Amino acid-based aggregate as well as preparation method and application thereof

Abstract

The invention discloses an amino acid-based aggregate as well as a preparation method and application thereof. The amino acid modified by at least two hydrophobic groups is added into water, the pH value is adjusted to dissolve the amino acid, and then the pH value is adjusted to a specific value to obtain the amino acid-based aggregate. The obtained amino acid-based aggregate can replace an existing peptide aggregate, the cost of drug delivery is reduced, in addition, a series of amino acid-based aggregates of pH windows are obtained by changing modification groups and modification positions, and the pH response requirements of various application scenes are met.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to an amino acid-based condensate, its preparation method and application, and more specifically to a method for preparing amino acid-based condensates using amino acids modified with hydrophobic groups and the application of the obtained amino acid-based condensates in the preparation of oral drug delivery systems. Background Technology

[0002] Peptides are polymers composed of amino acids. The formation of peptide condensates via liquid-liquid phase separation (LLPS) is a common process in biology. The existing sticker-and-spacers model posits that the liquid-liquid phase separation of peptides arises from a proper balance between attractive and non-attractive forces between their different amino acid residues. Amino acids with opposite charges, aromatic or hydrophobic residues exhibit attractive forces, referred to as "stickers," while amino acids with hydrophilic residues ensure that the condensate retains bound water to maintain a liquid state, referred to as "spacers." Peptide condensates offer advantages such as high drug loading capacity, high biocompatibility, simple formulation, absence of organic solvents, non-endocytic entry into cells, and pH regulation, thus gradually becoming a novel drug delivery carrier.

[0003] Most existing peptide condensates are formed from polypeptides because, compared to short peptides, the longer sequences of polypeptides increase the multivalent nature of interactions and facilitate phase separation. However, polypeptides are typically synthesized in a solid-phase environment, and the longer their sequences, the higher the cost. Therefore, pursuing the formation of condensates using minimal peptides can help reduce the application cost of peptide condensates. Existing short peptide condensate systems include tetrapeptide condensates (FFssFF), tripeptide condensates (WKY), and dipeptide condensates (FF-OMe). However, short peptide condensates still have limitations imposed by solid-phase synthesis. Developing amino acid condensates can overcome this limitation and further reduce application costs. Summary of the Invention

[0004] The purpose of this invention is to provide an amino acid-based condensate and its preparation method. This invention solves the technical problem of the difficulty in preparing condensates from single amino acids by modifying amino acids with hydrophobic groups and controlling the pH window for condensate formation. This invention can effectively reduce the cost of drug delivery via peptide condensates. The prepared amino acid-based condensates can enrich hydrophobic drug molecules, exhibiting high encapsulation efficiency and drug loading, making them a low-cost and promising drug delivery carrier.

[0005] The method for preparing amino acid-based aggregates provided by the present invention includes the following steps: adding hydrophobic group-modified amino acids to water, adjusting the pH value to dissolve them, and then adjusting the pH to a specific value to obtain amino acid-based aggregates.

[0006] The amino acid modified with the hydrophobic group contains at least two hydrophobic groups; The hydrophobic group may be selected from: C1-C6 straight-chain or branched alkyl, -CH2-Ar (Ar represents an aromatic group), C1-C6 straight-chain or branched alkoxy, -C(=O)-OR (R is an aromatic group or C1-C6 straight-chain or branched alkyl); The aromatic group may specifically be a phenyl group; The aromatic group is a substituted or unsubstituted aromatic group, and the substituent in the substituted aromatic group may be at least one of hydroxyl, amino, C1-C6 straight-chain or branched alkyl, and C1-C6 straight-chain or branched alkoxy. Furthermore, each of the two hydrophobic groups is independently selected from at least one of: Bn, Cbz; Boc, tBu, iPr, OMe; The amino acid in question may specifically be glycine.

[0007] Specifically, the hydrophobic group-modified amino acid is at least one of the following compounds:

[0008] in, , , , .

[0009] When a hydrophobic group is modified on the amino group of an amino acid, the pH window for the formation of aggregates is acidic (pH less than 7). When a hydrophobic group is modified at the carboxyl terminus of an amino acid, the pH window for the formation of aggregates is alkaline (pH greater than 7). Modifying both amino and carboxyl groups with hydrophobic groups allows for the formation of aggregates under both acidic and alkaline conditions, meaning the pH window for aggregate formation is 1-14.

[0010] For example, the amino acid modified by the hydrophobic group is Boc-G(-Bn), and the pH window for forming aggregates is 1.0-3.8; The hydrophobic group-modified amino acid is G(-Bn)-tBu, and the pH window for forming aggregates is 7.6-13.0; The hydrophobic group-modified amino acid is Boc-G(-Bn)-Bn, and the pH window for forming aggregates is 1.0-13.0; The hydrophobic group-modified amino acid is Boc-G(-Bn-OH)-Bn, and the pH window for forming aggregates is 1.0-10.4; The hydrophobic group-modified amino acid is Boc-G(-Bn-NH2)-Bn, and the pH window for forming aggregates is 4.7-13.0.

[0011] The amino acid-based condensates obtained by the above method are also within the scope of protection of this invention.

[0012] The application of the amino acid-based condensate in the preparation of oral drug delivery systems is also within the scope of protection of this invention.

[0013] The drug may be a hydrophobic drug, specifically curcumin.

[0014] This invention also provides a curcumin@amino acid-based condensate drug delivery system.

[0015] The amino acid-based condensate may specifically be a Boc-G(-Bn) condensate.

[0016] The application of the above-mentioned curcumin@amino acid-based condensate drug delivery system in the preparation of drugs for treating acute colitis is also within the scope of protection of this invention.

[0017] This invention develops a novel liquid-liquid phase separation system that modifies amino acids with hydrophobic groups to induce them to form aggregates within a suitable pH window. Figure 1 and Figure 2 We designed amino acid-based condensates with a series of pH windows to meet different pH response requirements.

[0018] The method for preparing amino acid-based condensates of the present invention can effectively reduce the cost of drug delivery via peptide condensates. Currently, the commercial synthesis of 100 mg of a 10-amino acid peptide (95% purity) costs approximately 2,000 yuan, while 100 g of Boc-G(-Bn) (98% purity) only costs approximately 100 yuan, a cost difference of approximately 20,000 times. The application of drugs such as curcumin is limited by their poor water solubility and low bioavailability. Boc-G(-Bn) aggregates can enrich hydrophobic drug molecules such as curcumin, with an encapsulation efficiency of up to 99% and a drug loading of up to 66%.

[0019] Boc-G(-Bn) condensates are resistant to strong acids, high salts, and proteases, and can be loaded with hydrophobic drug molecules. Due to their stability in gastric juice and drug release in intestinal juice, Boc-G(-Bn) condensates are suitable for oral drug delivery. Furthermore, by changing the modifying groups and their positions, a series of amino acid-based condensates with varying pH windows were obtained to meet the pH response requirements of different applications. Attached Figure Description

[0020] Figure 1(a) shows a schematic diagram and confocal micrograph illustrating the formation pattern of amino acid-based condensates. (b) shows the fluorescence recovery curve of G(-Bn)-Bn condensates after quenching (pH=10). (c) shows the fluorescence recovery curve of G(-Bn)-Bn condensates after quenching (pH=10). (d) shows the fusion of G(-Bn)-Bn condensates (pH=10).

[0021] Figure 2 The pH window represents the amino acid condensates with different modified groups (OH and NH2 are both para-substituted).

[0022] Figure 3 (a) Schematic diagram of Boc-G(-Bn) condensates used for oral drug delivery. (b) Confocal micrograph of CUR@Boc-G(-Bn). (c) Encapsulation efficiency and drug loading. (d) Distribution in mouse intestine. (e) Establishment of an acute colitis mouse model and drug administration regimen. (f) Changes in mouse body weight. (g) Length of mouse colon. (h) Actual image of mouse colon. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The reagents used in the following examples are from the following sources: Boc-G(-Bn), Macklin, B802305. G(-Bn)-Bn, Macklin, L816467. Cbz-G(-Bn), Macklin, N805031. Cbz-G-Bn, Macklin, B897513. Boc-G(-Bn)-Bn, Bid, BD1406. G(-Bn)-tBu, Bid, BD18804. Boc-G(-Bn)-OMe, Aladdin, B136644. Boc-G(-Bn-NH2)-OMe, Macklin, B891176. Boc-G(-Bn-OH)-OMe, Aladdin, B110968. Cbz-G, Macklin, Z820730. G-Bn, Macklin, G810327. G(-Bn), Aladdin, P110424. Curcumin (CUR), TCI, C0434. Sodium dextran sulfate (DSS), Macklin, D808272. Simulated gastric juice (SGF), Macklin, S769005. Simulated intestinal juice (SIF), Macklin, S769006.

[0026] The following examples use Boc-G(-Bn) as an example to illustrate the preparation of amino acid-based condensates.

[0027] Example 1: Preparation of amino acid-based condensates Taking Boc-G(-Bn) as an example Add Boc-G(-Bn) to water at a dosage of 10 mg / mL, and adjust the pH to >10 with 2 M NaOH to dissolve it. Then adjust the pH to 3 with 2 M HCl, and aggregates will form immediately.

[0028] Similarly, for amino acid derivatives modified with carboxyl terminus, the pH is first adjusted to acidic to dissolve them, and then the pH is adjusted to alkaline (pH=10) to form aggregates.

[0029] For amino acid derivatives modified at both the amino and carboxyl ends, it is impossible to make them soluble in water by adjusting the pH. They need to be dissolved in DMSO (100 mg / mL) first, and then diluted in water (10 mg / mL), where they will immediately form aggregates.

[0030] Characterization of amino acid condensates: The morphology, fusion, and recovery after fluorescence bleaching of the obtained aggregates were observed using laser scanning confocal microscopy (CLSM). The turbidity of the aggregates was detected by microplate reader. The size of the aggregates was measured by dynamic light scattering.

[0031] Figure 1(a) shows a schematic diagram and confocal micrograph illustrating the formation pattern of amino acid-based condensates. (b) shows the fluorescence recovery curve of G(-Bn)-Bn condensates after quenching (pH=10). (c) shows the fluorescence recovery curve of G(-Bn)-Bn condensates after quenching (pH=10). (d) shows the fusion of G(-Bn)-Bn condensates (pH=10).

[0032] Figure 1 Experimental results show that amino acids containing only one hydrophobic group (such as Cbz-G, G-Bn, G(-Bn)) cannot effectively form LLPS systems, while amino acids containing at least two hydrophobic groups (such as Cbz-G(-Bn), G(-Bn)-Bn, Cbz-G-Bn, Cbz-G(-Bn)-Bn) can effectively form LLPS systems, thus obtaining amino acid-based condensates.

[0033] Based on the above procedures, the pH window of amino acid condensates with different modified groups was investigated.

[0034] Figure 2 The pH window represents the amino acid condensates with different modified groups.

[0035] Depend on Figure 2 It is known that when a hydrophobic group is modified on the amino group of an amino acid, the pH window for the formation of aggregates is acidic (pH less than 7); for example, if the amino acid modified by the hydrophobic group is Boc-G (-Bn), the pH window for the formation of aggregates is 1.0-3.8.

[0036] When a hydrophobic group is modified at the carboxyl terminus of an amino acid, the pH window for the formation of aggregates is alkaline (pH greater than 7); the amino acid modified with the hydrophobic group is G(-Bn)-tBu, and the pH window for the formation of aggregates is 7.6-13.0.

[0037] Modifying both amino and carboxyl groups with hydrophobic groups allows for the formation of aggregates under both acidic and alkaline conditions, with a pH window of 1-14 for aggregate formation. For example, if the amino acid modified with the hydrophobic group is Boc-G(-Bn)-Bn, the pH window for aggregate formation is 1.0-13.0. By modifying the side-chain benzene ring with a pH-responsive group, the pH window can be finely adjusted. The amino acid modified with the hydrophobic group is Boc-G(-Bn-OH)-OMe, with a hydroxyl group modified at the para-position of the benzene ring. It becomes charged under strong basic conditions through deprotonation, thus forming aggregates with a pH window of 1.0-10.4. Alternatively, the amino acid modified with the hydrophobic group is Boc-G(-Bn-NH2)-OMe, with an amino group modified at the para-position of the benzene ring. It becomes charged under strong acid conditions through protonation, thus forming aggregates with a pH window of 4.7-13.0.

[0038] Example 2: Preparation of CUR@Boc-G(-Bn) Dissolve curcumin (CUR) in DMSO (100 mg / mL). Dissolve Boc-G(-Bn) in DMSO (200 mg / mL). Mix the CUR and Boc-G(-Bn) solutions at a mass ratio of 1:1. Add the CUR and Boc-G(-Bn) mixture to PBS with pH adjusted to 3 to obtain CUR@Boc-G(-Bn) (10 mg / mL).

[0039] It is visible to the naked eye that after CUR enrichment, it changes from white microdroplets to black microdroplets. Figure 3 (b)). Its encapsulation efficiency is as high as 99%, and its drug loading is as high as 66% ( Figure 3 (c)

[0040] Example 3: Construction and treatment of a mouse model of acute colitis Mice (BALB / c, female, 20 g) were given a 2.5% DSS solution via drinking water from day 0 to day 7, with the solution changed every two days. On day 8, the drinking water was changed to DSS-free water. On days 2, 4, and 6, mice were administered PBS (pH=3), Boc-G(-Bn) (100 mg / kg, pH=3), CUR (100 mg / kg, pH=3), and CUR@Boc-G(-Bn) (100 mg / kg) by gavage. On day 9, the mice were sacrificed and analyzed. The results showed that Boc-G(-Bn) aggregates maintained stability in simulated gastric fluid and were released in simulated intestinal fluid, indicating potential application as an oral drug delivery carrier. Figure 3 (a) Boc-G(-Bn) aggregates are resistant to strong acids, high salts, and proteases, and can enrich hydrophobic drug molecules, such as curcumin (CUR). Boc-G(-Bn) aggregates prolong the retention time of drugs in the intestine. Figure 3 (d)). Boc-G(-Bn) aggregates enhanced the alleviating effect of curcumin on acute colitis, mainly reflected in the fact that the mice in the CUR@Boc-G(-Bn) group did not show a significant decrease in body weight or a significant shortening of colon length. Figure 3 (f)-(h)).

[0041] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing amino acid-based aggregates, comprising the following steps: adding hydrophobic group-modified amino acids to water, adjusting the pH value to dissolve them, and then adjusting the pH value to a specific value to obtain amino acid-based aggregates; in, The amino acid modified with the hydrophobic group contains at least two hydrophobic groups; The hydrophobic group is selected from: C1-C6 straight-chain or branched alkyl, -CH2-Ar (Ar represents aromatic group), C1-C6 straight-chain or branched alkoxy, -C(=O)-OR (R is aromatic group or C1-C6 straight-chain or branched alkyl).

2. The method according to claim 1, characterized in that, The aromatic group is phenyl; The aromatic group is a substituted or unsubstituted aromatic group, and the substituent in the substituted aromatic group is at least one of hydroxyl, amino, C1-C6 straight-chain or branched alkyl, and C1-C6 straight-chain or branched alkoxy.

3. The method according to claim 1, characterized in that, The two hydrophobic groups are each independently selected from at least one of: Bn, Cbz; Boc, tBu, iPr, OMe; The amino acid in question is glycine.

4. The method according to claim 1, characterized in that, The hydrophobic group-modified amino acid is at least one of the following compounds: in, , , , .

5. The method according to claim 1, characterized in that, If a hydrophobic group is modified on the amino group of the amino acid, the pH window for the formation of aggregates will be acidic. When hydrophobic groups are modified at the carboxyl terminus of amino acids, the pH window for the formation of aggregates is alkaline. Modifying both amino and carboxyl groups with hydrophobic groups allows for the formation of aggregates under both acidic and alkaline conditions, meaning the pH window for aggregate formation is 1-14.

6. An amino acid-based condensate prepared by any one of claims 1-5.

7. The application of the amino acid-based condensate of claim 6 in the preparation of an oral drug delivery system; wherein the drug is a hydrophobic drug.

8. A curcumin@amino acid-based condensate drug delivery system, wherein, The amino acid-based condensate is a Boc-G(-Bn) condensate.

9. The use of the curcumin@amino acid-based condensate drug delivery system according to claim 8 in the preparation of a medicament for treating acute colitis.

Images