Construction and application of a class of nano-delivery system based on endoperoxide

Abstract

The application belongs to the technical field of biological medicine, and relates to construction and application of a kind of nano oxygen delivery system based on internal peroxide. The application designs and synthesizes a kind of nano drug which can be used as a molecular oxygen delivery agent. In the nano drug, active molecules are composed of internal peroxide compound and 1,4-diazabicyclo[2.2.2]octane (DABCO), and the two molecules are constructed on gold nanorods to improve water solubility, biocompatibility and targeting of the active molecules. Finally, the active molecule modified gold nanorods obtained by the application can be used to relieve the damage of tissues and organs caused by hypoxia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the construction and application of a type of nano-oxygen delivery system based on internal peroxides. Background Technology

[0002] Activated phagocytes consume oxygen to produce reactive oxygen species (ROS), which play a crucial role in the initial inflammatory response following tissue injury. Oxygen is the most direct requirement for angiogenesis and connective tissue formation during wound healing. However, edema, microcirculatory disturbances, and vasoconstriction in damaged tissues can affect the adequate supply of oxygen. Furthermore, in cases of acute anemia, external trauma, or other situations involving significant blood loss, blood transfusion becomes a vital solution, but it also presents numerous challenges during actual transfusion procedures. Therefore, there is a need to develop artificial oxygen carriers that pose no risk of infection, require no matching, and can be stored long-term to address these issues.

[0003] Currently, there are two main research approaches for artificial oxygen carriers. One is hemoglobin oxygen carriers, which modify hemoglobin through chemical methods such as cross-linking, polymerization, and coupling, or encapsulate it in artificially synthesized membranes. However, hemoglobin derivatives encapsulated by liposomes or polymers have a short half-life and can trigger inflammatory responses. Although perfluorinated carbon has a strong oxygen-carrying capacity, clinical studies have found that it often produces many side effects. Therefore, research on perfluorinated carbon oxygen-carrying drugs still needs to be addressed.

[0004] Therefore, it is particularly important to develop a water-soluble and biocompatible molecular oxygen supply agent with a targeted effect and low toxicity. Summary of the Invention

[0005] In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide a gold nanorod that releases molecular oxygen. This oxygen-carrying nanogroup has excellent repair effects on damaged skin, tissues and organs, and can also penetrate into the damaged site through blood circulation to release molecular oxygen.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing a type of nano-oxygen delivery system based on internal peroxides, the method comprising the following steps:

[0008] Gold nanorods (GNRs), NAPH-PEG-SH, and DABCO-PEG-SH were mixed in a buffer solution for 12-15 hours, centrifuged, dispersed in the buffer solution, and a catalytic amount of methylene blue was added. Then, 1-2 drops of TFA were added. The mixture was stirred at -8 to 0°C for 10-12 hours under oxygen and red light irradiation.

[0009] Specifically, the dosage of methylene blue is 0.5mg-2mg.

[0010] Specifically, the mass ratio of gold nanorods (GNRs), NAPH-PEG-SH, and DABCO-PEG-SH is 1:0.8-1.5:0.8-1.5.

[0011] The oxygen environment refers to opening the reaction vessel or connecting it to the outside air and introducing oxygen into the contents of the reaction vessel.

[0012] 1.1 The preparation method of the gold nanorods:

[0013] (1) Mix hexadecyltrimethylammonium bromide solution (CTAB) with gold tetrachloride (HAuCl4) solution, then quickly add cold sodium borohydride (NaBH4) solution and stir vigorously, then let stand for 30-40 minutes to make a seed solution;

[0014] (2) Mix hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL). Add a certain amount of warm water, then add AgNO3 solution. Keep the mixture undisturbed at 30-35℃ for 15-20 minutes, then add gold tetrachloroate (HAuCl4) solution. After stirring for 90 minutes (700 rpm), the solution becomes colorless. Then add hydrochloric acid to adjust the pH to 1.36±0.03. Stir slowly at 400 rpm for 15-20 minutes, then add ascorbic acid (AA) and stir vigorously for 30-40 seconds to prepare the growth solution. After stirring, add the seed solution and keep undisturbed at 30-32℃ for 10-12 hours to grow gold nanorods (GNRs). Centrifuge at 7000 rpm for 30 minutes, then remove the supernatant to obtain the gold nanorods loaded with active molecules.

[0015] 1.2 The structure of the NAPH-PEG-SH is as follows:

[0016] 1.3 The structure of the DABCO-PEG-SH:

[0017] 1.4. Mix gold nanorods, NAPH-PEG-SH, and DABCO-PEG-SH (10 mg) in 5 mL of HEPES buffer for 12-15 hours. Centrifuge to remove excess and unreacted NAPH-PEG-SH and DABCO-PEG-SH, and continue dispersing in HEPES buffer. Then add a catalytic amount of methylene blue. Subsequently, add 1-2 drops of TFA. Stir the mixture at -8 to 0°C under oxygen for 10-12 hours and irradiate with red light.

[0018] Where R is R 1-R 3 R 5 -R 7 R 9 -R 12 R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C10 alkyl, C1-C10 aryl, amino, and C1-C10 amino-substituted alkyl, where n is an integer from 1 to 500; R 4 R 8 R 13 R 17 Each and independent or The sums of n1 and n2 are independent integers between 0 and 6, and m is an integer between 0 and 6.

[0019] As a preferred technical solution of the present invention, in step (1), the concentrations of the hexadecyltrimethylammonium bromide (CTAB) solution, tetrachloroauric acid (HAuCl4) solution and sodium borohydride (NABH4) solution are 0.2M, 0.5mM and 0.01M, respectively, and the volume ratio is 8-10:8-10:1.

[0020] As a preferred embodiment of the present invention, in step (2), the concentrations of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NAOL) in the mixed solution are 0.0768M and 0.02M, respectively, and the concentrations of AgNO3 solution, tetrachloroauric acid (HAuCl4) solution, hydrochloric acid and ascorbic acid (AA) solution are 4mM, 1mM, 12.1M and 0.064M, respectively. The volume ratio of the mixed solution of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NAOL), AgNO3 solution, tetrachloroauric acid (HAuCl4) solution, hydrochloric acid and ascorbic acid (AA) solution is 200-250:9-12:200-250:1.5-2:1.

[0021] As a preferred technical solution of the present invention, in step (2), the volume ratio of the seed solution to the growth solution is 1:600-650.

[0022] The nano-oxygen delivery system prepared by the aforementioned method based on internal peroxides has the following structure for the singlet oxygen-releasing group connected to the gold nanorods:

[0023]

[0024] Where R0 is

[0025] R 1 -R3 R 5 -R 7 R 9 -R 12 R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C10 alkyl, C1-C10 aryl, amino, and C1-C10 amino-substituted alkyl, where n is an integer from 1 to 500;

[0026] R 4 R 8 R 13 R 17 Each and independent or

[0027] The sums of n1 and n2 are independent integers between 0 and 6, and m is an integer between 0 and 6.

[0028] In some specific nano-oxygen delivery systems, R 1 -R 3 R 5 -R 7 R 9 -R 12 R 14 -R 16 Each is independently selected from hydrogen, trimethylsilyl, C1-C6 alkyl, phenyl, and amino groups, where n is an integer from 50 to 200;

[0029] In some specific nano-oxygen delivery systems, n is an integer between 50 and 150.

[0030] In some specific compounds, n is an integer between 100 and 150.

[0031] In some specific compounds, n is an integer between 100 and 120.

[0032] R 1 -R 3 R 5 -R 7 R 9 -R 12 R 14 -R 16 Each of these components is independent of the others: hydrogen, methyl, ethyl, propyl, and isopropyl.

[0033] As a preferred embodiment of the present invention, in step 1.4, the mass ratio of gold nanorods, NAPH-PEG-SH, and DABCO-PEG-SH is 1:1:1. The centrifugation speed is 12000 rpm for 10 minutes.

[0034] The beneficial effects of this invention are as follows: This type of molecular oxygen delivery agent using gold nanorods as carriers simultaneously loads endogenous peroxide molecules and DABCO onto nanomaterials with good water solubility and biocompatibility. Simultaneously, the endogenous peroxide molecules and DABCO molecules can be tightly adjacent, allowing DABCO to efficiently convert the singlet oxygen released by the endogenous peroxide molecules into molecular oxygen for release, thereby increasing the molecular oxygen content and enhancing wound healing in tissues and organs. Due to the ease of surface modification and large specific surface area of ​​the nanomaterials, they can achieve higher drug loading compared to ordinary small molecules. Furthermore, the introduction of PEG significantly improves the water solubility of the system, resulting in this endogenous peroxide system exhibiting good water solubility and promoting the proliferation of NIH / 3T3 (mouse embryonic fibroblasts) under hypoxic conditions. Attached Figure Description

[0035] Figure 1 The image shows the appearance and ultraviolet absorption of gold nanorods under a transmission electron microscope.

[0036] Figure 2 The graph shows the change in singlet oxygen release intensity of GNRs-ENDO(10) and GNRs-ENDO-DABCO(9) over time in vitro.

[0037] Figure 3 The effect of GNRs-ENDO(10) and GNRs-ENDO-DABCO(9) on cell growth under hypoxic conditions is shown in the figure.

[0038] Specific implementation methods

[0039] The present invention will now be described in detail through embodiments. It should be noted that the following embodiments are only for further illustration of the present invention and are not intended to limit it, unless otherwise stated. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0040] The abbreviations involved in this invention are:

[0041] Trifluoroacetic acid: TFA

[0042] Hexadecyltrimethylammonium bromide: CTAB

[0043] Sodium oleate: NAOL

[0044] Silver nitrate: AgNO3

[0045] Tetrachloroauric acid: HAuCl4

[0046] Ascorbic acid: AA

[0047] Sodium borohydride: NaBH4

[0048] Gold nanorods: GNRs

[0049] DABCO's polyethylene glycol macromolecule: DABCO-PEG-SH

[0050] NAPH-PEG-SH, a naphthalene-based peroxide precursor, is a large molecule of polyethylene glycol.

[0051] Endo-peroxy polyethylene glycol macromolecule: ENDO-PEG-SH

[0052] Gold nanorods modified with endogenous peroxy polyethylene glycol macromolecules: GNRs-ENDO

[0053] Modified with both endogenous peroxide polyethylene glycol macromolecules and DABCO polyethylene glycol macromolecules.

[0054] Gold nanorods: GNRs-ENDO-DABCO

[0055] Blank control group: Blank

[0056] The specific embodiments of the present invention are described in detail below with reference to the technical solutions:

[0057] In the following embodiments, the structure of H2N-PEG-SH used is as follows: n is 110.

[0058] Example 1

[0059] Preparation method:

[0060] 1. Synthesis of gold nanorods (GNRs)

[0061] Preparation of seed solution: Mix 5 mL of 0.5 mM HAuCl4 solution with 5 mL of 0.2 M CTAB solution, then quickly add 0.6 mL of fresh 0.01 M NaBH4 solution. The solution color changes from yellow to brownish-yellow after vigorous stirring for 2 minutes. The seed solution is then aged at room temperature for 30 minutes before use.

[0062] Preparation of growth solution: Dissolve 7.0 g CTAB and 1.543 g NaOL in 250 mL of warm water (approximately 50 °C). After cooling the solution to 30 °C, add 12 mL of 4 mM AgNO3 solution. Maintain the mixture at 30 °C for 15 min, then add it to 250 mL of 1 mM HAuCl4 solution. After stirring for 90 min, the solution becomes colorless. Add 2.1 mL of hydrochloric acid (12.1 M) to adjust the pH. Stir slowly for another 15 min, then add 1.25 mL of 0.064 M ascorbic acid (AA).

[0063] Finally, 0.8 mL of seed solution was added, and the mixture was allowed to stand for 12 h to obtain gold nanorods (GNRs). Characterization was performed as follows: Figure 1

[0064] 2. Preparation of endogenous peroxide precursor polyethylene glycol macromolecules and DABCO polyethylene glycol macromolecules.

[0065] (1) Synthesis method of endogenous peroxide precursor polyethylene glycol macromolecule

[0066]

[0067] Step a: Preparation of compound 2

[0068] Under argon atmosphere and in the dark, 1,4-dimethylnaphthalene (1) (0.78 g, 5.0 mM) was dissolved in 8 mL of chloroform. Bromine (0.27 mL, 5.25 mM) was added to the reaction mixture at 0 °C over 10 minutes. The reaction mixture was stirred at room temperature for 4 hours, monitored by TLC during the reaction. The reaction mixture was then diluted with 25.0 mL of chloroform, quenched with 25.0 mL of saturated sodium thiosulfate solution, and washed with 25.0 mL of water and 25.0 mL of brine. The organic layers were combined and dried over anhydrous sodium sulfate. The solid was filtered off, the reaction mixture was concentrated, and the crude product was purified by silica gel column chromatography using n-hexane as eluent to give a white solid. 1 H NMR(400MHz,Chloroform-d)δ8.12–8.06(m,1H),8.04–7.99(m,1H),7.67(dd,J=6.4,3.4Hz,2H),7.60(s,1H),2.90(s,3H),2.71(s,3H); 13 C NMR (101MHz, Chloroform-d) δ132.79,132.35,130.59,130.11,129.37,125.28,124.40,123.83,123.67,121.23,17.88,17.55.

[0069] Step b: Preparation of compound 3

[0070] Compound 2 (470 mg, 2 mM) and (4-(2-ethoxy-2-oxoethoxy)phenyl)boronic acid (537 mg, 2.4 mM) were dissolved in 5 mL of a 9:1 mixture of methanol and water. Tetra(triphenylphosphine)palladium (115.5 mg, 0.1 mM) and K₂CO₃ (552.8 mg, 4 mM) were also added to this mixture. The mixture was refluxed at 115 °C for 9 hours, monitored by TLC. After completion, the reaction mixture was allowed to cool to room temperature. Then, 5 mL of 1 M hydrochloric acid was added to adjust the pH to 2–3. The reaction mixture was then extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude product. The crude product was purified by silica gel column chromatography using DCM:MeOH (20:1, v / v) to give a pale yellow powder. 1 H NMR(400MHz,Chloroform-d)δ8.15–8.08(m,1H),8.06–8.01(m,1H),7.62–7.51(m,2H),7.38 –7.32(m,2H),7.28–7.21(m,1H),7.06–6.99(m,2H),4.77(s,2H),2.69(s,3H),2.58(s,3H); 13 C NMR(101MHz,Chloroform-d)δ155.23,136.82,135.69,132.04,130.84,130.76, 130.03,128.10,127.87,124.84,124.25,124.04,123.53,63.92,18.25,15.13.

[0071] Step c: Preparation of compound 4 NAPH-PEG-SH

[0072] Compound 3 (42 mg, 0.14 mM) and H2N-PEG-SH (500 mg, 0.1 mM, MW: 5000 g / mol) were dissolved in 5 mL of THF. EDC (21.5 mg, 0.14 mM) and DMAP (23 mg, 0.175 mM) were added to the solution. After stirring for 2 hours, cold diethyl ether was added to the solution, resulting in the precipitation of a white precipitate. The precipitate was filtered to obtain a pure white solid, NAPH-PEG-SH. 1H NMR(400MHz,Chloroform-d)δ8.11(d,J=10.0Hz,1H),8.03(d,J=8.5Hz,1H),7.65–7.47(m,2H),7.32(dd,J=8.7,2H),7.22(s,1H),7.01(d,J=8.8Hz, 2H),4.57(s,2H),3.85–3.78(m,4H),3.64(s,PEG),3.50–3.42(m,4H),3.4 1–3.34(m,1H),3.29–3.14(m,2H),2.78(s,3H),2.69(s,3H),2.57(s,3H). 13 C NMR(101MHz,Chloroform-d)δ167.33,155.20,136.82,135.53,132.05,130.79,130.05,128.72,128.08,127.83,124.89,124.30 ,124.05,123.55,71.86,69.52,69.34(d,J=4.0Hz),69.20,68.77,66.50,37.82,23.24,18.29,15.15.MALDI-TOF:found:5257.8.

[0073] (2) Synthesis method of DABCO polyethylene glycol macromolecule

[0074]

[0075] Step d: Preparation of compound 6

[0076] Piperazine (5) (1.0 g, 11.61 mmol) was dissolved in 50 mL of toluene. Triethylamine (3.24 mL, 23.22 mmol) and ethyl 2,3-dibromopropionate (1.69 mL, 11.61 mmol) were then added to the reaction system. The dissolution was accelerated by sonication, and the mixture was then heated and stirred overnight at 80 °C. The reaction system was diluted with a large amount of toluene and filtered. The yellow solution was collected, and the filtrate was concentrated by rotary evaporation at 60 °C to obtain an oily substance. The crude product was purified by silica gel column chromatography (elution: dichloromethane containing 5%-10% methanol, gradient elution) to obtain a pure product. 1 HNMR(400MHz,Chloroform-d)δ4.25(q,J=7.1Hz,2H),3.48(t,J=8.5Hz,1H),3.1 5–3.05(m,2H),2.93(t,J=7.5Hz,3H),2.86–2.62(m,5H),1.31(t,J=7.1Hz,3H). 13C NMR (101MHz, Chloroform-d) δ171.60,60.81,57.04,49.20,48.56,46.64,46.06,42.64,14.11.

[0077] Step e: Preparation of compound 7

[0078] Ethyl 1,4-diazabicyclo[2.2.2]octane-2-carboxylate (6) (0.7 g) was dissolved in 1 mL of toluene and slowly added to a solution of 10 mL of ethylenediamine over 30 minutes at room temperature. The resulting solution was heated to 50 °C and stirred overnight. Excess ethylenediamine was distilled off under reduced pressure and could be dissolved and distilled repeatedly with methanol to finally obtain an oily product. 1 HNMR(400MHz,Chloroform-d)δ7.19(s,1H),3.35–3.23(m,2H),3.22–3.14(m,1H),3.08(d d,J=14.3,1H),2.99(dd,J=13.5,1H),2.78(td,J=11.2,10.4,5H),2.61(dt,J=18.5,6H). 13 C NMR (101MHz, Chloroform-d) δ170.83,56.19,47.77,47.48,45.67,45.36,41.98,40.90,40.69.

[0079] Step f: Preparation of compound 8 DABCO-PEG-SH

[0080] Compound 7 (59 mg, 0.3 mM) and NHS-PEG-SH (500 mg, 0.1 mM, MW: 5000 g / mol) were dissolved in 5 mL of LMF. DIPEA (38 mg, 0.3 mM) was added. After the mixture reacted for 2 hours, cold diethyl ether was added, resulting in the formation of a yellow precipitate. Filtration yielded a pure yellow solid, DABCO-PEG-SH (n = 110 in the structure). 1 H NMR (400MHz, Chloroform-d) δ3.90 (d, J = 8.2Hz, 2H), 3.77–3.72 (m, 3H), 3.58 (s, PEG), 3.43–3.36 (m, 5H). 13 C NMR(101MHz,Chloroform-d)δ170.99,169.71,69.98,69.53,68.57,56.10,47.67,47.38,45.60,45.25,41.85,38.07,37.81.MALDI-TOF:found:4920.

[0081] 3. Modification of gold nanorods by active molecules (step g: preparation of nanogroup 9)

[0082] (1) Modification of naphthalene compounds

[0083] GNRs (10 mg), NAPH-PEG-SH (10 mg), and DABCO-PEG-SH (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min to remove excess, unreacted compound 4 and DABCO-PEG-SH.

[0084] (2) Modification with internal peroxides

[0085] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. Two drops of TFA were then added. The mixture was stirred in an open container at 0°C for 12 hours, irradiated with 18 W, 630 nm red light during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0086] A special note: here, compound 4 and the singlet oxygen quencher group DABCO derivative are first modified onto gold nanorods, and then the naphthalene ring is ring-opened and connected to the peroxide bridge using the method of generating internal peroxide. There are two reasons for this linking order: First, if the internal peroxide precursor compound is ring-opened to form an internal peroxide compound, the thiol group has a lone pair of electrons, which affects the generation of internal peroxide. Second, if the internal peroxide compound is linked to the gold nanorods, the reaction time is too long, which can easily lead to the instability of the internal peroxide compound and the generation of the precursor compound, ultimately resulting in little or no singlet oxygen generation at room temperature or 37°C.

[0087] 4. Modification of gold nanorods by single-component peroxide precursor compounds (step h: preparation of nanogroup 10)

[0088] GNRs (10 mg) and compound 4 (10 mg) were mixed in 2 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 10 min to remove excess, unreacted compound 4.

[0089] (2) Modification with internal peroxides

[0090] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. The mixture was stirred in an open container at 0°C for 12 hours, with 18 W, 630 nm red light irradiation during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0091] It should be noted that the reason for first modifying the gold nanorods with compound 4 is the same as that mentioned above.

[0092] Example 2

[0093] 1. The synthesis method of gold nanorods is as described in Example 1.

[0094] 2. Preparation methods of endogenous peroxide precursor polyethylene glycol macromolecules and DABCO polyethylene glycol macromolecules

[0095] (1) Preparation method of endogenous peroxide precursor polyethylene glycol macromolecule

[0096]

[0097] Step a: Preparation of compound 11

[0098] Methoxy(cyclooctadiene)iridium(I) dimer (82.5 mg, 0.125 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (67 mg, 0.125 mmol), bis-pinacol diborane (697.5 mg, 2.75 mmol), and compound 1 (0.385 mL, 2.5 mmol) were added to anhydrous cyclohexane (10 mL). The solution was heated to 60 °C and reacted for 20 hours. After the reaction was stopped, the solvent was removed by rotary evaporation after cooling to room temperature. The crude product was purified by silica gel column chromatography using n-hexane:ethyl acetate (20:1, v / v) as the developing solvent to obtain a white solid.

[0099] Step b: Preparation of compound 12

[0100] Compound 11 (56.4 mg, 0.2 mmol), p-bromophenoxyacetic acid (23.1 mg, 0.1 mmol), and potassium carbonate (27.64 mg, 0.2 mmol) were dissolved in a mixture of tetrahydrofuran (6 mL) and water (1 mL). Tetraphenylphosphine palladium (5.8 mg, 0.005 mmol) was then added to the mixture. The mixture was refluxed at 80 °C for 9 h under argon protection. The reaction was cooled to room temperature, the pH was adjusted to 1 with 1 M HCl, and the mixture was extracted with ethyl acetate. The organic phase was collected, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography with DCM:MeOH (20:1, v / v) as the developing solvent to give a pale yellow solid.

[0101] Step c: Preparation of compound 13

[0102] Compound 12 (42 mg, 0.14 mM) and H2N-PEG-SH (500 mg, 0.1 mM, MW: 5000 g / mol) were dissolved in 5 mL of THF. EDC (21.5 mg, 0.14 mM) and DMAP (23 mg, 0.175 mM) were added to the solution. After stirring for 2 hours, cold diethyl ether was added to the solution, resulting in the precipitation of a white precipitate. The precipitate was filtered to obtain a pure white solid.

[0103] (2) Preparation method of DABCO compound polyethylene glycol macromolecule

[0104] The specific implementation method is the same as in Example 1.

[0105] 3. Modification of gold nanorods by active molecules (step g: preparation of nanogroup 14)

[0106] (1) Modification of naphthalene compounds

[0107] GNRs (10 mg), compound 13 (10 mg), and DABCO-PEG-SH (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min to remove excess and unreacted compound 13 and DABCO-PEG-SH.

[0108] (2) Modification with internal peroxides

[0109] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. One drop of TFA was then added. The mixture was stirred in an open container at 0°C for 12 hours, irradiated with 18 W, 630 nm red light during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0110] A special note: here, compound 13 and the singlet oxygen quencher group DABCO derivative are first modified onto gold nanorods, and then the naphthalene ring is ring-opened and connected to the peroxide bridge using the method of generating internal peroxide. There are two reasons for this linking order: First, if the internal peroxide precursor compound is ring-opened to form an internal peroxide compound, the thiol group has a lone pair of electrons, which affects the generation of internal peroxide. Second, if the internal peroxide compound is linked to the gold nanorods, the reaction time is too long, which can easily lead to the instability of the internal peroxide compound and the generation of the precursor compound, ultimately resulting in little or no singlet oxygen generation at room temperature or 37°C.

[0111] 4. Modification of gold nanorods by single-component peroxide precursor compounds (step h: preparation of nanogroup 15): GNRs (10 mg) and compound 13 (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 min. The mixture was then stirred for 12 h. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min to remove excess, unreacted compound 13.

[0112] (2) Modification with internal peroxides

[0113] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. The mixture was stirred in an open container at 0°C for 12 hours, with 18 W, 630 nm red light irradiation during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0114] It should be noted that the reason for first modifying the gold nanorods with compound 13 is the same as that mentioned above.

[0115] Example 3

[0116] Preparation method:

[0117] 1. The synthesis method of gold nanorods is as described in Example 1.

[0118] 2. Preparation methods of endogenous peroxide precursor polyethylene glycol macromolecules and DABCO polyethylene glycol macromolecules

[0119] (1) Preparation method of endogenous peroxide precursor polyethylene glycol macromolecule

[0120]

[0121] Step a: Preparation of compound 17

[0122] Compound 16 (24 mg, 0.23 mmol) was dissolved in 5 mL of dimethyl ether and cooled to 0 °C. Then, NaH (60% oil suspension, 9.5 mg, 0.24 mmol) was added. After stirring the mixture for 10 minutes, LiBr (40 mg, 0.46 mmol) was added, and the mixture was stirred for 20 minutes. Methyl bromoacetate (42 mg, 0.225 mmol) was added. The mixture was heated to 60 °C and maintained for 4 hours, then cooled to room temperature and filtered. The product was concentrated using a rotary evaporator, and the crude product was purified by silica gel column chromatography to obtain a colorless oil.

[0123] Step b: Preparation of compound 18

[0124] Compound 17 (0.6 g, 3.32 mmol) was dissolved in 10 mL of methanol in sodium hydroxide solution, and the mixture was heated and stirred for 3 hours. After cooling to room temperature, the pH was adjusted to 4. The reaction mixture was concentrated using a rotary evaporator, and the crude product was purified by silica gel column chromatography using dichloromethane:ethyl acetate (2:1, v / v) as eluent to give a yellow powder.

[0125] Step c: Preparation of compound 19

[0126] Compound 18 (21.4 mg, 0.14 mM) and H2N-PEG-SH (500 mg, 0.1 mM, MW: 5000 g / mol) were dissolved in 5 mL of THF. EDC (21.5 mg, 0.14 mM) and DMAP (23 mg, 0.175 mM) were added to the solution. After stirring for 2 hours, cold diethyl ether was added to the solution, resulting in the precipitation of a white precipitate. The precipitate was filtered to obtain a pure white solid.

[0127] (2) Preparation method of DABCO compound polyethylene glycol macromolecule

[0128] The specific implementation method is the same as in Example 1.

[0129] 3. Modification of gold nanorods by active molecules (step g: preparation of nanogroup 20)

[0130] (1) Modification of naphthalene compounds

[0131] GNRs (10 mg), compound 19 (10 mg), and DABCO-PEG-SH (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 min to remove excess and unreacted compound 19 and DABCO-PEG-SH.

[0132] (2) Modification with internal peroxides

[0133] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. Two drops of TFA were then added. The mixture was stirred in an open container at 0°C for 12 hours, irradiated with 18 W, 630 nm red light during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0134] A special note: here, compound 19 and the singlet oxygen quencher group DABCO derivative are first modified onto gold nanorods, and then the naphthalene ring is ring-opened and connected to a peroxide bridge using the method of generating internal peroxide. There are two reasons for this linking order: First, if the internal peroxide precursor compound is ring-opened to form an internal peroxide compound, the thiol group has a lone pair of electrons, which affects the generation of internal peroxide. Second, if the internal peroxide compound is linked to the gold nanorods, the reaction time is too long, which can easily lead to the instability of the internal peroxide compound and the generation of the precursor compound, ultimately resulting in little or no singlet oxygen generation at room temperature or 37°C.

[0135] 4. Modification of gold nanorods by single-component peroxide precursor compounds (step h: preparation of nanogroup 21)

[0136] GNRs (10 mg) and compound 19 (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 10 min to remove excess, unreacted compound 19.

[0137] (2) Modification with internal peroxides

[0138] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. The mixture was stirred in an open container at 0°C for 12 hours, with 18 W, 630 nm red light irradiation during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0139] The reason for first modifying the gold nanorods with compound 19 is the same as described above.

[0140] Example 4

[0141] Preparation method:

[0142] 1. The synthesis method of gold nanorods is as described in Example 1.

[0143] 2. Preparation methods of endogenous peroxide precursor polyethylene glycol macromolecules and DABCO polyethylene glycol macromolecules

[0144] (1) Preparation method of endogenous peroxide precursor polyethylene glycol macromolecule

[0145]

[0146] Step a: Preparation of compound 23

[0147] Compound 22 (0.96 g, 5 mmol) was dissolved in 7.5 mL of chloroform, and a bromine solution (0.27 mL, 5.25 mmol) was added to the reaction mixture under ice bath conditions at 0 °C. All reactions were performed under argon protection and in the dark. The reaction system was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. Then, the reaction mixture was diluted with 10 mL of chloroform and quenched with 17.5 mL of saturated Na₂S₂O₃ solution, washed with 20 mL of water and 20 mL of saturated NaCl, the organic layers were combined and dried over anhydrous Na₂SO₄, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography using n-hexane as the eluent to give a colorless oily product.

[0148] Step b: Preparation of compound 24

[0149] Compound 23 (59.4 mg, 0.22 mmol), 4-carboxylic acid phenylboronic acid (36.3 mg, 0.22 mmol), tetrakis(triphenylphosphine)palladium (30 mg, 0.022 mmol), and sodium carbonate (1155 mg, 11 mmol) were dissolved in a mixed solution of toluene (6 mL), ethanol (2 mL), and water (3 mL). The reaction system was carried out under argon protection at 100 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phase was collected, washed with saturated brine, and dried over anhydrous Na₂SO₄. The organic phases were combined, concentrated to remove the solvent, and the crude product was purified by silica gel column chromatography (3 / 1, DCM / Hex).

[0150] Step c: Preparation of compound 25

[0151] Compound 24 (43.6 mg, 0.14 mM) and H2N-PEG-SH (500 mg, 0.1 mM, MW: 5000 g / mol) were dissolved in 5 mL of THF. EDC (21.5 mg, 0.14 mM) and DMAP (23 mg, 0.175 mM) were added to the solution. After stirring for 2 hours, cold diethyl ether was added to the solution, resulting in the precipitation of a white precipitate. The precipitate was filtered to obtain a pure white solid.

[0152] (2) Preparation method of DABCO compound polyethylene glycol macromolecule

[0153] The specific implementation method is the same as in Example 1.

[0154] 3. Modification of gold nanorods by active molecules (step g: preparation of nanogroup 26)

[0155] (1) Modification of naphthalene compounds

[0156] GNRs (10 mg), compound 25 (10 mg), and DABCO-PEG-SH (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 10 min to remove excess and unreacted compound 25 and DABCO-PEG-SH.

[0157] (2) Modification with internal peroxides

[0158] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. One drop of TFA was then added. The mixture was stirred in an open container at 0°C for 12 hours, irradiated with 18 W, 630 nm red light during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0159] A special note: here, compound 25 and the singlet oxygen quencher group DABCO derivative are first modified onto gold nanorods, and then the naphthalene ring is ring-opened and connected to a peroxide bridge using the method of generating internal peroxide. There are two reasons for this linking order: First, if the internal peroxide precursor compound is ring-opened to form an internal peroxide compound, the thiol group has a lone pair of electrons, which affects the generation of internal peroxide. Second, if the internal peroxide compound is linked to the gold nanorods, the reaction time is too long, which can easily lead to the instability of the internal peroxide compound and the generation of the precursor compound, ultimately resulting in little or no singlet oxygen generation at room temperature or 37°C.

[0160] 4. Modification of gold nanorods by single-component peroxide precursor compounds (step h: preparation of nanogroup 27)

[0161] GNR (10 mg) and compound 25 (10 mg) were mixed in 5 mL of HEPES buffer (pH 7.2, 20 mM) and sonicated for 1 minute. The mixture was then stirred for 12 hours. After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 10 min to remove excess, unreacted compound 19.

[0162] (2) Modification with internal peroxides

[0163] The precipitate obtained after centrifugation was dispersed in 2 mL of HEPES buffer (pH 7.2, 20 mM), and then a catalytic amount (approximately 0.5 mg) of methylene blue was added. The mixture was stirred in an open container at 0°C for 12 hours, with 18 W, 630 nm red light irradiation during the reaction. The methylene blue was removed using a low-temperature centrifuge.

[0164] It should be noted that the reason for first modifying the gold nanorods with compound 25 is the same as that mentioned above.

[0165] Example 5

[0166] In vitro singlet oxygen release experiment of internal peroxides.

[0167] SOSG was used as a singlet oxygen trapping reagent. GNRs-ENDO(10) and the nanogroup GNRs-ENDO-DABCO(9) were mixed with SOSG, and the release of singlet oxygen was detected using excitation light at 504 nm. Figure 2 b and Figure 2 As shown in Figure c, the horizontal axis represents wavelength, and the vertical axis represents the change in singlet oxygen release intensity. SOSG was used as a control group, and the singlet oxygen release of the control group is shown below. Figure 2 As shown in Figure a. Furthermore, using the absorption intensity at 504 nm as the ordinate, the changes in singlet oxygen release over time were compared between GNRs-ENDO (10), the nanogroup GNRs-ENDO-DABCO (9), and the control experiment (e.g., ). Figure 2 (As shown in d). Comparison revealed that GNRs-ENDO can release singlet oxygen even without a singlet oxygen quencher group, while GNRs-ENDO-DABCO with introduced quencher groups showed almost no detectable release of singlet oxygen. These experiments demonstrate that, in the designed molecular oxygen carrier, DABCO can efficiently convert toxic singlet oxygen into molecular oxygen for release, thus improving molecular oxygen yield while eliminating the damage of singlet oxygen to cells.

[0168] Example 6

[0169] Effects of hypoxia on cell proliferation.

[0170] NIH / 3T3 fibroblasts were seeded at 5000 cells per well into 96-well plates and incubated for 24 hours in a normoxic cell incubator at 37°C, 5% CO2. Different concentrations of GNRs, GNRs-ENDO (10), and GNRs-ENDO-DABCO (9) were then added. Afterward, incubation was continued for 24 hours in a cell incubator at 37°C, 5% CO2, and 0.1% O2. Cell viability was determined using the MTT assay. Figure 3 As can be seen, GNRs-ENDO-DABCO has a more positive effect on cell proliferation under hypoxic conditions than GNRs-ENDO, proving that the gold nanorod system has great application potential as an oxygen transport agent.

[0171] Example 7

[0172] Following the methods in Examples 5 and 6, in vitro singlet oxygen release experiments and experiments on the effects of hypoxia on cell proliferation were also performed on GNRs-ENDO-DABCO(14), GNRs-ENDO-DABCO(20), and GNRs-ENDO-DABCO(26). It was found that the release of singlet oxygen was not detected in gold nanoparticle oxygen carriers 14, 20, and 26, and they had a more positive effect on cell proliferation under hypoxia conditions.

Claims

1. A method for constructing a type of nano-oxygen delivery system based on internal peroxides, characterized in that, The method includes the following steps: Gold nanorods (GNRs), NAPH-PEG-SH, and DABCO-PEG-SH were mixed in a buffer solution for 12-15 h. After centrifugation, the mixture was dispersed in a buffer solution, methylene blue was added, followed by 1-2 drops of TFA. The mixture was stirred at -8 to 0 °C for 10-12 h under oxygen and red light irradiation. The methylene blue was removed by centrifugation. The structure of NAPH-PEG-SH is ; The structure of DABCO-PEG-SH is as follows: ; Where R is , or ; R 5 -R 7 R 9 -R 12 R 14 -R 16 Each is independently hydrogen or C1-C10 alkyl group; R 8 R 13 R 17 Each and independent , or ; The sums of n1 and n2 are independent integers between 0 and 6, and m is an integer between 0 and 6.

2. The method for constructing a nano-oxygen delivery system based on internal peroxides according to claim 1, characterized in that, The mass ratio of gold nanorods (GNRs), NAPH-PEG-SH, and DABCO-PEG-SH was 1:0.8-1.5:0.8-1.

5.

3. The method for constructing a nano-oxygen delivery system based on internal peroxides according to claim 1, characterized in that, Remove methylene blue by centrifugation at -4 to 0 ℃.

4. The method for constructing a nano-oxygen delivery system based on internal peroxides according to claim 2, characterized in that, The mass ratio of gold nanorods (GNRs), NAPH-PEG-SH, and DABCO-PEG-SH was 1:1:

1.

5. The method for constructing a nano-oxygen delivery system based on internal peroxides according to claim 1, characterized in that, The preparation method of the gold nanorods GNRs is as follows: 1) Mix hexadecyltrimethylammonium bromide solution with gold tetrachloride solution, add sodium borohydride solution and stir, let stand for 30-40 minutes to make seed solution; 2) Mix hexadecyltrimethylammonium bromide and sodium oleate and add to warm water, then add AgNO3 solution. Keep the mixture undisturbed at 30-35 ℃ for 15-20 minutes, then add gold tetrachloride solution and stir until the solution becomes colorless. Add hydrochloric acid to adjust the pH to 1.36±0.03, stir for another 15-20 minutes, then add ascorbic acid solution and stir the solution for 30-40 seconds as the growth solution. Add the seed solution from step 1 and keep undisturbed at 30-32 ℃ for 10-12 hours. Centrifuge at 7000 rpm for 30 minutes, remove the supernatant, and obtain the gold nanorods GNRs.

6. The method according to claim 5, characterized in that, In step 1, the concentrations of the hexadecyltrimethylammonium bromide solution, tetrachloroauric acid solution, and sodium borohydride solution are 0.2 M, 0.5 mM, and 0.01 M, respectively, with a volume ratio of 8-10 : 8-10 :

1.

7. The method according to claim 5, characterized in that, In step 2, the concentrations of hexadecyltrimethylammonium bromide and sodium oleate in the mixed solution are 0.0768 M and 0.02 M, respectively. The concentrations of AgNO3 solution, gold tetrachloroauric acid solution, hydrochloric acid and ascorbic acid solution are 4 mM, 1 mM, 12.1 M and 0.064 M, respectively. The volume ratio of the mixed solution of hexadecyltrimethylammonium bromide and sodium oleate, AgNO3 solution, gold tetrachloroauric acid solution, hydrochloric acid and ascorbic acid solution is 200-250 : 9-12 : 200-250 : 1.5-2 :

1.

8. The method according to claim 5, characterized in that, In step 2, the volume ratio of the seed solution to the growth solution is 1:600-650.

9. A molecular oxygen transporter with gold nanorods as a carrier prepared by the method according to any one of claims 1-8.

10. The use of the molecular oxygen delivery agent with gold nanorods as a carrier prepared by the method according to any one of claims 1-8 in the preparation of drugs to alleviate or treat organ and tissue damage caused by hypoxia.

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