A composition and method for modulating tumor cell stiffness

Abstract

The application discloses a composition and a regulation method for regulating the hardness of tumor cells, and the composition comprises: a low-pH insertion peptide, the low-pH insertion peptide can at least partially pass through a cell membrane under the condition of pH < 7.0; a peptide nucleic acid capable of being connected with the low-pH insertion peptide to form pHLIP-PNA; the low-pH insertion peptide in the pHLIP-PNA at least partially passes through the cell membrane, and the peptide nucleic acid enters the inside of the cell; a tetrahedron, the tetrahedron has a first end complementary to at least part of the peptide nucleic acid, and the tetrahedron comprises at least one of a DNA tetrahedron, an RNA tetrahedron or a PNA tetrahedron. The composition of the application can harden tumor cells.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a method based on low pH response and intracellular K+. + A method for regulating tumor cell stiffness through mediated dynamic assembly of DNA tetrahedra. Background Technology

[0002] Compared to healthy cells, tumor cells exhibit unique mechanical properties, including Young's modulus, viscoelasticity, membrane tension, cell contractility, and adhesion. The low stiffness of tumor cells has been shown to be closely related to their biological behaviors, such as invasiveness, metastasis, and immune evasion. This characteristic stems from abnormal morphology, cytoskeleton remodeling, and changes in cell-cell and cell-matrix interactions. Cancer cells reduce cellular mechanical forces by lowering cholesterol levels in their cell membranes, making them more susceptible to migration and spread within tissues, increasing the likelihood of transformation and malignant metastasis. Furthermore, cancer cells utilize their low stiffness to reduce the mechanical stress exerted on T lymphocytes, thereby decreasing their ability to recognize and attack cancer cells and inhibiting the killing effect of immune cells. Increasing the stiffness of tumor cells may limit their migration and invasiveness, thus inhibiting tumor spread. By regulating tumor cell stiffness, it is possible to slow or prevent tumor spread and metastasis, improving treatment efficacy.

[0003] Previous research has proposed several feasible methods to regulate tumor cell stiffness. Among these, the regulation of extracellular matrix (ECM) components, gene editing, and drug intervention have been proven to effectively alter tumor cell stiffness. Extracellular matrix (ECM) regulation primarily alters tumor cell stiffness by regulating the composition and structure of the ECM. Cross-linking agents such as glutaraldehyde can enhance ECM stiffness, thereby affecting tumor cell stiffness. Gene editing, utilizing technologies such as CRISPR-Cas9 gene editing and siRNA or shRNA silencing of genes related to cell softening, can precisely modify genes affecting the cytoskeleton or extracellular matrix (ECM), thereby changing tumor cell stiffness. Drug intervention to regulate tumor cell stiffness refers to altering the mechanical properties of tumor cells, particularly cell stiffness or rigidity, using specific drugs. Drugs such as lovastatin and methyl-β-cyclodextrin (MeβCD) can increase cell membrane stiffness by extracting cholesterol from the cell membrane. Cholesterol is an important component of the cell membrane, affecting membrane fluidity and stiffness. Reducing cholesterol in the membrane can increase membrane rigidity, thereby increasing cell stiffness. Casein kinase inhibitors (such as CKI-7) can increase cell stiffness by affecting intracellular signaling pathways and cytoskeleton dynamics. Summary of the Invention

[0004] One objective of this invention is to provide a composition for regulating tumor cell stiffness, comprising:

[0005] A low-pH intercalation peptide, wherein at least a portion of the low-pH intercalation peptide is capable of crossing the cell membrane at pH < 7.0;

[0006] A peptide nucleic acid capable of being linked with the low-pH insert peptide to form a pHLIP-PNA; at least a portion of the low-pH insert peptide in the pHLIP-PNA crosses the cell membrane, and the peptide nucleic acid enters the cell interior;

[0007] A tetrahedron having a first end complementary to at least a portion of the peptide nucleic acid, the tetrahedron including at least one of a DNA tetrahedron, an RNA tetrahedron, or a PNA tetrahedron.

[0008] Furthermore, the tetrahedron includes a state in which it has been assembled into a tetrahedron, or a single-chain or multi-chain state in which it has not been assembled into a tetrahedron.

[0009] Furthermore, the tetrahedron binds to the peptide nucleic acid in the pHLIP-PNA within the cell membrane.

[0010] Furthermore, the concentration range of pHLIP-PNA is 0.1–10.0 μmol / L.

[0011] Furthermore, the concentration range of the tetrahedron is 0.1–10.0 μmol / L.

[0012] Furthermore, the tetrahedron also possesses a high concentration of K within the cell. + Under certain environmental conditions, G-quadruplexes are formed, leading to aggregation at the binding ends. The aforementioned "high intracellular concentration of K..." + The "environment" refers to the high concentration of potassium that naturally forms under the intrinsic physiological conditions of the cell. + environment.

[0013] Furthermore, the binding terminus includes at least one of guanine-rich single-stranded DNA, peptide nucleic acid, or oligonucleotide analog; the first terminus comprises at least one of single-stranded DNA, peptide nucleic acid, or oligonucleotide analog.

[0014] Another object of the present invention is to provide a method for regulating tumor cell stiffness, comprising the following steps:

[0015] Step 1: Connect the low-pH insert peptide and peptide nucleic acid to form pHLIP-PNA, wherein the low-pH insert peptide is capable of at least partially crossing the cell membrane under pH < 7.0 conditions;

[0016] Step 2, synthesizing a tetrahedron having a first end that is complementary to at least a portion of the peptide nucleic acid, wherein the tetrahedron includes at least one of a DNA tetrahedron, an RNA tetrahedron, or a PNA tetrahedron;

[0017] Step 3: The products synthesized in steps 1) and 2) are added to a sample of tumor cells under acidic conditions, and the tetrahedrons aggregate within the cells.

[0018] Furthermore, in step one, the reaction in which the low-pH insert peptide pHLIP and peptide nucleic acid PNA are linked to form pHLIP-PNA includes at least one of an amide reaction, a Michael addition reaction, or a click chemistry reaction.

[0019] Furthermore, in step three, the acidic condition involves placing the tumor cells in PBS or cell culture medium with a pH below 7.0.

[0020] Furthermore, in step three, the concentration range of the added pHLIP-PNA is 0.1–10.0 μmol / L; the concentration range of the tetrahedron is 0.1–10.0 μmol / L.

[0021] This invention provides a method based on low pH response and intracellular K + A method for regulating tumor cell stiffness through mediated dynamic assembly of DNA tetrahedra, utilizing the acidic environment responsiveness of low-pH insertion peptides and the high intracellular K+ concentration of tetrahedra. + Under certain conditions, G-quadruplex aggregates are formed. The peptide nucleic acid at the C-terminus of the low-pH inserted peptide can complementarily pair with the single-stranded DNA at the apex of the DNA tetrahedron, causing the DNA tetrahedron to aggregate on the inner side of the cell membrane to form a scaffold structure to regulate the stiffness of tumor cells. This can effectively achieve targeted regulation of the stiffness of tumor cells.

[0022] This invention utilizes the acid-environment responsiveness of low-pH insertion peptides and the high intracellular K+ concentration of DNA tetrahedra. + Under certain conditions, G-quadruplex aggregates are formed, creating a scaffold structure on the inner side of the cell membrane to regulate cell stiffness. Regulating tumor cell stiffness can effectively target the tumor microenvironment, utilizing its slightly acidic nature to specifically act on tumor cells and precisely control their stiffness. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0025] Figure 2 Circular dichroism spectroscopy was used to characterize the formation of G-tetrachain.

[0026] Figure 3 The assembly characterization of pHLIP-PNA and G4-TDN on cells is shown below. A represents the insertion of pHLIP-PNA into the cell membrane under acidic conditions (pH < 7.0), B represents the aggregation of DNA tetrahedra within the cell, and C represents the assembly of pHLIP-PNA and DNA tetrahedra under acidic conditions.

[0027] Figure 4 The cell stiffness and immunotherapy characteristics are shown in Figure A, where A represents the cell stiffness characterization by atomic force microscopy, B represents the cell stiffness characterization by optical tweezers, and C represents the cancer cell killing effect mediated by the tetrahedral assembly of pHLIP-PNA and DNA. Detailed Implementation

[0028] Low pH inserted peptide (pHLIP): N-terminus – AEQNPIYWARYADWLFTTPLLLLDLALLVDADEG – C-terminus (SEQ ID NO: 1) N-terminus modified with biotin, C-terminus modified with maleimide group

[0029] Peptide-nucleic acid chain (PNA) linked to a low-pH intercalation peptide: 5'-CATCTAGTGA-3' (5' end modified with a thiol group)

[0030] SSDNA1:5'TTGGGTTGGGAATACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAG TA-3' (SEQ ID NO: 2)

[0031] SSDNA2:5'TTGGGTTGGGAATTATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAAT AC-3' (SEQ ID NO: 3)

[0032] SSDNA3:5'TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTCTTATCACTAGA TG-3 (SEQ ID NO: 4)

[0033] SSDNA4:5'TTGGGTTGGGTTATTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGC AT-3 (SEQ ID NO: 5)

[0034] The main principle of the acid response of pHLIP-PNA is:

[0035] pHLIP-PNA can form a transmembrane α-helix to penetrate the cell membrane and insert into the cell at low pH (<7.0), but it does not insert at normal physiological pH. When pHLIP-PNA is inserted into the cell, the C-terminus is inside the cell membrane and the N-terminus is outside the cell membrane.

[0036] Intracellular K + The principles of mediated dynamic assembly of DNA tetrahedra can be summarized as follows:

[0037] Intracellular K + At higher concentrations, the guanine-rich single-stranded DNA at the base of the DNA tetrahedron will react with high concentrations of K. + G-quadruplexes are formed inside the cells and aggregate.

[0038] The principle of regulating tumor cell stiffness can be summarized as follows: it is achieved through complementary pairing of the PNA sequence at the C-terminus of pHLIP-PNA and the single-stranded DNA sequence at the apex of the DNA tetrahedron.

[0039] like Figure 1 As shown, the principle of this invention is as follows.

[0040] pHLIP-PNA can form a transmembrane α-helix at low pH (<7.0) to penetrate the cell membrane and insert into the cell. The PNA sequence at the C-terminus of pHLIP-PNA is complementary to the single-stranded DNA sequence at the apex of the DNA tetrahedron in the cell membrane. The guanine-rich single-stranded DNA at the basal end of the DNA tetrahedron, under high K+ concentrations... + G-quadruplexes are formed in the solution. This causes DNA tetrahedra to aggregate on the cell membrane, thereby regulating the stiffness of tumor cells.

[0041] The sample containing tumor cells includes PBS (containing Mg). 2+ ( ), at least one of the cell culture media.

[0042] Example 1

[0043] 1. Preparation of pHLIP-PNA

[0044] The low-pH intercalated peptide (pHLIP) and peptide nucleic acid (PNA) were linked by amide reaction, Michael addition reaction, click chemistry reaction to synthesize crude pHLIP-PNA, and pHLIP-PNA was purified by high performance liquid chromatography.

[0045] In a preferred embodiment of the invention, the pHLIP-PNA synthesis is achieved by linking a maleimide-modified low-pH insert peptide (pHLIP) and a thiol-modified peptide nucleic acid (PNA) via an amide reaction to synthesize pHLIP-PNA.

[0046] In this embodiment, the C-terminus of the low-pH inserted peptide (pHLIP) is modified with a maleimide group, and the 5' end of the peptide nucleic acid is modified with a thiol group.

[0047] 2. Preparation of DNA tetrahedra

[0048] DNA tetrahedra (G4-TDNs) were synthesized by denaturing oligonucleotide chains SSDNA1, SSDNA2, SSDNA3, and SSDNA4 at 50-100℃ for 5-10 min, followed by cooling to 4℃. The resulting single-stranded DNA was rich in guanine at the base and had a PNA-complementary sequence at the tip. The final annealed product was stored at 4℃.

[0049] In a preferred embodiment of the present invention, oligonucleotide chains SSDNA1, SSDNA2, SSDNA3, and SSDNA4 are denatured at 95°C for 5 minutes, and then gradually cooled down at a temperature of 4°C, decreasing by 1 degree every 30 seconds.

[0050] In a preferred embodiment of the present invention, the PNA sequence at the C-terminus of pHLIP-PNA is complementary to the single-stranded DNA sequence at the apex of the DNA tetrahedron.

[0051] 3. Characterization of pHLIP-PNA insertion into the cell membrane under acidic conditions.

[0052] MC38 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in confocal culture dishes containing 2 mL of DMEM. The medium was aspirated, and the cells were washed twice with PBS. Then, 1 mL of DMEM medium containing pH 6.5 and pHLIP-PNA was added. The cells were incubated at 37°C for 1 h, followed by two washes with PBS. The washed samples were characterized using a high-sensitivity confocal microscope (Zeiss LSM880 + Airyscan). The optimal concentration range of pHLIP-PNA was 0.1–10.0 μmol / L.

[0053] In this embodiment, the acidic condition is PBS (containing Mg) at pH 6.5. 2+ (or in cell culture medium.)

[0054] In this embodiment, the concentration of pHLIP-PNA is 2 μmol / L.

[0055] 4. Characterization of G4-TDNs aggregation within the cell membrane

[0056] MC38 cells were spaced at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well in 24-well DMEM plates (2 mL each) and cultured for 24 h. The original medium was replaced with DMEM at pH 6.5. Then, G4-TDNs containing only the FAM fluorescent group, G4-TDNs containing only the BHQ group, and G4-TDNs containing both the FAM and BHQ groups were added. The plates were then incubated at 37°C for 1 h. After washing three times with PBS, the cells were stained with DAPI and analyzed using an inverted fluorescence microscope (Nikon, Japan). The optimal concentration range for G4-TDNs was 0.1–10.0 μmol / L.

[0057] In this embodiment, the concentration of G4-TDNs is 0.2 μmol / L.

[0058] 5. pHLIP-PNA-TDN assembly characterization

[0059] MC38 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells / mL in 2 mL DMEM confocal culture dishes. The culture medium was aspirated, and the cells were washed twice with PBS. Then, 1 mL of pH 6.5 DMEM medium containing pHLIP-PNA was added, and the cells were incubated at 37°C for 1 h, followed by two washes with PBS. Next, pH 6.5 DMEM medium containing G4-TDNs was added, and the cells were incubated at 37°C for 1 h, followed by two washes with PBS. The cells were characterized using a high-sensitivity confocal microscope (Zeiss LSM880 + Airyscan). The optimal concentration range for pHLIP-PNA was 0.1–10.0 μmol / L. The optimal concentration range for G4-TDNs was 0.1–10.0 μmol / L.

[0060] In this embodiment, the concentration of pHLIP-PNA is 2 μmol / L. The concentration of G4-TDNs is 0.2 μmol / L.

[0061] 6. pHLIP-PNA-TDN assembly regulates cell stiffness characterization

[0062] 1) Atomic force microscopy (AFM) method for measuring cell stiffness.

[0063] AFM force profiles were recorded using a custom Dimension Icon AFM (Bruker) mounted on top of an inverted optical microscope. The sample was moved using the XY series until the target cell was positioned under the AFM tip through the optical microscope. Force profiles of the cells were recorded using a PNP-TR-B cantilever beam (Nanoworld4). Single cells were measured at 37°C before and after assembly treatment with pHLIP-PNA-TDN. The force profiles were processed using nanoanalysis software (Bruker), and Young's modulus of the sample was calculated by fitting approximate curves at cell indentations.

[0064] 2) The optical tweezer (OT) method was used to measure cell stiffness.

[0065] Indentation experiments were performed using streptavidin-coated silica microspheres. A custom-designed sample chamber containing adherent cells was mounted on a microscope, and the microspheres were added to the chamber. One microsphere was trapped above the bottom of the sample chamber by an optical trap. The microsphere faced the most perpendicular side of the cell membrane, i.e., the side without plate-like pseudopodia. The microsphere was then laterally pushed into the cell by moving the position of the optical trap at a constant speed, followed by contraction at a predetermined point at the same speed. The stiffness of the cell was quantified as the slope of the force-deformation curve within a linear range of 200–500 nm for the indentation.

[0066] 7. After assembly of pHLIP-PNA-TDN, CD8 was incubated in a co-incubation system. + The effect of T cell-mediated killing of cancer cells

[0067] 1) CD8 + Extraction and activation of T cells

[0068] Spleens from OT-I mice were harvested and homogenized using a 70µm cell filter on day 0. Red blood cells were lysed with erythrocyte lysis buffer for 5 minutes at room temperature, washed twice with PBS, and cultured for 2 days in RPMI 1640 medium containing SIINFEKL peptide and recombinant mouse IL-2. The medium was supplemented with FBS (10 v / v%), HEPES (1 v / v%), penicillin / streptomycin (1 v / v%), and β-mercaptoethanol (0.1 v / v%). EasySep was used for further analysis. TM Mouse CD8 + T Cell Isolation Kit extracts CD8 + After T cells are activated, OT-I CD8 will be activated. +T cells were preserved in a culture medium containing recombinant mouse IL-2 and used for in vitro or in vivo studies 4 to 8 days after spleen cell collection. The optimal concentration range for SIINFEKL peptide was 0.1–5.0 μmol / L, and the optimal concentration range for IL-2 was 0.1–20 ng / ml. -1 ).

[0069] 2) Experimental study on the in vitro killing of cancer cells by T cells

[0070] In the MC38 cancer cell killing assay, cancer cells were first pulsed with SIINFEKL for 30 min and washed twice with PBS. Next, the cancer cells were treated with DMEM medium at pH 6.5 containing pHLIP-PNA and DMEM medium at pH 6.5 containing G4-TDNs. Activated OT-I CD8... + T cell suspensions were added to intact RPMI 1640 medium at an E:T ratio of 1:1 or 10:1. After co-culturing at 37°C for 5–24 hours, CytoTox was used. The Non-Radioactive Cytotoxicity Assay quantifies cell death in MC38 cancer cells. The optimal concentration range for SIINFEKL peptide is 0.1–5.0 μmol / L.

[0071] Results analysis:

[0072] like Figure 2 As shown, circular dichroism (CD) spectroscopy was used to investigate whether guanine-rich DNA tetrahedra could form G tetrahedra in a high-concentration K solution. The results showed that at 140 mM K... + In the presence of (cytoplasmic concentration), the positive peak at ~277nm blue shifted to ~274nm, indicating that G-tetraplexes were formed between G4-TDNs.

[0073] like Figure 3 As shown, Figure 3 A indicates that pHLIP-PNA inserts into the cell membrane only at pH 6.5, but not at pH 7.4, suggesting that pHLIP-PNA has an acid-responsive capability. Figure 3 B indicates that the fluorescence intensity on the cell surface was significantly reduced after the simultaneous addition of G4-TDNs with FAM fluorescent groups and G4-TDNs with BHQ groups compared to the control group with only FAM fluorescent groups added. This suggests that TDNs aggregate intracellularly and form G4-strands. Figure 3C indicates that pHLIP-PNA-TDN assembles on the cell membrane and exhibits good co-localization only at pH 6.5, while no assembly occurs at pH 7.4, and TDN is uniformly dispersed within the cell membrane. These results demonstrate that pHLIP-PNA-TDN possesses good targeting properties in the tumor microacidic environment and can aggregate within the cell membrane to form G4 chains.

[0074] like Figure 4 As shown, atomic force microscope (AFM) Figure 4 A) The results showed that, only under pH 6.5 conditions, assembly of pHLIP-PNA-TDN significantly increased the stiffness of mouse MC38 cells, and was significantly higher than that of the control group. To confirm the results, another well-established cellular biomechanics measurement technique, optical tweezers, was used to probe the stiffness of cancer cells. Consistent with the results of atomic force microscopy, assembly of pHLIP-PNA-TDN only under pH 6.5 conditions significantly increased the stiffness of mouse MC38 cancer cells. Figure 4 B). This indicates that pHLIP-PNA-TDN assembly can effectively regulate tumor cell stiffness. Figure 4 The results showed that MC38 cells exhibited significantly increased sensitivity to T cell-mediated killing after pHLIP-PNA-TDN assembly, enhancing T cell lysis of cancer cells. This indicates that pHLIP-PNA-TDN assembly enhances T cell-mediated cytotoxicity by hardening cancer cells.

[0075] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A composition for regulating the stiffness of tumor cells, comprising: a low-pH intercalating peptide, which is capable of penetrating at least partially into a cell membrane under a condition of pH < 7.0; the sequence of the low-pH intercalating peptide is shown as SEQ ID NO: 1; a peptide nucleic acid, which is capable of being linked to the low-pH intercalating peptide to form a pHLIP-PNA, wherein the low-pH intercalating peptide in the pHLIP-PNA penetrates at least partially into the cell membrane, and the peptide nucleic acid enters the interior of the cell; the sequence of the peptide nucleic acid is CATCTAGTGA; a tetrahedron, which has a first end complementary to at least a part of the peptide nucleic acid, and the tetrahedron is a DNA tetrahedron; the sequence of the DNA tetrahedron is shown as SEQ ID NO: 2 to SEQ ID NO:

5.

2. The composition for regulating the stiffness of tumor cells according to claim 1, wherein: The tetrahedron includes a state of being assembled into a tetrahedron, or a single-stranded or multi-stranded state of not being assembled into a tetrahedron.

3. The composition for regulating the stiffness of tumor cells according to claim 1, wherein: The concentration of the pHLIP-PNA ranges from 0.1 to 10.0 μmol / L.

4. The composition for regulating the stiffness of tumor cells according to claim 1, wherein: The concentration of the tetrahedron ranges from 0.1 to 10.0 μmol / L.

5. A composition for modulating the stiffness of tumor cells according to any one of claims 1 to 4, characterized in that: The tetrahedron has a G-quadruplex formed in a cell at a high concentration of K + The G-quadruplex is formed in a cell at a high concentration of K+; the binding end is a guanine-rich single-stranded DNA; and the first end is a single-stranded DNA.

6. A method for preparing the composition for regulating the stiffness of tumor cells according to claim 1, comprising the following steps: Step 1, linking a low-pH intercalating peptide and a peptide nucleic acid to form a pHLIP-PNA, wherein the low-pH intercalating peptide is capable of penetrating at least partially into a cell membrane under a condition of pH < 7.0; Step 2, synthesizing a tetrahedron, which has a first end complementary to at least a part of the peptide nucleic acid, and the tetrahedron is a DNA tetrahedron; Step 3, adding the products synthesized in steps 1) and 2) into a sample of tumor cells with an acidic condition, and the tetrahedron aggregates in the cells.

7. The method of claim 6, wherein: In step 1, the reaction of linking the low-pH intercalating peptide pHLIP and the peptide nucleic acid PNA to form the pHLIP-PNA includes at least one of an amide reaction, a Michael addition reaction, or a click chemistry reaction.

8. The method of claim 6, wherein: In step 3, the acidic condition is placing the tumor cells in PBS or cell culture medium with a pH lower than 7.

0.

9. The method of claim 6, wherein: In step 3, the concentration of the added pHLIP-PNA ranges from 0.1 to 10.0 μmol / L; and the concentration of the tetrahedron ranges from 0.1 to 10.0 μmol / L.

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