Logic responsive biomacromolecule carrier based on phase separation system, and construction method and application thereof

Abstract

This invention discloses a logic-responsive biomolecule carrier based on a phase separation system, its construction method, and its applications. The carrier is based on the attraction between cationic peptides with different logic responses and negatively charged biomolecules such as DNA or RNA, driving liquid-liquid phase separation and forming droplet-like particle structures. The carrier of this invention can selectively use protein kinase A (PKA) to phosphorylate peptides and use matrix metalloproteinases (MMPs) or glutathione (GSH) to cleave the peptide chain, thereby changing the charge density of the peptide to control the controlled release of biomolecules. It exhibits excellent biocompatibility, resistance to enzymatic degradation, and transfection efficiency, enabling effective delivery of biomolecules.

Description

Technical Field

[0001] This invention belongs to the field of delivery carriers for biological macromolecules, and relates to a logic-responsive biological macromolecule carrier based on a phase separation system, its construction method, and its application. Background Technology

[0002] Biological macromolecules (such as DNA, RNA, proteins, and ribonucleoprotein complexes, RNPs) have shown great potential in the precision treatment of tumors and genetic diseases due to their specific regulatory functions on biological pathways. The core bottleneck in their therapeutic efficacy lies in achieving efficient, safe, and precise delivery. Existing delivery vectors are divided into viral and non-viral vectors: while viral vectors offer high delivery efficiency, they suffer from strong immunogenicity, limited loading capacity, and potential insertion mutation risks. Non-viral vectors (such as liposomes and cationic polymer nanoparticles) offer controllable biocompatibility but suffer from limitations such as single response, insufficient binding stability, and inherent cytotoxicity, making them difficult to adapt to the complex tumor microenvironment.

[0003] Phase separation technology for biomolecules offers a new direction for carrier construction. The resulting droplet-like condensates possess good biocompatibility, high loading capacity, and environmental responsiveness potential, and can stably encapsulate biomolecules through charge neutralization. Existing literature reports that most phase-separated carriers are single-response (e.g., responding only to pH or a single protease), lacking the ability to logically recognize complex microenvironments. They cannot achieve precise targeted release regulation based on combinations of multiple characteristic signals, leading to non-specific release of the carriers in normal tissues, poor therapeutic effects, and a high risk of cytotoxicity. ([1] Sun, Y.; Lau, SY; Lim, ZW; et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat. Chem. 2022, 14, 274-283. [2] Hong, Y, Najafi, S., Casey, T. et al. Hydrophobicity of arginine leads to reentrantliquid-liquid phase separation behaviors of arginine-richproteins. Nat. Commun. 2022, 13, 7326. [3] Chen, R.; Jiang, Y.; Lv, M.; et al. Redox-Regulated Phase Switchable Peptide Droplets with Degradation Resistance for Intravenous Delivery of Biopharmaceuticals. Adv. Mater. 2026, 38, e11828.). Based on this, the present invention aims to provide a logic-responsive biomacromolecule carrier based on a phase separation system, its construction method and application. By designing cationic peptides with different logic response functions, they are self-assembled with biomacromolecules through phase separation to form composite particles. Relying on the combined recognition of disease characteristic signals such as PKA, MMP, and GSH, efficient targeted delivery and responsive precise release of biomacromolecules can be achieved, solving the problems of single response, poor targeting and insufficient delivery efficiency of existing carriers, and providing technical support for the clinical translation of biomacromolecules. Summary of the Invention

[0004] The purpose of this invention is to provide a logic-responsive biomacromolecule carrier based on a phase separation system that is easy to synthesize, highly controllable, and has low cytotoxicity, as well as its construction method and application.

[0005] A logic-responsive biomacromolecule carrier based on a phase-separation system is a micron-sized polypeptide-biomacromolecule composite particle. The composite particle is formed by charge-driven phase separation self-assembly of negatively charged biomacromolecules and cationic polypeptides with different logic-responsive functions. The negatively charged biomacromolecules include DNA, RNA, and proteins. The cationic polypeptides have specific response sites, specifically including: a serine phosphorylation site specifically recognized by PKA, a PLGLAG peptide cleavage site specifically recognized by MMP, and a -Cs-sC- disulfide bond cleavage site specifically recognized by GSH. Each site is adapted to the response functions of PKA, MMP, and GSH, thereby enabling the cationic polypeptide to form different logic gate response types. The logic gate types include: AND gates (AM-AND gates with dual responses to PKA and MMP, and AC-AND gates with dual responses to PKA and PKC), OR gates (AM-OR gates with dual responses to PKA and MMP, and AC-OR gates with dual responses to PKA and PKC), and multi-input gates (OR-AND gates with triple responses to PKA, MMP, and GSH, and AND-AND gates with triple responses to PKA, MMP, and GSH). By phosphorylating peptides with protein kinase A (PKA) or cleaving peptide chains with matrix metalloproteinases (MMP) or glutathione (GSH), the charge density of the peptides is altered, thereby achieving precise regulation of the release of biomolecules.

[0006] Preferably, the amino acid sequences of the cationic polypeptides with different logical responses are as follows:

[0007] AP-AND: GRRRASLPLGLAGRR (SEQ ID No. 1);

[0008] AC-AND: GRRRASLGRGGSKK (SEQ ID No. 2);

[0009] AP-OR: GGRRASLPLGLAGRR (SEQ ID No. 3);

[0010] AC-OR: GGRRASLRGGGSVK (SEQ ID No. 4);

[0011] T-OR-AND: GRRRASLRCs-sCRRPLGLAGRR (SEQ ID No. 5);

[0012] T-AND-AND: GGRRASLPLGLAGRRCs-sCRRPLGLAGGGRRASL (SEQ ID No. 6).

[0013] The above-mentioned method for preparing a logic-responsive biomacromolecule carrier based on a phase-separated system includes the following steps:

[0014] Negatively charged biomacromolecules such as DNA or RNA are dissolved in a solution containing ATP. Then, cationic peptides and biomacromolecules are mixed at 37°C and phase separation occurs to form droplet-like particle solutions, thus obtaining peptide-biomacromolecule composite particle carriers that transport biomacromolecules.

[0015] Preferably, the mixing time of the cationic polypeptide and the biomacromolecule is 30 min.

[0016] Preferably, the final concentration of the biological macromolecules is 200 nM, and the final concentration of the cationic polypeptide is 100 μM.

[0017] Preferably, the molar ratio of the biomacromolecule to the cationic polypeptide is 1:500.

[0018] Preferably, the solution containing ATP can be a buffer solution with pH=7.4 consisting of 20mM HEPES and 2.5mM ATP, or a buffer solution with pH=7.4 consisting of 20mM Tris and 2.5mM ATP.

[0019] Furthermore, the present invention provides the application of the above-mentioned carrier in the preparation of drugs for the prevention and / or treatment of cancer, through which biological macromolecules are delivered into cells and disease treatment is achieved.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The phase-separation system of the present invention provides a logic-responsive biomolecular carrier that is easy to synthesize and design, and exhibits good biocompatibility and stability. This biomolecular carrier also possesses low cytotoxicity and high controllability and stability. By designing cationic peptides with different logic-responsive functions, these peptides are self-assembled with biomolecular molecules through phase separation to form composite particles. Relying on the combined recognition of disease characteristic signals such as PKA, MMP, and GSH, the charge density of the peptides is altered, achieving precise targeted delivery and responsive release of biomolecular molecules. This solves the problems of existing carriers having single response, poor targeting, and insufficient delivery efficiency. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the construction and multi-input logic response release of a logic-responsive biomacromolecule carrier based on a phase-separated system.

[0023] Figure 2 This is a confocal image of the construction results of a logic-responsive biomacromolecule carrier based on a phase-separated system in Example 2;

[0024] Figure 3 This is a confocal result diagram and particle number statistics diagram of the multi-input logical response decomposition of the logic-responsive biomacromolecule carrier based on the phase separation system in Example 3;

[0025] Figure 4 This is a confocal image showing the results of using the multi-input logic response vector treated with different inhibitors for intracellular mRNA delivery in Example 4. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or in the product manual.

[0027] Example 1

[0028] Figure 1 This is a schematic diagram of a logic-responsive biomolecule carrier in a phase-separated system. Negatively charged biomolecules and cationic peptides with different logic-responsive functions self-assemble into aggregates via liquid-liquid phase separation. Because cationic peptides selectively possess phosphorylation sites, enzyme cleavage sites, and disulfide bond cleavage sites, the release of biomolecules is controlled by altering the charge density of the peptides through phosphorylation using protein kinase A (PKA) and cleavage of the peptide chain using matrix metalloproteinases (MMPs) or glutathione (GSH).

[0029] Example 2

[0030] Preparation and confocal imaging of logic-responsive biomacromolecule carriers in phase-separated systems.

[0031] Take 2 μL of 1 μM Plasmid / ssDNA / mRNA / micRNA / siRNA and mix them in 2.5 μL of buffer (80 mM HEPES, 10 mM ATP, pH 7.4). Add 4.5 μL of ultrapure water, and then add 1 μL of each of the six 1 mM cationic peptides. Incubate at 37 °C for 30 min. Transfer to a confocal dish and image using a Nikon confocal microscope.

[0032] Example 3

[0033] Multi-input logic release, confocal imaging, and particle count of a logic-responsive biomacromolecule carrier in a phase-separated system. 2 μL of 1 μM siRNA was mixed in 2.5 μL of buffer (80 mM HEPES, 10 mM ATP, pH 7.4), followed by 4.5 μL of ultrapure water. Then, 1 μL of each of six cationic peptides (1 mM) were added, and the mixture was incubated at 37 °C for 30 min. 1.5 μL of a logic combination of PKA, MMP, and GSH was added, and the reaction was allowed to proceed for 1 h. The mixture was then transferred to a confocal dish and imaged using a Nikon confocal microscope.

[0034] Example 4

[0035] The multi-input logic response vectors treated with different inhibitors were used for intracellular mRNA delivery experiments. The specific steps are as follows:

[0036] (1) Seeding: After digesting the adherent HeLa cells with trypsin, prepare them to a suitable cell density. Take 100 μL of suspension cells and add them to a four-well confocal dish. Culture for one day or observe the cells after they have adhered.

[0037] (2) Inhibitor treatment: Glutathione inhibitor BSO was dissolved in PBS to prepare a working solution with a final concentration of 500 μM; PKA inhibitor H-89 was dissolved in PBS to prepare a working solution with a final concentration of 20 μM; MMP inhibitor GM6001 was dissolved in PBS to prepare a working solution with a final concentration of 20 μM. The culture medium in the confocal culture dish was aspirated, and 100 μL of the corresponding logical combination of inhibitor working solution was added to incubate the cells. BSO was treated for 24 h, and H-89 and GM6001 were treated for 2 h. After incubation, the inhibitor working solution was aspirated, and the cells were washed 3 times with PBS to remove residual inhibitors.

[0038] (3) Vector preparation and cell incubation: 2 μL of 10 μM mcherry mRNA expressing red fluorescent protein was mixed in 2.5 μL of buffer (80 mM HEPES, 10 mM ATP, pH = 7.4), 4.5 μL of ultrapure water was added, followed by 1 μL of 10 mM of each of the six cationic peptides mentioned above. 90 μL of cell culture medium was added, and the mixture was incubated at 37°C for 30 min. Six peptide-mRNA particles were obtained. 100 μL of each peptide-mRNA particle was added to a four-well dish and incubated for another 24 h. The mixture was then imaged using a confocal microscope.

[0039] Example 5

[0040] from Figure 2 The results of the laser confocal imaging experiment show that the cationic polypeptide described in this invention can bind to a variety of negatively charged biomacromolecules and successfully self-assemble into a complex aggregate, verifying the feasibility of the carrier construction.

[0041] from Figure 3 The results of the laser confocal imaging experiment and its statistical graphs show that the polypeptide-biomacromolecule composite particles described in this invention can only dissociate and release biomacromolecules under the stimulation of the corresponding logical combination signal, demonstrating good logical response specificity.

[0042] from Figure 4 The results of the laser confocal imaging experiment showed that the blank group (without inhibitor treatment) could successfully express red fluorescent protein, indicating that the composite particles could smoothly enter the cells and release mRNA; in some groups treated with inhibitors, the expression level of red fluorescent protein was significantly reduced, indicating that the composite particles were difficult to dissociate after the corresponding logic response was inhibited, which verified that the vector can achieve multi-input logic response regulation in cells.

Claims

1. A logic-responsive biomacromolecule carrier based on a phase-separated system, characterized in that, These are micron-sized polypeptide-biomacromolecule composite particles. The composite particles are formed by the self-assembly of negatively charged biomacromolecules and cationic polypeptides with different logical response functions through phase separation. The negatively charged biomacromolecules include DNA, RNA, and proteins. The cationic polypeptides have specific response sites, specifically including: a serine phosphorylation site specifically recognized by PKA, a PLGLAG peptide cleavage site specifically recognized by MMP, and a -Cs-sC- disulfide bond cleavage site specifically recognized by GSH. Each site has a responsive function to PKA, MMP, and GSH, respectively. This allows cationic peptides to form different logic gate response types, including: AND logic gates (AM-AND logic gates with dual responses to PKA and MMP, and AC-AND logic gates with dual responses to PKA and PKC), OR logic gates (AM-OR logic gates with dual responses to PKA and MMP, and AC-OR logic gates with dual responses to PKA and PKC), and multi-input logic gates (OR-AND logic gates with triple responses to PKA, MMP, and GSH, and AND-AND logic gates with triple responses to PKA, MMP, and GSH).

2. The carrier according to claim 1, characterized in that, The amino acid sequences of the cationic polypeptides with different logical responses are as follows: AP-AND: GRRRASLPLGLAGRR; AC-AND: GRRRASLGRGGSKK; AP-OR: GGRRASLPLGLAGRR; AC-OR: GGRRASLGRGGSVK; T-OR-AND: GRRRASLRCs-sCRRPLGLAGRR; T-AND-AND:GGRRASLPLGLAGRRCs-sCRRPLGLAGGGRRASL.

3. The method for preparing the carrier according to claim 1 or 2, characterized in that, Includes the following steps: Negatively charged biomacromolecules such as DNA or RNA are dissolved in a solution containing ATP. Then, cationic peptides and biomacromolecules are mixed at 37°C and phase separation occurs to form droplet-shaped particle solutions, thus obtaining a carrier for transporting biomacromolecules, namely peptide-biomacromolecule composite particles.

4. The method for preparing the carrier according to claim 3, characterized in that, The mixing time for cationic peptides and biomacromolecules was 30 min.

5. The method for preparing the carrier according to claim 3, characterized in that, The final concentration of all biological macromolecules was 200 nM, and the final concentration of cationic polypeptides was 100 μM.

6. The method for preparing the carrier according to claim 3, characterized in that, The molar ratio of biological macromolecules to cationic polypeptides is 1:

500.

7. The method for preparing the carrier according to claim 3, characterized in that, The solution containing ATP is a buffer solution with pH=7.4 consisting of 20 mM MEPES and 2.5 mM ATP, or a buffer solution with pH=7.4 consisting of 20 mM Tris and 2.5 mM ATP.

8. The use of the carrier according to claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of cancer.

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