reported has extremely excellent encapsulation capacity for curcumin.

[0055] Example 3. Evaluation of antioxidant activity

[0056] The pre-dissolved DPPH solution (0.2 mM, 75% ethanol) was diluted to an ultraviolet absorbance of 0.800 at 517 nm. Subsequently, the sample after standing for 2 h was centrifuged at 4 °C and 10000 r / min for 10 min, the supernatant (Cur 10 μg / mL) was mixed with the DPPH solution at a volume ratio of 1:2, and incubated in the dark for 30 min. DPPH mixed with pure water was used as a control. The DPPH free radical scavenging activity was calculated according to the following formula:

[0057]

[0058] In the formula, A C and A S are the absorbance values of the control group and the experimental group, respectively.

[0059] ABTS free radicals were prepared by mixing a 7.0 mM ABTS aqueous solution and a 2.45 mM potassium persulfate mixture at room temperature in the dark for 12 h. Then, by diluting the mixture with PBS (0.2 M, pH 7.4), an ABTS working solution was prepared until the absorbance at 734 nm reached 0.700. Next, 180 μL of the working solution was added to the wells in a 96-well microplate, followed by the addition of 20 μL of the sample (Cur 10 μg / mL), incubation in the dark for 5 min, and then measurement of the absorbance at 734 nm using an enzyme marker (Spark TECAN, Switzerland). The ABTS free radical scavenging activity was calculated using the following formula:

[0060]

[0061] In the formula, A C , A S and A B are the absorbance values of the control group, the experimental group and the blank group, respectively.

[0062] Figure 3The results showed that the DPPH and ABTS radical scavenging activities of the self-assembly encapsulating curcumin were more than 35.4% and 19.4%, respectively, which were about 11.8 times and 2.6 times of the free curcumin under the same experimental conditions.

[0063] Example 4. Biocompatibility evaluation

[0064] Hemolysis analysis: Fresh sterile defibrinated rabbit blood was washed with physiological saline for several times, and then diluted to obtain a 4% red blood cell suspension. Based on the highest curcumin concentration of 100 μg / mL, the sample was mixed with an equal volume of red blood cell suspension and shaken at 37 °C for 3 h. Deionized water and physiological saline were used as positive and negative control groups, respectively. After incubation, the sample was centrifuged at 3000 r / min for 10 min. The absorbance value of the supernatant at 540 nm was detected using a multi-mode enzyme marker (Spark, TECAN, Switzerland), and the hemolysis rate was calculated using the following formula:

[0065]

[0066] wherein A S , A B and A C represent the absorbance values of the sample, negative control group and positive control group, respectively.

[0067] Cytotoxicity analysis: Caco-2 (human colon cancer cells), L02 (human liver cells) were seeded in a 96-well plate (90 μL) and cultured for 12 h. Then 10 μL of sample was added to each well. After 24 h of incubation, 20 μL of MTS was added for 2 h. The absorbance value at 490 nm was recorded using a multi-mode enzyme marker (Spark, TECAN, Switzerland). The blank group used 90 μL of DMEM instead of the cell suspension, and the control group used 10 μL of DMEM instead of the sample. The cell viability was calculated according to the following formula:

[0068]

[0069] wherein A S , A B and A C represent the absorbance values of the sample group, blank group and control group, respectively.

[0070] Figure 4 The results of cytotoxicity and hemolysis rate fully demonstrated the high biocompatibility of the self-assembly and the co-assembly formed by the self-assembly and curcumin. Even when the content of curcumin in the sample reached 100 μg / mL, there was still no obvious cytotoxicity and hemolysis (cell viability > 85%, hemolysis rate < 1%). This provided a solid safety guarantee for the reported application of self-assembly as a delivery carrier in vivo.

[0071] Example 5. Bioaccessibility Gain Assessment

[0072] Caco-2 cells were seeded in 12-well Transwell plates (pore size 0.4 μm, growth surface area 1.12 cm², Corning Incorporated) and cultured for 21 days to form a monolayer. The integrity of the monolayer was examined by measuring the transepithelial electrical resistance (TEER) value using an EVOM epithelial voltammeter / resistometer (Millicell-ERS, Millipore, USA). Only cells with a TEER value greater than 600 Ω / cm² were used for transport studies. Different samples with the same curcumin content were added to the top of the monolayer and incubated for 2 hours before sampling from the other side. The curcumin content in the samples was quantified using a multimode microplate reader (Spark, TECAN, Switzerland, 425 nm), and the apparent permeability coefficient was calculated according to the following formula.

[0073]

[0074] Where Q is the total amount of sample permeated (μg), A is the membrane area (cm²), C is the initial concentration of the sample in the donor region (μg / mL), and t is the total time of the experiment (s).

[0075] Figure 5 The experimental results showed that free curcumin can cross the Caco-2 cell monolayer membrane app The value is only 3.4 × 10 -8 cm / s, and the mixture of pure peptide chain QIGLF and curcumin is only 5.0 × 10 -8 cm / s, and the comparison showed that the self-assembly encapsulated curcumin could significantly improve the transport level of curcumin on the Caco-2 monolayer cell membrane, P app Reached 4.1× 10 -7 cm / s, which were 12 times and 8.2 times that of free curcumin and the mixture of QIGLF and curcumin, respectively, fully verifying the bioaccessibility gain of curcumin by the self-assembly.

Claims

1. A Schiff base-mediated peptide self-assembly, characterized in that: The basic peptide sequence of the self-assembly is Gln-Ile-Gly-Leu-Phe (QIGLF); the preparation method of the peptide self-assembly comprises the following steps: (a) 1.0–4.0 mg / mL QIGLF was dissolved in ultrapure water, and 1.0%–3.0% (v / v) glutaraldehyde (GA) solution was added under continuous magnetic stirring. (b) adjusting the pH of the mixed solution obtained in step (a) to 5.0-7.0 and reacting in a water bath at 30-45°C for 18-36 hours; (c) dialyzing the system obtained in step (b) through a 3.5 kDa dialysis bag for 24 to 36 hours and then freeze-drying to obtain the peptide self-assembly; The self-assembly is assembled from a set of mixed building blocks mediated by Schiff base reaction.

2. The self-assembly according to claim 1, characterized in that The hybrid building blocks that constitute the self-assembly are 2QIGLF-3GA as the main component, and the molecular formula is: C 71 H 104 N 12 O 16 In addition, 2QIGLF-2GA, 2QIGLF-4GA, and 2QIGLF-5GA participated in the formation of the self-assembly, and the content ratio of the four was 11:3:6:

2.

3. The self-assembly according to claim 1, characterized in that The loading rate of hydrophobic curcumin in the self-assembly was 18% to 26%.

4. The self-assembly according to claim 1, characterized in that After curcumin was encapsulated in the self-assembly, the DPPH and ABTS free radical scavenging activities reached more than 35.4% and 19.4%, respectively, which were 11.8 times and 2.6 times that of free curcumin under the same experimental conditions.

5. The self-assembled body according to claim 1, characterized in that After the self-assembly encapsulated 100 μg / mL of curcumin, the corresponding self-assembly concentration was 250 μg / mL. The results of the cytotoxicity experiment were: Caco-2 cell survival rate was 83.4% ~88.2%, L02 cell survival rate was 89.2% ~ 91.5%; the hemolysis rate was 0.414% ~ 0.307%.

6. The self-assembled body according to claim 1, characterized in that After the self-assembly encapsulated curcumin, the apparent permeability coefficient of curcumin on the Caco-2 cell monolayer was P aap The value increased to 3.996 × 10 -7 ~ 4.223×10 -7 cm / s, which is 11.23 to 12.68 times that of free curcumin under the same experimental conditions.

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