CN115844892B - Disulfide bond-modified nanoaggregates and preparation method and application thereof - Google Patents

Abstract

The invention discloses a disulfide bond modified nano aggregate, a preparation method and application thereof, wherein the nano aggregate comprises CPUL1 and a disulfide bond-containing compound, and the nano aggregate is formed by modifying CPUL1 and the disulfide bond-containing compound through non-covalent interaction. The preparation method comprises the following steps: (1) CPUL1 and a compound containing disulfide bond are respectively dissolved in an organic solvent to obtain CPUL1 solution and a compound solution containing disulfide bond; (2) Adding water into the compound solution containing disulfide bonds, stirring for the first time to obtain a mixed solution, adding the CPUL1 solution in the step (1) into the mixed solution, and stirring uniformly again to obtain the disulfide bond modified nano aggregate. The nano-aggregate provided by the invention has the advantages of good stability, low toxicity, rapid cell uptake, specific response to TrxR and improved anti-tumor effect on HUH7 liver cancer cells.

Description

Disulfide bond-modified nanoaggregates and preparation method and application thereof

Technical Field

The present invention relates to the field of medicine, and more specifically, to disulfide bond-modified nano aggregates and a preparation method and application thereof.

Background technique

The thioredoxin reductase-thioredoxin (TrxR-Trx) system and the glutathione reductase-glutathione-glutaredoxin (GR-GSH-Grx) system are two major regulatory systems for redox balance in organisms. TrxR mainly maintains the reduced state of the endogenous substrate Trx by providing electrons from NADPH and regulates various redox-based signaling pathways, which are involved in almost all aspects of cell function, such as differentiation, proliferation and death.

Disulfides are natural substrates for various redox systems, but different configurations may exhibit different specificities. Specific thiol/disulfide exchange reactions are the basis of many important pathways in biological systems. Linear disulfides exhibit irreversibility and nonspecificity in the exchange and reduction of thiol-disulfides in cells, where cyclic disulfides often exhibit different kinetics and thermodynamics. Linear disulfides undergo irreversible cleavage upon entry into cells, resulting in the release of the compounds to which they are attached.

In the applicant's previous work, the TrxR inhibitor CPUL1 was screened out. The structure of CPUL1 is recorded in patent CN201510894070.0. The pyrano[3,2-α]phenazine derivative in the patent is CPUL1, but the poor solubility of CPUL1 greatly limits its further anti-tumor application.

Summary of the invention

Purpose of the invention: The purpose of the present invention is to provide a disulfide bond-modified nanoaggregate with high drug loading rate and uniform particle size; another purpose of the present invention is to provide a method for preparing disulfide bond-modified nanoaggregates; another purpose of the present invention is to provide an application of disulfide bond-modified nanoaggregates.

Technical solution: A disulfide bond-modified nanoaggregate of the present invention comprises CPUL1 and a compound containing a disulfide bond, and the nanoaggregate is formed by modifying CPUL1 and the compound containing a disulfide bond through non-covalent interaction.

Disulfide bonds (-S-S-) have good biological activity (such as anti-tumor activity) and unique chemical properties. Disulfide bonds can undergo biodegradation reactions in the presence of thiol and are responsive to TrxR and GSH stimulation. Disulfide bonds can also undergo oxidation reactions inside tumor cells with high reactive oxygen to enhance the hydrophilicity of the drug delivery system. Based on this characteristic, disulfide bonds are used as prodrug molecules or carriers to promote the disintegration and release of nanoparticles through hydrolysis reactions. Nanomedicines modified with disulfide bonds can exist stably when they do not enter the cell. Under the action of high concentrations of TrxR, GSH and ROS in tumor cells, they can be rapidly degraded and released, which can effectively avoid the toxic side effects of premature release of drugs on normal cells.

Furthermore, the compound containing a disulfide bond is lipoic acid or dithiodipropionic acid.

Furthermore, the particle size of the nanoaggregates is 90-700 nm.

On the other hand, the present invention provides a method for preparing the above-mentioned disulfide bond-modified nanoaggregates, comprising the following steps:

(1) CPUL1 and a compound containing a disulfide bond are dissolved in an organic solvent respectively to obtain a CPUL1 solution and a compound containing a disulfide bond solution;

(2) adding water to the disulfide bond-containing compound solution, stirring for the first time to obtain a mixed solution, adding the CPUL1 solution in step (1) to the mixed solution, and stirring again to obtain disulfide bond-modified nanoaggregates.

Furthermore, in step (1), the molar concentration ratio of the CPUL1 solution to the disulfide bond compound solution is 1:0.5-1:4.

Furthermore, in step (1), the organic solvent is any one of ethyl acetate, acetone, methanol, dichloromethane, chloroform, ethyl propionate, propyl acetate, dimethyl sulfoxide or ethanol.

Furthermore, in step (2), the initial stirring and/or secondary stirring is magnetic stirring.

Furthermore, in step (2), the temperature of the initial stirring and/or the secondary stirring is 20-60° C., the stirring speed is 50-1000 rpm, and the stirring time is 0.5-3 h.

Furthermore, in step (2), the molar concentration of CPUL1 and/or the compound containing a disulfide bond in the nanoaggregate is 0.16-16 mM/L.

On the other hand, the present invention provides an application of the above-mentioned nano-aggregates in the preparation of tumor therapeutic drugs. The particle size of the nano-aggregates of the present invention is at the nanometer level, which can prolong the in vivo circulation period. The nanoparticles have a small particle size, have a good EPR effect, and are easy to accumulate at the tumor site. At the same time, the disulfide bonds in the nanoparticle structure have targeting capabilities, which can specifically react to TrxR, increase the intracellular ROS level, and more effectively promote cancer cell apoptosis.

The present invention constructs nanoaggregates modified with lipoic acid (LA) containing cyclic 1,2-dithiolane units or dithiodipropionic acid (DA) containing linear disulfide units by means of the EPR effect and the mitochondrial targeting of disulfide bonds, and compares them with disulfide-free nanoaggregates CPUL1-AA NAs prepared with adipic acid (AA) without disulfide bonds. It is found that the nanoaggregates modified with disulfide bonds are more stable and controllable.

Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

(1) The drug loading rate reaches 100%. There is no other carrier material in the nanoaggregate, which avoids the problem of low drug loading rate requiring large-scale injection and the additional toxicity that may be caused by the carrier material;

(2) Nanoparticles with uniform particle size can prolong the circulation cycle in the body, improve the bioavailability of hydrophobic drugs, and easily accumulate in the tumor site through the EPR effect at the tumor site, reducing exposure to normal tissues and better exerting anti-tumor effects;

(3) It is targeted and can specifically target TrxR, inducing the production of excessive ROS, leading to mitochondrial dysfunction and triggering cell apoptosis;

(4) The preparation method of nanomedicine is simple and easy, which can save costs and is safe and pollution-free.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the hydrated particle size distribution of CPUL1-LA NAs in Example 1, (A) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:0.5; (B) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:0.8; (C) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:1; (D) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:1.5; (E) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:2; (F) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:3;

FIG2 shows the stability of CPUL1-LA NAs in Example 1, and the data are expressed as mean ± SD (n = 3);

FIG3A is a transmission electron microscopy image of CPUL1-LA NAs at a concentration of 0.05 mg/mL in Example 1;

FIG3B is a diagram showing the hydrated particle size distribution of CPUL1-LA NAs (molar ratio of 1:1) in Example 1;

FIG4 is the UV spectra of CPUL1, CPUL1-LA NAs and LA in Example 1;

FIG5 is the fluorescence spectra of CPUL1 and CPUL1-LA NAs in Example 1;

Figure 6 is the hydrated particle size distribution of CPUL1-DA NAs in Example 2, (A) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:0.5; (B) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:1; (C) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:1.5; (D) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:2; (E) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:3; (F) is the hydrated particle size distribution of the molar ratio of CPUL1 to LA of 1:4;

FIG7 is a hydrated particle size distribution diagram of CPUL1-DA NAs (molar ratio of 1:2) in Example 2;

FIG8 is a zeta potential distribution diagram of CPUL1-DA NAs (molar ratio of 1:2) in Example 2;

FIG9 shows the stability of CPUL1-DA NAs (molar ratio of 1:2) in Example 2, and the data are expressed as mean ± SD (n = 3);

FIG10 is a transmission electron microscopy image of CPUL1-DA NAs at a concentration of 0.05 mg/mL in Example 2;

FIG11 is the UV spectra of CPUL1, CPUL1-DA NAs and DA in Example 2;

FIG12 is the fluorescence spectra of CPUL1 and CPUL1-DA NAs in Example 2;

Figure 13 is a hydrogen spectrum of LA in DMSO-d6/D2O (5:1), CPUL1-LANAs in DMSO-d6/D2O (1:5), CPUL1-LA NAs in DMSO-d6/D2O (5:1), CPUL1 in DMSO-d6/D2O (5:1), and CPUL1 in DMSO-d6 (5:1) in Example 3;

Figure 14 is a hydrogen spectrum of DA in DMSO-d6/D2O (5:1), CPUL1-DANAs in DMSO-d6/D2O (1:5), CPUL1-DA NAs in DMSO-d6/D2O (5:1), CPUL1 in DMSO-d6/D2O (5:1), and CPUL1 in DMSO-d6 in Example 3;

FIG15 shows the stability of CPUL1-AA NAs in comparative examples expressed by hydrodynamic size, and the data are expressed as mean ± SD (n = 3);

FIG16 shows the size changes of CPUL1-LA NAs (100 μM) in 1 mM GSH aqueous solution at different time points in Example 4;

FIG17 shows the size change of CPUL1-LA NAs (100 μM) in response to TrxR (50 nM)/NADPH (200 μM) in Example 4;

18 is a graph showing the killing effects of samples of different concentrations (LA, DA, AA, CPUL1, CPUL1-LA NAs (molar ratio of 1:1), CPUL1-DA NAs (molar ratio of 1:2), and CPUL1-AA NAs (molar ratio of 1:2)) on HUH7 cells in Example 5;

FIG19 is a graph showing the killing effect of CPUL1-LA NAs in different ratios on HUH7 cells in Example 5;

FIG20 is a graph showing the killing effect of CPUL1-DA NAs in different ratios on HUH7 cells in Example 5;

21 is a graph showing the killing effects of samples of different concentrations (LA, DA, AA, CPUL1, CPUL1-LA NAs (molar ratio of 1:1), CPUL1-DA NAs (molar ratio of 1:2), and CPUL1-AA NAs (molar ratio of 1:2)) on L02 cells in Example 5;

FIG22A is a curve showing the change in fluorescence intensity of free CPUL1 and CPUL1-LA NAs in HUH7 cells over time in Example 6;

FIG22B is a comparison of the fluorescence intensity of free CPUL1 and CPUL1-LA NAs in HUH7 cells at the same time point using flow cytometry in Example 6;

FIG23A is a curve showing the change in fluorescence intensity of free CPUL1 and CPUL1-D NAs in HUH7 cells over time in Example 6;

FIG23B is a comparison of the fluorescence intensity of free CPUL1 and CPUL1-DANAs in HUH7 cells at the same time point using flow cytometry in Example 6;

Figure 24 shows the apoptosis ratio of HUH7 cells induced by CPUL1 and CPUL1-LA NAs at different concentrations (Q1, dead cells; Q2, late apoptotic or necrotic cells; Q3, early apoptotic cells; Q4, living cells) detected by flow cytometry in Example 7;

Figure 25 shows the apoptosis ratio of HUH7 cells induced by CPUL1 and CPUL1-LDA NAs at different concentrations (Q1, dead cells; Q2, late apoptotic or necrotic cells; Q3, early apoptotic cells; Q4, living cells) detected by flow cytometry in Example 7;

Figure 26 is a picture of the mitochondrial localization of CPUL1 and CPUL1-LA NAs in HUH7 cells at different time points detected by laser confocal microscopy in Example 8;

Figure 27 is a picture of the mitochondrial localization of CPUL1 and CPUL1-DANAs in HUH7 cells at different time points detected by laser confocal microscopy in Example 8;

Figure 28 shows the production of cellular ROS in Example 9: the fluorescence intensity of CPUL1 and CPUL1-LA NAs in HUH7 cells was analyzed by fluorescence images and flow cytometry;

Figure 29 shows the production of cellular ROS in Example 9: the fluorescence intensity of CPUL1 and CPUL1-DANAs in HUH7 cells was analyzed by fluorescence images and flow cytometry;

Detailed ways

The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

Example 1: Preparation and characterization of CPUL1-LA NAs

The nano-aggregates provided in this embodiment include CPUL1 and LA, and the preparation method is as follows:

(1) 4.34 mg of CPUL1 was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution A with a CPUL1 molar concentration of 100 mM/L; 2.06 mg of LA was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution B with a LA molar concentration of 100 mM/L;

(2) 100 μL of solution B was injected into 4.14 mL of deionized water, and magnetically stirred at 500 rpm at 25°C to obtain solution C; 100 μL of solution A was injected into solution C to obtain solution D, and magnetically stirred at 500 rpm at 25°C for 0.5 h to obtain nanoaggregates.

(3) The amount of CPUL1 was kept unchanged and the amount of LA was adjusted to 1.03 mg, 1.64 mg, 3.09 mg, 4.12 mg, and 6.18 mg to prepare nanoaggregates with other molar ratios (1:0.5, 1:0.8, 1:1.5, 1:2, and 1:3).

Dynamic light scattering (DLS) was used to study the size and size distribution of nanoaggregates with different molar ratios of CPUL1 to LA. As shown in Figure 1, nanoaggregates with molar ratios of CPUL1 to LA of 1:0.5, 1:0.8, and 1:1 showed more suitable particle sizes and more uniform size distributions than those with molar ratios of 1:1.5, 1:2, and 1:3. The surface charge of CPUL1-LA NAs was +34.15 mV. As shown in Figure 2, CPUL1-LA NAs showed good stability within 10 days at a temperature of 25°C. As shown in Figures 3A and 3B, when the molar ratio was 1:1, the hydrodynamic diameter and polydispersity index were 160 nm and 0.128, respectively, and transmission electron microscopy showed that CPUL1 and LA self-assembled into uniform spherical particles. As shown in Figure 4, the UV-Vis spectrum of CPUL1-LA NAs showed the typical absorption peak of CPUL1. As shown in Figure 5, the emission spectrum of CPUL1-LA NAs is similar to that of CPUL1 in DMSO, and the maximum emission peak is slightly red-shifted to 552 nm. However, at the same concentration, the fluorescence intensity of CPUL1-LA NAs in water is much lower than that in DMSO.

Example 2: Preparation and characterization of CPUL1-DA NAs

The nano-aggregates provided in this embodiment include CPUL1 and DA, and the preparation method is as follows:

(1) 4.34 mg of CPUL1 was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution A with a CPUL1 molar concentration of 100 mM/L; 2.10 mg of DA was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution B with a DA molar concentration of 100 mM/L;

(2) 100 μL of solution B was injected into 4.14 mL of deionized water, and magnetically stirred at 500 rpm at 25°C to obtain solution C; 100 μL of solution A was injected into solution C to obtain solution D, and magnetically stirred at 500 rpm at 25°C for 0.5 h to obtain nanoaggregates.

(3) Keeping the amount of CPUL1 unchanged, nanoaggregates with other molar ratios (1:0.5, 1:1.5, 1:2, 1:3, 1:4) were prepared by adjusting the amount of DA to 1.05 mg, 1.68 mg, 3.15 mg, 4.20 mg, 6.30 mg, and 8.40 mg.

As shown in Figure 6, all nanoaggregates showed suitable particle size and relatively uniform size distribution. As shown in Figures 7-12, when the molar ratio was 1:2, the hydrodynamic diameter was 97nm and the polydispersity index was 0.102. The surface charge of CPUL1-DA NAs was +25.65mV, and CPUL1-DA NAs showed good stability within 10 days at a temperature of 25°C. The UV-visible spectrum and fluorescence spectrum of CPUL1-DA NAs showed the typical absorption peaks and emission peaks of CPUL1.

Example 3: NMR Characterization of CPUL1-LA NAs and CPUL1-DA NAs

The structures of CPUL1-LA NAs and CPUL1-DA NAs were characterized by H NMR spectroscopy. As shown in Figures 13 and 14, in a DMSO-d ₆ /D ₂ O (1:5) mixed solvent, the H NMR spectra of CPUL1-LA NAs and CPUL1-DA NAs only showed LA and DA peaks. In a DMSO-d ₆ /D ₂ O (5:1) mixed solvent, with the increase of DMSO-d ₆ content, the signal peak of CPUL1 gradually appeared, indicating that the nanoaggregates of CPUL1 were destroyed and the CPUL1 molecules were released. Due to the presence of D ₂ O in the system, the signal peaks 1' and 2' of the CPUL1 amino group exchanged with D ₂ O and disappeared completely. These results confirmed that the CPUL1 molecules were indeed tightly wrapped inside the nanoparticles.

Comparative Example: Preparation and Characterization of CPUL1-AA NAs

The nano-aggregates provided in this comparative example include CPUL1 and AA (the molar ratio is CPUL1:AA=1:2), and the preparation method is as follows:

(1) 4.34 mg of CPUL1 was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution A with a CPUL1 molar concentration of 100 mM/L; 2.92 mg of AA was dissolved in 100 μL of dimethyl sulfoxide to obtain a solution B with an AA molar concentration of 200 mM/L;

(2) 100 μL of solution B was injected into 4.14 mL of deionized water, and magnetically stirred at 500 rpm at 25°C to obtain solution C; 100 μL of solution A was injected into solution C to obtain solution D, and magnetically stirred at 500 rpm at 25°C for 0.5 h to obtain nanoaggregates.

As shown in Figure 15, although CPUL1-AA NAs can form uniform spherical particles with a diameter of about 100 nm, aggregation and precipitation occurred after 48 h, indicating that the system is unstable. The above results show that the disulfide bond unit is the key to constructing stable and controllable nanoaggregates.

Example 4: Interaction of CPUL1-LA NAs and CPUL1-DA NAs with TrxR and GSH

The present invention mainly explores the selective response of nanoaggregates to GSH and TrxR from two aspects. The first aspect is the change in particle size, and the second aspect is the absorbance test at 340nm. First, DLS was used to monitor the particle size changes of CPUL1-LA NAs, CPUL1-DANAs and CPUL1-AA NAs in a simulated tumor cytoplasmic environment (10mM GSH); secondly, the absorbance at 340nm of CPUL1-LA NAs, CPUL1-DA NAs and CPUL1-AA NAs when they acted with TrxR and GSH respectively was measured by ultraviolet spectrometer. The corresponding compounds (100mM) and GSH (1Mm)/GR (0.5U/mL) or TrxR (50nM)/NADPH (200mM) were dissolved in sodium phosphate buffer, and the absorbance change at 340nm was measured at 37°C for 30 consecutive minutes. Each group was tested three times in parallel, and then the decay rates of _A340 of CPUL1-LA NAs, CPUL1-DA NAs and CPUL1-AA NAs to GSH and TrxR in the first 30 min were calculated respectively.

As shown in Figures 16 and 17, the interaction of CPUL1-LA NAs with TrxR and GSH was studied by DLS. The particle size of nanoaggregates incubated with GSH did not change significantly within 24 h, while CPUL1 was released in the presence of TrxR/NADPH, and the nanoaggregates began to collapse and swell from 3 h, with the release amount exceeding 1 μM. It was further investigated whether CPUL1-LA NAs, CPUL1-DA NAs, and CPUL1-AA NAs could selectively reduce TrxR. As shown in Table 1, the absorbance change at 340 nm caused by NADPH consumption was measured, and the decay rate within the first 30 min of the reaction was calculated. In the GSH system, the background decay rate of A ₃₄₀ was 1.12×10 ^-3 min ^-1 , while the oxidation rate of NADPH did not change significantly with the addition of nanoaggregates, indicating that GSH could not reduce the nanoaggregates. When CPUL1-LA NAs, CPUL1-DANAs, and CPUL1-AA NAs were added to the mixed system of TrxR and NADPH, the oxidation rates of NADPH were 17.3×10 ^-3 min ^-1 , 8.3×10 ^-3 min ^-1 , and 6.6×10 ^-3 min ^-1 , respectively, which were 34, 16, and 13 times the background attenuation value of 0.54×10 ^-3 min ^-1 , respectively. It is worth noting that the effectiveness of CPUL1-AANAs is mainly attributed to the inherent TrxR inhibitory ability of CPUL1. Therefore, these results indicate that the disulfide bond units in the present nanoaggregates can react specifically with TrxR and have a synergistic effect with the TrxR inhibitor CPUL1.

Table 1: Consumption rate of NADPH by different nanoaggregates

Example 5: Cytotoxicity evaluation of CPUL1-LA NAs and CPUL1-DA NAs

In this example, the killing effects of CPUL1-LA NAs, CPUL1-DA NAs and CPUL1-AA NAs as well as LA, DA and AA on HUH7 liver cancer cells and normal L02 cells were detected by tetramethylrhodamine colorimetry, and the method is as follows:

(1) Cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum, and L02 cells were cultured in RPMI-1640 containing 10% fetal bovine serum in an incubator at 37° C. and 5% carbon dioxide.

(2) Cell viability assay: The cytotoxic effects of LA, DA, AA, CPUL1, CPUL1-LA NAs (molar ratio of 1:1), CPUL1-DA NAs (molar ratio of 1:2), and CPUL1-DA NAs (molar ratio of 1:2) on HUH7 and L02 cells were detected by MTT colorimetric assay. HUH7 and L02 cells were seeded in 96-well plates at 6000 cells/well. After incubation for 12 h, 10 μL of LA, DA, AA, CPUL1, CPUL1-LA NAs (molar ratio of 1:1), CPUL1-DA NAs (molar ratio of 1:2), and CPUL1-AA NAs (molar ratio of 1:2) samples of different concentrations (2.5, 5, 10, 20, 40 μM) were added to the two cells, respectively. After 48 h of culture, 10 μL of MTT (5.0 mg/mL) was added. After incubation for 4 hours, remove the culture medium and add 150 μL of dimethyl sulfoxide to dissolve the purple crystals. Use a multifunctional microplate reader to measure the absorbance of the solution at 490 nm. Cells cultured without any treatment were used as controls. Three parallel wells were set for each group.

As shown in Figure 18, all monomers have almost no cytotoxicity. The IC 50 values of CPUL1-LA NAs (molar ratio of 1:1), CPUL1-DA NAs (molar ratio of 1:2), and CPUL1-AA NAs ( _molar ratio of 1:2) are 4.78±0.23μM, 4.30±0.25μM, and 10.11±0.35μM, respectively. Except for CPUL1-AA NAs, the cytotoxicity of the other two nanoaggregates is about twice that of free CPUL1 (IC ₅₀ =9.30±0.66μM), which may be attributed to the mitochondrial targeting ability of the nanoaggregates and the specific selection of TrxR response, indicating that the self-assembled nanoaggregates prepared by this method can achieve better tumor inhibition effects at lower drug concentrations. In addition, as shown in Figures 19 and 20, the cytotoxicity of CPUL1-LA NAs and CPUL1-DA NAs at different molar ratios was also tested, but there was no significant difference in the IC ₅₀ values. It is noteworthy that, as shown in Figure 21, all nanoaggregates showed very weak cytotoxicity to normal L02 cells, which means that they have great biosafety potential.

Example 6: Tumor Cell Uptake of CPUL1-LA NAs and CPUL1-DA NAs

In this example, the endocytosis of CPUL1-LA NAs and CPUL1-DA NAs by HUH7 cells was observed by flow cytometry, and the method was as follows:

The cultured cells were inoculated in a 12-well plate, with 50,000 cells per well, and 1 mL of DMEM liquid culture medium (containing 10% fetal bovine serum) was added. After shaking, the cells were placed in a 37°C, 5% carbon dioxide incubator for 24 hours to allow them to adhere completely. HUH7 cells were treated with CPUL1 and the provided nanoaggregates for 1 hour, 2 hours, 4 hours and 6 hours, respectively, where the drug concentrations of CPUL1 were 2.5 μM, 5 μM and 10 μM, respectively. After 4 hours, the cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde, and then washed three times with PBS, and then the nuclei were stained with DAPI. Cell fluorescence images were obtained under a fluorescence microscope. Since CPUL1 itself has green fluorescence, when Ex = 490nm, the drug molecules can be directly imaged in the cells without adding other dyes.

In the flow cytometer test, HUH7 cells with good growth status were selected, inoculated into 12-well plates (300,000 cells per well), placed in a 37°C, 5% carbon dioxide incubator for 24 hours to make them completely adhere to the wall, and 2.5μM CPUL1, CPUL1-LA NAs and CPUL1-DA NAs sample solutions were added and incubated for 1h, 2h, 4h and 6h, respectively. The culture medium of each well was removed, and each well was washed once with PBS. 100μL TP was used to digest the cells to cause cell detachment, and TP was neutralized with culture medium. The obtained cell suspension was centrifuged at 400g for 5min, and the obtained cell pellet was washed twice with PBS before being tested on the machine.

As shown in Figure 22A, the enhanced fluorescence signal shows that the nanoaggregates are quickly localized in the cytoplasm after incubation for 1 hour, and emit stronger green fluorescence after incubation for 2 hours, 4 hours and 6 hours under the same conditions. The fluorescence intensity after CPUL1-LA NAs treatment gradually increases with time. As shown in Figure 22B, at the same time point, cells treated with CPUL1-LA NAs showed a greater fluorescence peak shift than cells treated with CPUL1. Also, as shown in Figures 23A and 23B, the results of CPUL1-DA NAs are similar to those of CPUL1-LANAs. Therefore, these results show that compared with the same concentration of CPUL1, the nanoaggregates provided by the present invention can be taken up more by HUH7 cells, which may benefit from the positive charge on the surface of the nanoaggregates. The fluorescence intensity is proportional to the number of single drug molecules in the cell and is therefore directly related to their cytotoxicity.

Example 7: CPUL1-LA NAs and CPUL1-DA NAs induce apoptosis of tumor cells

In this example, the Annexin-V-APC/7AAD apoptosis detection kit was used to detect the killing effect of CPUL1, CPUL1-LA NAs and CPUL1-DA NAs on HUH7 cells, and the method is as follows:

(1) Cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37° C. and 5% carbon dioxide.

(2) Observation of cell activity: HUH7 cells in good condition in step (1) were selected and inoculated into 12-well plates at a cell density of 1.5×10 ⁵ cells/well. The plates were placed in a 5% CO ₂ incubator and cultured at 37°C for 24 h to allow the cells to completely adhere to the wall. Sample solutions of CPUL1, CPUL1-LA NAs and CPUL1-DA NAs at concentrations of 2.5 μM, 5 μM and 10 μM were added for treatment. After incubation for another 24 h, the old culture medium was carefully discarded, the cells were washed twice with preheated PBS, and the cells were digested with 100 μL of trypsin (TP). The obtained cell suspension was centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended with PBS and the centrifugation step was repeated. The cell pellet obtained in each centrifuge tube was added with 500 μL Bing Buffer to suspend the cells, and 5 μL Annexin-V-APC and 5 μL 7-AAD dye solutions were added respectively and mixed. Incubate in the dark for 15 min. The BD AccuriC6 flow cytometer was used for on-machine detection within 1 hour. Annexin-V-APC (Ex = 633nm, Em = 660nm) was detected using the FL4 channel, and 7-AAD (Ex = 546pm, Em = 647nm) was detected using the FL3 channel. During the detection process, cells treated with apoptosis were used for fluorescence compensation adjustment to eliminate experimental errors caused by spectral overlap. Ten thousand cells were collected and divided into zones, and the percentage of cells in each zone was counted to analyze the mode of cell death.

The results showed that HUH7 cells treated with CPUL1-LA NAs had a higher apoptosis rate compared to cells treated with free CPUL1 at the same concentration. As shown in Figure 24, after treating cells with CPUL1-LA NAs (10 μM), 44.2% of the cells were in the apoptotic phase. More precisely, 12.7% of the cells were in the early apoptotic phase, 31.5% of the cells were in the late apoptotic phase, while only 24.9% of the cells treated with free CPUL1 were in the apoptotic phase. When the concentrations were 2.5 and 5 μM, the results were consistent with those at 10 μM. In addition, the apoptosis rate of HUH7 cells induced by CPUL1-DA NAs was detected by flow cytometry, and the results were similar to those of CPUL1-LA NAs, as shown in Figure 25.

Example 8: Mitochondrial targeting of CPUL1-LA NAs and CPUL1-DA NAs

In this example, the targeting effect of CPUL1, CPUL1-LA NAs and CPUL1-DA NAs on HUH7 cell mitochondria was tested as follows:

(1) Cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37° C. and 5% carbon dioxide.

(2) Observation of nanoaggregates targeting mitochondria: HUH7 cells in good condition in step (1) were selected and inoculated into a 24-well plate covered with a coverslip at a cell density of 1×10 ⁵ cells/well. The cells were placed in a 5% CO ₂ incubator and cultured at 37°C for 24 h to allow the cells to fully adhere to the wall. The cells were treated with sample solutions of CPUL1, CPUL1-LA NAs and CPUL1-DA NAs at a concentration of 2.5 μM and incubated at 37°C for 2 h and 4 h. After the addition and incubation, the culture medium was discarded, the cells were washed twice with PBS buffer solution, and the mitochondrial red fluorescent probe MitoTracker Red (100 nM) was added to stain the HUH7 cells in the dark for 30 min. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min, and finally washed twice with PBS buffer solution to remove excess PFA. The coverslip was removed and placed on a slide with an antifade agent. The sealing solution was dripped around the coverslip, and the localization of mitochondria in the cells was detected using a laser confocal microscope (CLSM). The green fluorescence of CPUL1 overlaps with the red fluorescence of the dye to produce yellow fluorescence. The degree of mitochondrial colocalization is expressed by the Pearson correlation coefficient.

As shown in Figure 26, in HUH7 cells, CPUL1-LA NAs and CPUL1 can co-localize with the dye in mitochondria after incubation for 2h or 4h, and the Pearson correlation coefficient (PCC) value of cells incubated with nano-aggregates for 2h or 4h is higher than that of CPUL1. Similarly, the results of CPUL1-DA NAs are similar to those of CPUL1-LA NAs, as shown in Figure 27. In addition, the co-localization fluorescence intensity of the nano-aggregates increases significantly over time. These experimental results show that the nano-aggregates provided by the present invention have good mitochondrial targeting and rapid time-dependent uptake characteristics.

Example 9: CPUL1-LA NAs and CPUL1-DA NAs induce mitochondrial damage

In this example, the damage effect of the nano-aggregates of the present invention on mitochondria was determined by testing the level of ROS increase in CPUL1, CPUL1-LA NAs and CPUL1-DA NAs in HUH7 cells, and the HUH7 cells were stained with the commercial dye MitoSOX Red. MitoSOX red mitochondrial superoxide indicator is a new dye for highly selective detection of superoxide in mitochondria of living cells. The method is as follows:

(1) Cell culture: HUH7 cells were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37° C. and 5% carbon dioxide.

(2) Observation of intracellular ROS levels: HUH7 cells in good condition in step (1) were selected and inoculated into a 24-well plate covered with a coverslip at a cell density of 5×10 ⁴ cells/well. The cells were placed in a 5% CO ₂ incubator and cultured at 37°C for 24 hours to allow the cells to fully adhere to the wall. The cells were treated with sample solutions of CPUL1, CPUL1-LA NAs and CPUL1-DA NAs at concentrations of 2.5 μM, 5 μM and 10 μM. After further incubation for 12 hours, the old culture medium was carefully discarded, and the cells were washed twice with preheated PBS. 1 mL of staining solution MitoSOX Red (5 μM) was used for staining at 37°C in the dark for 15 minutes. The staining solution was aspirated and washed twice with PBS. The coverslip was removed, placed on a slide with an antifade agent, and sealed. The generation of intracellular ROS was observed under an inverted fluorescence microscope and the results were recorded and photographed.

Fluorescence was quantitatively analyzed by flow cytometry. HUH7 cells in good condition in step (1) were selected and inoculated into 12-well plates (2.5×10 ⁵ cells/well). They were placed in a 5% CO ₂ incubator and cultured at 37°C for 24 hours to allow the cells to fully adhere to the wall. CPUL1, CPUL1-LA NAs and CPUL1-DA NAs were co-incubated with 2.5 μM, 5 μM and 10 μM concentrations for 24 hours. After dye treatment for 30 minutes, the cells were digested and centrifuged and collected, and immediately tested on the machine. The interference of the compound's own fluorescence was eliminated by adjusting the compensation.

As shown in Figure 28, CPUL1-LA NAs induced the generation of ROS in a dose-dependent manner. The mitochondria of cells treated with CPUL1-LA NAs showed stronger red fluorescence, and MitoSOX Red was oxidized by superoxide. Compared with free CPUL1, the superoxide content in mitochondria increased significantly after cells were incubated with the same concentration of CPUL1-LA NAs for 24 hours, resulting in superoxide accumulation and mitochondrial membrane damage. Similarly, the results of CPUL1-DA NAs were similar to those of CPUL1-LA NAs, as shown in Figure 29.

In summary, the CPUL1-LA NAs and CPUL1-DA NAs nanoaggregates of the present invention have high drug content, good aqueous solution stability and great biosafety potential. It is noteworthy that the cyclic disulfide of LA and the linear disulfide of DA endow the nanoaggregates with targeting ability, reacting specifically to TrxR through GSH. In addition, both CPUL1-LANAs and CPUL1-DA NAs exhibit rapid cellular uptake properties, can be internalized by cancer cells, and can produce more abundant ROS than free CPUL1 to induce cell apoptosis, thereby significantly improving the anti-tumor effect on HUH7 cells. The present invention will provide new opportunities for personalized treatment and diagnosis of disulfide-modified self-releasing drugs and cell imaging systems.

Claims (9)

1. A disulfide bond-modified nanoaggregate, characterized in that the nanoaggregate comprises CPUL1 and a compound containing a disulfide bond, and the nanoaggregate is modified by CPUL1 and the compound containing a disulfide bond through non-covalent interaction; the preparation method of the nanoaggregate comprises the following steps: (1) CPUL1 and a compound containing a disulfide bond are respectively dissolved in an organic solvent to obtain a CPUL1 solution and a compound containing a disulfide bond solution; (2) Add water to the disulfide bond-containing compound solution, stir for the first time to obtain a mixed solution, add the CPUL1 solution in step (1) to the mixed solution, stir again to obtain disulfide bond-modified nanoaggregates. 2. The disulfide bond-modified nanoaggregate according to claim 1, wherein the disulfide bond-containing compound is lipoic acid or dithiodipropionic acid. 3. The disulfide bond-modified nanoaggregate according to claim 1, wherein the particle size of the nanoaggregate is 90-700 nm. 4. A method for preparing a disulfide bond-modified nanoaggregate according to any one of claims 1 to 3, characterized in that it comprises the following steps: (1) CPUL1 and a compound containing a disulfide bond are respectively dissolved in an organic solvent to obtain a CPUL1 solution and a compound containing a disulfide bond solution; (2) Add water to the disulfide bond-containing compound solution, stir for the first time to obtain a mixed solution, add the CPUL1 solution in step (1) to the mixed solution, stir again to obtain disulfide bond-modified nanoaggregates. 5. The preparation method according to claim 4, characterized in that in step (1), the molar concentration ratio of the CPUL1 solution to the disulfide bond compound solution is 1:0.5-1:4. 6. The preparation method according to claim 4, characterized in that in step (1), the organic solvent is any one of ethyl acetate, acetone, methanol, dichloromethane, chloroform, ethyl propionate, propyl acetate, dimethyl sulfoxide or ethanol. 7. The preparation method according to claim 4, characterized in that in step (2), the initial stirring and/or the secondary stirring is magnetic stirring. 8. The preparation method according to claim 4, characterized in that in step (2), the initial stirring and/or secondary stirring temperature is 20-60°C, the stirring speed is 50-1000 rpm, and the stirring time is 0.5-3h. 9 . The preparation method according to claim 4 , characterized in that in step (2), the molar concentration of CPUL1 and/or the compound containing a disulfide bond in the nanoaggregate is 0.16-16 mM/L.