Preparation method of small-size water-phase liquid metal nanomaterial

Abstract

The application relates to a preparation method of small-size water-phase liquid metal nanomaterials, which comprises the following steps: S1, preparing a mixed solution containing liquid metal and DOX.HCl and performing ultrasonic treatment; S2, after the ultrasonic treatment, centrifuging the mixture, removing the precipitate and obtaining small-size water-phase liquid metal nanomaterials; the liquid metal is gallium or a gallium-based alloy. Through a simple sonochemical method, the application realizes the simple synthesis of 20nm water-phase Ga-based LMNDs by using degradable ligands for the first time, does not need complicated synthesis / purification processes or specific storage conditions, and the ligands can stabilize the newly-formed small-size LMNDs. The LMNDs can selectively trigger breast cancer cell apoptosis and anti-angiogenesis in vitro and in vivo, can effectively inhibit the growth of cancer cells, and do not have obvious cytotoxicity to normal cells.

Description

Technical Field

[0001] This application belongs to the field of liquid metal technology, and particularly relates to a method for preparing small-sized aqueous liquid metal nanomaterials. Background Technology

[0002] To optimize the treatment of cancer, researchers have been striving to develop drugs with better pharmacological properties, enhanced selectivity, and fewer side effects. Typically, many drug design theories rely on the protection of the active agent (e.g., drug carriers, structural modifications), followed by selective release of the drug through physiological parameter selection (e.g., pH, redox environment, hypoxia, and enzymes) or external energy input (ultrasound, photo / electrochemical).

[0003] Prodrug strategies have greatly advanced the selective treatment of tumors, but due to problems such as the need for precise screening, complicated synthesis and purification processes, non-degradable inorganic components, low biocompatibility, and unsatisfactory reactivity, the therapeutic effects of prodrugs are full of uncertainty.

[0004] Nanomedicine has further advanced selective tumor therapy. Among them, liquid metal nanodroplets (LMNDs), as an emerging drug delivery system, have attracted widespread interest in various biomedical applications. LMNDs possess excellent biocompatibility, deformability, elasticity, and no cross-resistance, enhancing their ability to cross blood vessels and penetrate tumor parenchyma, and providing additional small capillary transport pathways. This novel drug delivery technology can improve the effectiveness of delivered drugs, helping doctors to locate tumors, and theoretically exhibits extremely low biotoxicity. Furthermore, Ga-based LMNDs do not exhibit significant cytotoxicity in normal cells. In the tumor microenvironment (TME), they undergo a series of degradation processes, becoming free anticancer Ga ions. This selective carrier-drug conversion triggered by endogenous biostimulation can address issues such as low drug loading rates, explosive drug release, multi-system toxicity, and carrier immune responses. Despite these unique advantages, the current application of LMNDs in tumor therapy is limited to passive drug carriers or mediators. In the rational design and construction of liquid metal-based nanoplatforms for chemotherapy, limitations such as low drug loading efficiency, poor aqueous phase stability, weak surface ligand modifiability, and difficulty in morphology control result in insignificant therapeutic effects. Furthermore, it is difficult to design bulk LMs into small-sized aqueous LMNDs (<40 nm, which is the threshold size for complete removal from the liver and spleen). Small-sized LMNDs are superior to large-sized LMNDs in overcoming diffusion resistance caused by tumor cell density, blood vessels, and tumor extracellular matrix (ECM). Based on previous research reports, adjusting ligand structure (functional groups, surface potential, molecular weight, size, and topology) and optimizing ultrasound parameters (ligand / LM feed ratio, ligand concentration, ultrasound time, and temperature) cannot significantly reduce the overall size of aqueous LMNDs to below 100 nm. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of existing liquid metal nanodroplets (LMNDs), such as low drug loading efficiency, poor aqueous phase stability, weak surface ligand modifiability, difficulty in morphology control, and difficulty in designing small-sized aqueous LMNDs, thereby providing a method for preparing small-sized aqueous liquid metal nanomaterials.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A method for preparing small-sized aqueous liquid metal nanomaterials includes the following steps:

[0008] S1: Prepare a mixed solution containing liquid metal and DOX·HCl, and then subject it to ultrasonic treatment;

[0009] S2: After ultrasonic treatment, the mixture is centrifuged to remove the precipitate and obtain small-sized aqueous liquid metal nanomaterials;

[0010] The liquid metal is gallium or a gallium-based alloy.

[0011] Preferably, liquid metal and DOX·HCl are mixed in deionized water to obtain a mixed solution containing liquid metal and DOX·HCl.

[0012] Preferably, the liquid metal gallium-based alloy is a gallium-indium eutectic alloy EGaIn or a gallium-indium-tin alloy GaInSn.

[0013] Preferably, the average particle size of the small-sized aqueous liquid metal nanomaterial is 20 nm.

[0014] Preferably, the ultrasonic treatment is performed in an ice bath.

[0015] Preferably, the ultrasonic treatment time is 12 minutes or more.

[0016] Preferably, the mass-to-volume ratio of DOX·HCl and liquid metal in step S1 is not higher than 0.06 g / mL.

[0017] Preferably, the volume ratio of the liquid metal to deionized water in step S1 is 0.6%-1%.

[0018] Preferably, the centrifugation conditions are 800-1200 rpm and the centrifugation time is 3-8 min.

[0019] The beneficial effects of this invention are:

[0020] (1) This invention achieves the simple synthesis of 20nm aqueous Ga-based LMNDs for the first time using a simple sonochemical method and a degradable ligand, without the need for a complicated synthesis / purification process or specific storage conditions. At the same time, the ligand can stabilize the newly formed small-sized LMNDs.

[0021] (2) The LMND nanomedicine prepared in this invention can enhance tumor penetration without introducing additional adjuvants or drugs, and obtain a dual complementary anticancer pathway through the electrodisplacement reaction (GRR) in the tumor microenvironment, that is, simultaneously consuming Cu in an irreversible manner. 2+ Metal ions such as ions and the release of Ga 3+ The ions, combined with the large amount of reactive oxygen species (ROS) generated in situ, selectively trigger apoptosis and anti-angiogenesis in breast cancer cells both in vitro and in vivo, effectively inhibiting cancer cell growth without cytotoxicity to normal cells. Compared with preclinical / clinical anticancer drugs tetrathiomolybdate (TM) and gallium nitrate Ga(NO3)3, the Ga-based LMND prepared in this invention significantly improves the therapeutic effect and survival rate of BCap-37 xenograft mouse models without significant side effects. LMND20 not only has a lower IC50 value (30.4 μg / mL) against BCap-37 breast cancer cells than TM (91.1 μg / mL) through apoptosis and anti-angiogenesis, but also enhances the tumor-suppressive effect in BCap-37 tumor-bearing mice. This is the first reported Ga-based LMND nanomedicine with intrinsic antitumor activity, and this GRR strategy complements existing cancer treatment modalities. Attached Figure Description

[0022] The technical solution of this application will be further described below with reference to the accompanying drawings and embodiments.

[0023] Figure 1 a and 1b are high-resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM) images of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 of this application;

[0024] Figure 2 Figure a shows the statistical analysis results of the size of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 of this application, based on field emission transmission electron microscopy (FE-TEM). Figure 2 b is a graph showing the analytical results of ζ and PDI of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 of this application based on DLS method;

[0025] Figure 3 This is the small-angle XRD pattern of the small-sized aqueous liquid metal nanomaterial prepared in Example 1 of this application;

[0026] Figure 4a is the FTIR spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 1 of this application and DOX. Figure 4 b is the Ga 3d high-resolution XPS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in the embodiments of this application;

[0027] Figure 5 This is the O1s high-resolution XPS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 1 of this application;

[0028] Figure 6 This is a chromatogram of the solution before and after ultrasound analysis in Example 1 of this application;

[0029] Figure 7 a-7k is the positive mode LC-MS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 1 of this application;

[0030] Figure 8 a-8d is the negative mode LC-MS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 1 of this application;

[0031] Figure 9 The absorbance and fluorescence spectra of LMND synthesized by different EGaIn / DOX·HCl feed ratios are shown.

[0032] Figure 10 This is a comparison chart of the size, surface zeta potential, and polydispersity index (PDI) of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 and Comparative Examples 1-3 of this application;

[0033] Figure 11 This is a comparison chart of the size, surface zeta potential, and polydispersity index (PDI) of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 and Comparative Examples 4-7 of this application;

[0034] Figure 12 This is an analysis chart of the particle size and PDI of the small-sized aqueous liquid metal nanomaterials prepared in Example 1 of this application after being stored in deionized water for 1 month;

[0035] Figure 13 a is a dynamic microPET imaging (0-60 min) and quantitative ROI analysis of LMND20 biodistribution statistics (n=4) of small animals; Figure 13 b and 13c show the plasma half-life (T1 / 2) of LMND20 calculated using microPET and ICP-MS methods, respectively (n=4).

[0036] Figure 14This is an analysis of fluorescence signals captured by IVIS imaging after injecting Cy5.5-labeled LMND20 (10 mg / kg) into BALB / c nude mice (n=5) carrying BCap-37.

[0037] Figure 15 Figure a is a comparative analysis of serum biochemical indicators (ALT, AST, BUN, CRE, LDH, CK, and CK-MB) related to liver and kidney function in the LMND20 treatment group and the control group (normal saline). Figure 15 b shows sections of various organs (heart, liver, spleen, lung, and kidney) from BCap-37 tumor-bearing BALB / c nude mice stained with hematoxylin and eosin as the main control and LMND20 treated.

[0038] Figure 16 For LMNDs and Cu 2+ Sunlight images of GRR mixtures with different LMNDs / CuCl2 feed ratios and reaction times;

[0039] Figure 17 a is the UV-Vis spectrum of LMND20 (3 mg / mL) aqueous solution after incubation with different concentrations of CuCl2 (0.8, 1.6, 3.2, 6.4, 8, 16 mM) for 30 min; Figure 17 b is the dynamic UV-Vis spectrum after incubation of LMND20 aqueous solution with 8mM CuCl2; Figure 17 c represents an aqueous solution of LMND20 and 8 mM of other metal ions (Ca). 2+ Mg 2+ Zn 2+ Fe 3+ The UV-Vis spectrum after 6 hours of incubation;

[0040] Figure 18 a represents the FTIR spectra of LMND20 and LMND20 nanodroplets (NDs) after incubation with CuCl2 (8mM) for 6 hours; Figure 18 b shows the high-resolution Ga 3d XPS spectra of LMND20 and LMND20 nanodroplets (NDs) after incubation with CuCl2 (8mM) for 6 h; Figure 18 c represents the high-resolution Cu2p XPS spectra of LMND20 and ND after incubation with CuCl2 (8mM) for 6 hours;

[0041] Figure 19 Cu-LMM Auger XPS spectra of CuCl2 before and after GRR reaction with LMND20;

[0042] Figure 20 a is the BCS detection of Cu in the substitution reaction between LMND20 and CuCl2 (8mM). + Absorbance spectra of the corresponding substances; Figure 20 b shows the XRD patterns of LMND20 and LMND20 nanodroplets (NDs) after incubation with CuCl2 (8mM) for 6 hours;

[0043] Figure 21 HRTEM and SAED spectra of NDs prepared with different LMND / CuCl2 feed ratios and reaction times, with a scale bar of 5 nm inserted;

[0044] Figure 22 a and 22b are STEM images and EDS spectra of ND obtained after incubating 1.5 mg and 3.0 mg LMND20 with 8 mM CuCl2 for 12 h, respectively. Scale bar. Figure 22 a is 90nm, Figure 22 b is 500nm.

[0045] Figure 23 The permeation of LMND20 prepared in Example 1 in vitro and in vivo. Figure 23 a, Z-stack CLSM images of BCap-37 tumor spheres incubated with LMND20 and LMND100, LMNDs pre-labeled with FITC, images acquired at 10 μm intervals from the equatorial plane of the top of the tumor sphere, scale bar, 250 μm; Figure 23 fluorescence intensity of BCap-37 tumor spheroids in cross-section of LMNDs, scale bar, 250 μm; Figure 23 Three-dimensional fluorescence intensity maps of f, g, and LMNDs; Figure 23 h, i, LMND20, LM / S, LMND100 and saline-treated BCap-37 cells were measured and analyzed by flow cytometry. Scale bar, 200 μm. Figure 23 TEM images of LMNDs distribution in BCap-37 tumors of BCap-37-bearing BALB / c nude mice at the same concentration of 5 mg / kg. -1 Mice were treated with LMND20 and LMND100 at multiple sites within the tumor. The mice died 48 hours after injection. Scale bar, 2 μm. Figure 23 k, CLSM images of LMNDs distributed in BCap-37 tumors excised from BCap-37-bearing BALB / c nude mice, LMNDs pre-labeled with FITC, and blood vessels and cell nuclei stained with CD31 and DAPI, respectively, in 10 μm frozen sections, scale bar, 100 μm;

[0046] Figure 24Cytotoxicity of a-24c, LMND20, LM / S, TM and Ga(NO3)3 to different cancer cell lines (BCap-37, MDA-MB-231, MCF-7); Figure 24 d represents the cytotoxicity of LMND20 against different cancer cell lines (SKOV-3 and PC-9 cells) and normal cells (293T cells); all data are expressed as mean ± sd, n = 4; Figure 24 e. Flow cytometry analysis of the apoptotic effects of LMND20, LM / S, TM, and Ga(NO3)3 at the same concentration of 50 μg / mL for 24 h on BCap-37 cells; Q4, live cells; Q3, early apoptotic cells; Q2, late apoptotic cells; Q1, necrotic cells. The percentage of cells in each region is expressed as a specific number. Figure 24 f. Histogram of BCap-37 cell cycle distribution after treatment with LMND20, LM / S, TM, and Ga(NO3)3 at the same concentration of 50 μg / mL for 24 h. Figure 24 g, Western blot was used to detect the expression level of caspase-3 in BCap-37 cells induced by the same concentration of 50 μg / mL LMND20, LM / S, TM and Ga(NO3)3 for 24 h. Cells treated with physiological saline were used as controls and actin was used as the internal reference protein. Figure 24 hj, free Cu 2+ The effects of adding LMND20 (0.5mM, 1mM) on the cytotoxicity of BCap-37, MDA-MB-231, and MCF-7 cells were shown. All data are expressed as mean ± sd, n = 4.

[0047] Figure 25 a, 1×10 6 The diagram shows the treatment method of subcutaneously inoculating 10 BCap-37 cells into the buttocks of BALB / c mice starting from day 7 (D0); Figure 25 b-25e, Comparison of the effects of different treatments (LMND20 (5mg / kg), TM (5mg / kg), Ga(NO3)3 (40mg / kg) and physiological saline) on tumor volume, body weight, survival rate and tumor weight in mice; Figure 25 f, Bright-field images of tumors removed from mice after sacrifice following different treatments (LMND20 (5 mg / kg), TM (5 mg / kg), Ga(NO3)3 (40 mg / kg) and saline); Figure 25 g, Distribution profile of differentially expressed genes (DEGs) in BCap-37 tumor tissues treated with different saline, TM, Ga(NO3)3 and LMND20; Figure 25h, RNA-seq hierarchical clustering diagram of DEG (differentially expressed genes) in BCap-37 tumor tissues treated with different saline, TM, Ga(NO3)3 and LMND20; Figure 25 i. Bubble diagram of DEGs significantly enriched pathways obtained by GO analysis (P<0.05); Figure 25 j. Representative IHC staining and TUNEL assay images of BCap-37 tumor sections after mouse sacrifice, scale bar, 50 μm; Figure 25 k, statistical analysis of immunohistochemical parameters (Ki-67, caspase-3, VEGF, CD31) and average fluorescence intensity by TUNEL assay, n=3, all data are expressed as mean±sd;

[0048] Figure 26 a-26j is the positive mode LC-MS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 2 of this application;

[0049] Figure 27 a-27d is the negative mode LC-MS spectrum of the small-sized aqueous liquid metal nanomaterial prepared in Example 2 of this application;

[0050] Figure 28 a-28d is the high-resolution XPS spectrum of C-1s, O-1s, N-1s and Ga-3d of the small-sized aqueous liquid metal nanomaterials prepared in Example 2 of this application. Detailed Implementation

[0051] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0052] The technical solution of this application will now be described in detail with reference to the accompanying drawings and embodiments.

[0053] The experimental reagents used in the following examples are as follows: gallium indium eutectic (EGaIn, Alfa Aesar, 99.99%, Ga:In; 75.5:24.5wt%), gallium indium tin eutectic (Alfa Aesar, 99.99%, Ga:In:Sn; 62:22:16wt%), doxorubicin hydrochloride (Ark Pharmacies, >98%), ammonium tetrathiomolybdate (Acros, 99.95%, metal grade), gallium nitrate (Acros, 99.9998%, metal grade), copper chloride dihydrate (Acros, 99.999%, metal grade), disodium sulfonate hydrate (Alfa Aesar, 97%), anthocyanin-5.5 (Cy5.5), NHS ester (95%, Lumiprobe) The following reagents were purchased from Sinopharm Chemical Reagent Corporation: fluorescein isothiocyanate isomer I (FITC) (Sigma-Aldrich, ≥90%), dextran (Acros, MW: 100-300kDa), anhydrous ethanol (Aladdin, >99.5%, anhydrous), and other metal salts (CuCl, CaCl2, FeCl3, MgCl2, ZnCl2). 68 GaCl3 was obtained from isotope technology by Garching GmbH (ITG). 68 Ge / 68 Ga was generated by a 0.1M HCl elution column (1110MBq). 89 Zr(ox)2 was purchased from Andico Pharmaceutical Group Co., Ltd.; ultrapure water was provided by... The water produced by the purification system has a resistivity of 18.2 MΩ·cm (25℃).

[0054] The experimental instruments used in the following examples are as follows: TEM images of LMNDs were obtained on a JEOL 2000FX or Tecnai G2 F20 (FEI) analytical electron microscope at an accelerating voltage of 200 kV; dynamic light scattering (DLS) was performed using a Malvern Zetasizer NanoZS to measure the hydrodynamic diameter and surface potential; scanning electron microscopy (SEM) was performed on a Zeiss Supra 55 electron microscope at ETH = 5 kV; single-particle energy dispersive spectroscopy (EDS) elemental maps were obtained on a FEI Titan 80-300 probe aberration-corrected scanning electron microscope (STEM) equipped with a chemstem system; X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo ESCALAB 250XI multi-functional imaging electron spectrometer; X-ray powder diffraction (XRD) analysis was performed on a Bruker D8 ADVANCE powder X-ray diffractometer; and ultrasound was performed using a fishbrand microscope. TMThe experiment was conducted on a Model 505 ultrasonic separator (power: 500 watts; frequency: 20 kHz); fluorescence was recorded on a Perkin Elmer LS55 fluorescence spectrometer; UV measurements were performed on a Lambda25 UV / Vis spectrophotometer.

[0055] Example 1

[0056] This embodiment provides a method for preparing small-sized aqueous liquid metal nanomaterials, including the following steps:

[0057] Mix 80 μL of EGaIn (gallium indium eutectic) and 5 mg of DOX·HCl in 12 mL of deionized water and sonicate in an ice bath for 12 min.

[0058] After ultrasonic treatment, the mixture was centrifuged at 1000 rpm for 5 minutes, and the large precipitated particles were discarded to obtain small-sized aqueous liquid metal nanomaterials (LMND).

[0059] High-resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM) Figure 1 As shown in (a, 1b), the small-sized LMND has an irregular morphology, according to the FE-TEM images ( Figure 2 Statistical results from (a, 2b) show that the average size of the small-sized LMND is 20.3 ± 4.3 nm (this size of LMND is referred to as LMND20 and used in subsequent experiments). DLS analysis shows that ζ is 36.5 ± 2.0 mV and PDI is 0.064 ± 0.013. In the small-angle XRD pattern, a peak at 0.85° indicates the nanoporous structure of the LMND20 prepared in this embodiment. Figure 3 It can serve as additional space and vacancy for drug loading and catalysis, respectively.

[0060] The interaction between the polyol degradation product of DOX·HCl and the LMND surface was investigated using Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). In the FTIR spectrum (… Figure 4 a) DOX is at 1731cm -1 The C=O signal disappears at 1017 and 1260 cm⁻¹ in LMND20. -1 Two new peaks were generated, belonging to the bending band of Ga-OH and the stretching band of C-OH, respectively. In XPS measurements, Ga... 0 and Ga 3+ All were detected in the high-resolution spectrum of Ga 3d. Figure 4 b). Furthermore, a primary peak at 531.5 eV in the O1s spectrum confirms the formation of the O-Ga bond. Figure 5 These data indicate that the polyol was successfully chelated onto the LMND20 surface.

[0061] Furthermore, liquid chromatography analysis of the solutions before and after ultrasound showed that DOX in deionized water was completely degraded into polyols after 12 minutes of ultrasound. Figure 6 Further analysis of the results from the positive and negative modes of high-resolution mass spectrometry (HRMS) was conducted. Figure 7 , 8 This confirmed the presence of a series of DOX fragments containing multiple hydroxyl groups (polyols).

[0062] Example 2

[0063] This embodiment provides a method for preparing small-sized aqueous liquid metal nanomaterials, including the following steps:

[0064] 120 μL of liquid gallium metal and 5 mg of DOX·HCl were mixed in 12 mL of deionized water and ultrasonicated in an ice bath for 12 min.

[0065] After ultrasonic treatment, the mixture was centrifuged at 1200 rpm for 3 minutes, and the large precipitated particles were discarded to obtain small-sized aqueous liquid metal nanomaterials (LMND20) with an average particle size of about 20 nm.

[0066] Small-sized aqueous liquid metal nanomaterials were analyzed using high-resolution mass spectrometry (HRMS) in both positive and negative modes. The results ( Figure 26 The presence of a series of DOX fragments containing polyhydroxyl groups (polyols) was confirmed by a-26j and 27a-27d. Furthermore, high-resolution XPS spectra of the C-1s, O-1s, N-1s, and Ga-3d phases of the liquid metal nanomedicine were obtained. Figure 28 As shown in a-28d, polyols were successfully combined with small-sized aqueous liquid metal nanomaterials, and the small-sized aqueous liquid metal nanomaterials retained some elemental gallium (Ga). 0 This is beneficial for subsequent metal replacement reactions with copper ions.

[0067] Example 3

[0068] This embodiment provides a method for preparing small-sized aqueous liquid metal nanomaterials, including the following steps:

[0069] 100 μL of liquid gallium indium tin alloy GaInSn and 5 mg DOX·HCl were mixed in 12 mL of deionized water and ultrasonically treated in an ice bath for 12 min.

[0070] After ultrasonic treatment, the mixture was centrifuged at 800 rpm for 8 minutes, and the large precipitated particles were discarded to obtain small-sized aqueous liquid metal nanomaterials (LMND) with an average particle size of about 20 nm.

[0071] Comparative Example 1

[0072] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses 20 μL of EGaIn (gallium indium eutectic).

[0073] Comparative Example 2

[0074] The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 uses 40 μL of EGaIn (gallium indium eutectic).

[0075] Comparative Example 3

[0076] The only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses 60 μL of EGaIn (gallium indium eutectic).

[0077] Comparative Example 4

[0078] The only difference between Comparative Example 4 and Example 1 is that Comparative Example 4 was subjected to ultrasonic treatment in an ice bath for 4 minutes.

[0079] Comparative Example 5

[0080] The only difference between Comparative Example 5 and Example 1 is that Comparative Example 5 was subjected to ultrasonic treatment in an ice bath for 6 minutes.

[0081] Comparative Example 6

[0082] The only difference between Comparative Example 6 and Example 1 is that Comparative Example 6 was subjected to ultrasonic treatment in an ice bath for 8 minutes.

[0083] Comparative Example 7

[0084] The only difference between Comparative Example 7 and Example 1 is that Comparative Example 7 was subjected to ultrasonic treatment in an ice bath for 10 minutes.

[0085] Test Example 1

[0086] This test example performed absorbance and fluorescence spectrometry analysis on the LMNDs prepared in Example 1 and Comparative Examples 1-3. With the increase of the EGaIn / DOX·HCl feed ratio, the characteristic absorption peak of the obtained LMNDs broadened, while the typical emission wavelength Em485 remained unchanged. Figure 9 Furthermore, with the increase of the EGaIn / DOX·HCl feed ratio, the size of the LMND steadily decreased, the surface zeta potential gradually increased and stabilized below +40mV, and the PDI stabilized below 0.15. Figure 10 ).

[0087] Test Example 2

[0088] This test case analyzed the LMNDs prepared in Example 1 and Comparative Examples 4-7. With increasing sonication time, the surface zeta potential of the LMNDs increased from 6.5 ± 0.6 mV (4 min sonication time) to over 35 mV (12 min sonication time), while the polydispersity index (PDI) remained below 0.1, indicating that the dynamic DOX fragment is a good ligand for stabilizing LMNDs (see...). Figure 11 Under dynamic ultrasound, polyol-mediated LMNDs transform bulk LM into LMND. After ultrasound treatment for 4 min, 6-10 min, and 12 min, the LMND sizes obtained are approximately 100 nm, 60-90 nm, and 20 nm, respectively.

[0089] Test Example 3

[0090] This test example examines the stability of LMND20 prepared in Example 1. Specifically, after 30 days of observation, the size of LMND20 slightly increased to 26.3 ± 5.7 nm, while the PDI remained at 0.075 ± 0.021, indicating that the changes in particle size and PDI were not significant. Figure 12 ).

[0091] Test Example 4

[0092] Radioactive labeling method is used. 68 GaCl3 (~100 μCi) was reacted with LMND20 (100 μL, 2.0 mg / mL in water) prepared in Example 1 at 30 °C for 15 min. Free ions were removed by three cycles of ultrafiltration-redispersion (4000 rpm, 5 min) and washing with water. 68 GaCl3, to obtain 68 Ga-LMND20. 68 The identification of Ga-LMND20 was performed using radio-iTLC with citrate buffer (50 mM, pH = 5) as the mobile phase. Free bands were observed on Varian iTLC-SA bands. 68 GaCl3 was eluted with solvent (R) f =0.8), and 68 Ga-LMND20 moved a little from the starting point (R) f =0.2), which is recorded on the BIOSCAN Mini-Scan TLC, indicating 68 GaCl3 (~100 μCi) completely binds to LMND20 (100 μL, 2.0 mg / mL).

[0093] BCap-37 tumor-bearing BALB / c nude mice (n=4) were intravenously injected 68Ga-LMND20 (100 μL, ~100 μCi) was used for dynamic small animal PET imaging (0–60 min). Quantitative ROI analysis was performed to obtain biodistribution statistics of LMND20 prepared in Example 1, and the attenuation-corrected half-life (T1 / 2) based on the dynamic small animal PET imaging data was calculated using DAS2.1 software. Dynamic small animal PET imaging and region of interest (ROI) analysis showed that LMND20 significantly enhanced... 68 GaCl3 is circulated in the blood and has a high hepatic uptake (>20% ID g-1 40 minutes after intravenous injection). Figure 13 a). In the microPET method, T1 / 2 is based on dynamic microPET imaging data. Attenuation correction and calculation are performed using DAS2.1 software, resulting in a decay-corrected half-life (t1 / 2) of 315 min for the LMND20 prepared in Example 1. Figure 13 (b) Using ICP-MS, 50 μL of blood was collected at different time points (2, 5, 10, 30, 60, 120, 240, 480, and 1440 min) after intravenous injection. Ga excretion was quantitatively analyzed using ICP-MS. Inductively coupled plasma mass spectrometry (ICP-MS) measurements showed a longer plasma half-life of 468 min. Figure 13 c).

[0094] Test Example 5

[0095] Long-term metabolic monitoring of LMND20 prepared in Example 1 was performed using the Cy5.5 labeling method. For fluorescent labeling, Cy5.5 NHS ester (50 μg) and LMND20 (1 mL, 1.0 mg / mL water) were incubated overnight at 30°C. Free dye was removed by three ultrafiltration-redispersion cycles (4000 rpm, 5 min) and washing with water to obtain Cy5.5-labeled LMND20. The fluorescence measurement was satisfactory, and it was stored in the dark for later use.

[0096] BALB / c nude mice (n=5) carrying BCap-37 were injected with Cy5.5-labeled LMND20 (10 mg / kg), and fluorescence signals were captured by IVIS imaging. Figure 14 The results showed that LMND20 still had significant accumulation in the kidneys and liver on day 3 after intravenous injection, and retained more than 80% and 55% of the original fluorescence signal intensity in the kidneys and liver on day 7 (day 3), respectively, and gradually decreased to 10-20% on day 14.

[0097] In addition, this test case attempted multiple administrations (5 mg / kg every other day for a total of 5 times) to assess the toxicity of the mouse model. Blood samples were collected 24 hours after the final treatment, and the data are expressed as mean ± standard deviation (n = 5). There were no statistically significant differences in serum biochemical indicators involving liver and kidney function between the LMND20 treatment group and the control group (normal saline). Figure 15 a). Furthermore, H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of mice treated with LMND20 showed no obvious damage or lesions. Figure 15 b).

[0098] Test Example 6

[0099] This test example uses LMND20 prepared in Example 1 and Cu 2+ GRR (metal substitution reaction) was performed. Initially, 3.0 mg LMNDs were added to CuCl2 aqueous solutions of different concentrations (2, 4, 8 mM), and the results are shown in the figure. Figure 16 With a 2mM CuCl2 addition, the yellow-green color of the LMND20 suspension rapidly darkened within 5 minutes, turned light wine-red after 6 hours, and yielded a clear wine-red solution after 12 hours, indicating a sustained Gross Restriction-Rate (GRR) between LMND20 and CuCl2. When the CuCl2 concentration increased to 4mM and 8mM, LMND20 underwent a similar process, except the solution color became lighter.

[0100] In order to obtain LMND20 and Cu 2+ More detailed information was collected during the GRR analysis by mixing 1 mL of LMND20 (3 mg / mL) with different concentrations of CuCl2 (0.8, 1.6, 3.2, 6.4, 8, 16 mM) for 30 minutes. As Cu... 2+ With increasing concentration, the typical UV absorption characteristic peaks of LMND20 (272, 295-470 nm) decreased more significantly, and the concentration at Cu... 2+ The concentration reached a plateau at 8 mM. Figure 17 a) This concentration was set as the standard metal concentration for further dynamic monitoring of GRR. For example... Figure 17 As shown in b, at 8mM Cu 2+ Under the influence of [specific action / condition], the typical absorbance of LMND20 at 272 nm disappeared rapidly within 2 minutes, and the absorbance at 373 nm continued to decrease over the following 300 minutes. Furthermore, the effects of LMND20 on different metal ions (Ca) were also tested. 2+ Mg 2+ Zn 2+ Fe 3+ and Cu 2+ The reactivity of LMND20 aqueous solution with 8 mM of other metal ions (Ca) was investigated. 2+ Mg2+ Zn 2+ Fe 3+ Incubate for 6 hours; within this timescale, Cu 2+ Among all metal ions, LMND20Abs272 has the best ability to reduce its concentration. Figure 17 c) This selectivity may contribute to metal homeostasis in vivo.

[0101] Next, this test example characterized the nanoparticles after GRR reaction of CuCl2 and LMND20. FTIR spectroscopy ( Figure 18 a) shows that after GRR, the levels from LMND20 (1,023, 1,260, and 2,930 cm⁻¹) were... -1 The significantly reduced signal indicates a change in the interaction between the polyol and the generated nanodroplets (NDs). In XPS analysis, the Ga spectrum shows that Ga… 0 The metal is completely converted to Ga 3+ ion( Figure 18 b). Cu 2p spectra show Cu at 963.1 eV and 942.5 eV. 2+ The satellite peak has completely disappeared. Figure 18 c) Two new signals are generated at 952.0 eV and 932.1 eV, while there is no Cu near 945 eV. + The weak satellite peaks. Meanwhile, the CuLMM peak broadens and intensifies in the range of 916.2–920.7 eV. Figure 19 To further confirm whether LMND20 produces Cu during the GRR process. + Ions, this test example uses Cu + Chelating agent copper bath chelate (BCS), Cu + - The BCS complex exhibits a typical signal at 483 nm and a shoulder peak at 572 nm. Figure 20 a) For the LMND20+CuCl2+BCS group, the intrinsic absorbance of LMND20 (400~525nm) and Cu + The characteristic peaks of the -BCS complex coincide, but not with the shoulder peaks, indicating that LMND20 and Cu 2+ During ion GRR, the main product is Cu. 0 Metal. Furthermore, XRD measurements also confirmed Cu. 0 The presence of metals ( Figure 20 b) No characteristic peaks of amorphous Ga, CuCl and CuCl2 were observed.

[0102] After incubating 1.5 mg of LMND20 prepared in Example 1 with 8 mM CuCl2 for 6 hours, the overall size of the resulting ND (21.9 ± 7.5 nm) did not change significantly, but lattice structures and sharp spots of Cu and In were observed in HRTEM and SAED images, respectively. Extending the reaction time to 12 h further increased the size of the nanoparticles to 46.4 ± 9.8 nm. When the LMND20 / CuCl2 addition was increased to 3.0 mg / 8 mM, the size of the ND significantly increased to 1,157.1 ± 359.7 nm, and new sharp spots of GaOOH appeared in the SAED region. These results indicate that higher initial LMND20 concentration, higher LMND20 / CuCl2 feed ratio, and longer reaction time lead to greater ND aggregation. Figure 21 The corresponding EDS elemental mapping confirmed the presence of Ga oxide and Cu in the final ND after GRR. Figure 22 a,22b).

[0103] Test Example 7

[0104] This test case primarily tests the size-dependent penetration capability of the LMNDs prepared in Example 1.

[0105] In the BCap-37 three-dimensional multicellular tumor spheroid model, from the outer shell to the core of the tumor spheroid, LMND20 exhibited stronger fluorescein isothiocyanate (FITC) fluorescence than dextran-stabilized LMND (>100 nm, which we hereby name LMND100). Figure 23 a). Quantitative fluorescence intensity and 3D fluorescence intensity maps of BCap-37 tumor spheroids confirmed the importance of size in the penetration ability and distribution of LMNDs within BCap-37 tumor spheroids. Figure 23 Interestingly, the highest ROS concentration was detected in LMND20-treated BCap-37 cells using confocal laser scanning microscopy (CLSM) and flow cytometry with 2'-7'-dichlorofluorescein (DCFH-DA). Figure 23 (h,i). Although the supernatant of LMND20 (LM / S) mainly consists of small molecules (polyols), the generated ROS level is even lower than that of LMND100. Therefore, we infer that ROS generation in LMND-treated tumor cells may be related to Ga... 3+ Ions are involved in intracellular diffusion, which can synergistically inhibit the division and proliferation of tumor cells.

[0106] In this test case, tumor tissue sections from LMND-treated mice were further collected and subjected to biological TEM analysis. Although administered intratumorally, LMND20 and LMND100 exhibited different biodistributions and accumulations. LMND20 was uniformly distributed in the tumor tissue, while LMND100 accumulated in specific areas. Figure 23 j). Immunohistochemical staining (IHC) showed that FITC-labeled LMND20 was widely distributed in the tumor tissue, and CD31-stained blood vessels showed abundant clustered differentiation (red fluorescence). Figure 23 However, almost no green fluorescence was observed in tumor tissue treated with LMND100. These results indicate that LMND20 has improved penetration, diffusion, and accumulation in tumor tissue compared to LMND100.

[0107] Test Example 8

[0108] This test case mainly studies the in vitro antitumor activity of LMND20 prepared in Example 1.

[0109] The effect of LMND20 on the proliferation of breast cancer cells was evaluated using the MTT assay and compared with tetrathiomolybdate (TM), Ga(NO3)3, and LM / S (the supernatant of LMND20). Results are as follows: Figure 24 As shown in figure a, LMND20 exhibited the highest cytotoxicity against BCap-37 breast cancer cells, with a half-maximal inhibitory concentration (IC50) of 30.4 μg / mL. -1 The concentrations were significantly lower than those of TM (IC50: 91.1 μg / mL), while the concentrations of Ga(NO3)3 and LM / S exceeded 250 μg / mL. -1 At that time, more than 75% of BCap-37 cells remained viable. The anticancer activity of LMND20 has also been demonstrated in other breast cancer cell lines involving MDA-MB-231 and MCF-7 cells. Figure 24 (b, c). However, LMND20 did not show significant cytotoxicity against other tumor cell lines with low copper levels (such as SKOV-3 and PC-9 cells) and normal cells. Figure 24 d) This selectivity can reduce side effects on healthy tissues and organs.

[0110] Then, the FITC-Annexin V / PI method was used to verify whether LMND20 activates cell death through apoptosis, which is the killing mechanism of many Ga-based anticancer drugs. Figure 24As shown in Figure e, flow cytometry results indicated that the proportions of apoptotic cells activated by TM, Ga(NO3)3, and LM / S were 30.96%, 19.95%, and 9.98%, respectively, while incubation with LMND20 significantly increased these proportions to 63.01%. Simultaneously, treatment with the same concentration of LMND20 significantly altered the cell cycle of BCap-37 cells, with the G0 / G1 ratio decreasing to a maximum of 31.7%, exhibiting marked sub-G1 phase apoptosis at a rate of 16.6%. Figure 24 f). The activating effect of caspase-3 protein, an important effector of apoptosis, was further confirmed. Western blot results showed that, compared with other groups, the expression of cleaved caspase-3 was significantly increased in cells treated with LMND20. Figure 24 These results confirm that LMND20 has anticancer activity by triggering apoptosis.

[0111] To determine whether the anticancer activity of LMND20 is also related to Cu depletion via GRR, this study investigated the effect of Cu status on LMND20 cytotoxicity. Compared to the severe cytotoxicity of LMND20 on BCap-37 cells, co-incubation with 0.5 mM CuCl2 significantly reduced cytotoxicity, with over 68% of BCap-37 cells surviving. Figure 24 h). This may be due to the large extracellular consumption of LMND20, resulting in large NDs size and poor cellular uptake, thus leading to very little Ga. 0 Metals can be used to consume Cu within cells. 2+ Ions. However, higher concentrations (1.0 mM) of CuCl2 still retain some Cu after GRR reaction with LMND20. 2+ The ions thus exert the inherent cytotoxicity of CuCl2. Similar cell viability trends were also observed in MDA-MB-231 and MCF-7 cells. Figure 24 i,j).

[0112] Test Example 9

[0113] This test case mainly examines the therapeutic effect of LMND20 prepared in Example 1 on a breast cancer xenograft model.

[0114] In this test, mice treated with saline (0.9%), TM (5 mg / kg), and Ga(NO3)3 (40 mg / kg) served as positive controls. Figure 25a) The anticancer activity of LMND20 in BCap-37 tumor-bearing mice was evaluated at a dose of 5 mg / kg. Besides severe toxic side effects, systemic administration of nanomaterials can induce intracellular / extracellular invasion of breast cancer cells, promoting new metastases; therefore, all formulations employed multi-site intratumoral administration. To optimize dosing frequency, ICP-MS was used to determine copper excretion after a single dose in this test case. Because Cu... 2+ The ions transform into metallic Cu 0 The solubility decreased after treatment, and the concentration of metabolized Cu in feces and urine was significantly reduced on the first day after treatment (D1). However, on day 4 after treatment, the excreted copper concentration in BCap-37 tumor-bearing mice returned to normal levels (D4). Therefore, the drug was administered once every 3 days for a total of 4 times to evaluate efficacy. During the treatment of BCap-37 tumor-bearing mice, Ga(NO3)3 (40 mg / kg) showed limited anticancer properties, consistent with clinical data in breast cancer patients. LMND20 (5 mg / kg) inhibited tumor growth more effectively than the same concentration of TM. Figure 25 b, 25e, 25f). No significant weight loss was observed in any group throughout the treatment process. Figure 25 c) indicates no serious systemic toxicity. In a 40-day survival study, all mice in the saline and Ga(NO3)3 treatment groups died, while the survival rate in the TM treatment group was 2 / 6, showing limited anticancer effect. All mice in the LMND20 treatment group survived. Figure 25 d) No abnormal behavior was observed, indicating an improvement in the anti-cancer effect of LMND20.

[0115] To further explore the anticancer mechanism of LMND20, we performed RNA sequencing (RNA-seq) on BCap-37 tumor tissues after treatment with saline, Ga(NO3)3, TM, and LMND20 alone. Distribution profiles of differentially expressed genes (DEGs) were also analyzed. Figure 25 In g), the vertical axis represents the up- and down-regulation results of genes, and the horizontal axis represents the differences between groups (P < 0.05). Taking L vs C as an example, the more genes that are up- or down-regulated, the more significant the treatment difference is between L (LMND20 group) and C (control group). Similarly, the fewer genes that are up- or down-regulated in G vs C, the less significant the difference is between G group (gallium nitrate) and the control group, i.e., the treatment effect is not significant. Figure 25 g indicates that, compared with other treatments, LMND20 treatment has a more significant effect on the transcriptome, with 568 and 327 genes upregulated and downregulated, respectively. Figure 25Table 1 and h show key genes with representative expression alterations, including those involved in apoptosis, angiogenesis, cell population proliferation, and response to ROS. Furthermore, GeneOntology (GO) enrichment analysis confirmed that, in addition to apoptosis, the angiogenesis pathway may also play a role in the anticancer properties of LMND20. Figure 25 i, Table 2), which is of great significance in the treatment of breast cancer.

[0116] Following treatment, xenografted BCap-37 tumor tissue was excised for H&E and IHC analysis. In these mice, the LMND20-treated group showed the highest apoptotic activity upon immunostaining with activated caspase-3 protease. Figure 25 (j). Simultaneously, its apoptotic effect was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence assay, further validating the anticancer mechanism of LMND20. H&E staining showed that most tumor cells were replaced by normal tissue after LMND20 treatment. Furthermore, Ki-67 antigen staining showed that LMND20 treatment significantly reduced the proliferation of BCap-37 tumor cells (j). Figure 25 j,k). Due to Cu in the cells 2+ The absence of VEGF significantly inhibits tumor angiogenesis, tumor growth, and metastasis, potentially leading to downregulation of VEGF expression and reduced microvascular formation. Figure 25 j,k showed that in mice treated with LMND20, the expression of VEGF and the number of microvessels in the tumors removed were significantly reduced, which was superior to the antitumor activity of the phase II anticancer drug TM.

[0117] Table 1 lists representative DEGs.

[0118]

[0119]

[0120] Table 2 shows a representative list of GOs.

[0121]

[0122] Based on the above-described preferred embodiments according to this application, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing small-sized aqueous liquid metal nanomaterials, characterized in that, Includes the following steps: S1: Prepare an aqueous mixed solution containing liquid metal and DOX·HCl, and then subject it to ultrasonic treatment; S2: After ultrasonic treatment, the mixture is centrifuged to remove the precipitate and obtain small-sized aqueous liquid metal nanomaterials; The liquid metal is gallium or a gallium-based alloy.

2. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1, characterized in that, The liquid metal is a gallium-indium eutectic alloy EGaIn.

3. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1 or 2, characterized in that, The average particle size of the small-sized aqueous liquid metal nanomaterial is 20 nm.

4. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1, characterized in that, The ultrasonic treatment was performed in an ice bath.

5. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1 or 4, characterized in that, The ultrasonic treatment time is 12 minutes or more.

6. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of DOX·HCl and liquid metal shall not exceed 0.06 g / mL.

7. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1, characterized in that, The centrifugation conditions are 800-1200 rpm.

8. The method for preparing small-sized aqueous liquid metal nanomaterials according to claim 1 or 7, characterized in that, The centrifugation time is 3-8 minutes.

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