A synthesis method and application of a composite nanomedicine capable of efficiently loading and temperature-controlled releasing sodium citrate

Abstract

The present invention discloses a synthesis method and application of a composite nanomedicine with efficient loading and temperature-controlled release of sodium citrate. The synthesis method comprises the following steps: dissolving DMPC and DPPC, the raw materials for synthesizing liposomes, and obtaining a thermosensitive liposome film after rotary evaporation; adding anhydrous ethanol to the formed liposome film, and then adding a sodium citrate aqueous solution containing ammonia water, and after the solution turns milky white, adding tetraethyl orthosilicate dropwise, stirring the reaction to obtain the product SiO2@SCT@lip, adding indocyanine green, stirring, and centrifuging and drying to finally obtain the SSLINP nanomedicine. The SSLINP synthesized by the present invention greatly increases the window concentration of SCT for treating tumors and significantly reduces the half-lethal concentration (IC50) of SCT for treating tumors. 50 ), with excellent safety and anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for synthesizing a composite nano drug loaded with sodium citrate and its application in the field of anti-tumor. Background Art

[0002] Citrate is a key metabolic factor and key metabolic regulator linking glycolysis and lipid metabolism in cellular energy homeostasis. Citrate treatment (sodium citrate and citric acid) has been reported to significantly inhibit tumor cell proliferation and growth in various tumor types. Mechanistically, citrate promotes excessive lipid biosynthesis and induces disruption of tumor cell lipid metabolism, leading to tumor cell senescence and growth inhibition. Furthermore, the serine-threonine protein kinase (ATM)-associated DNA damage response collaborates with the mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase (mTOR) signaling pathways to control citrate-induced tumor cell growth arrest and senescence. Furthermore, studies have demonstrated that citrate administration not only significantly inhibits tumor growth and progression in vivo but also exhibits synergistic antitumor effects with conventional chemotherapeutic drugs in combination with in vivo in a colon cancer xenograft model. These findings suggest that citrate administration is an effective and promising cancer treatment strategy.

[0003] Although citrate therapy has become an effective cancer treatment strategy, there are currently two main problems with sodium citrate (SCT) therapy: 1. SCT has an IC effect on tumors. 50 The IC of SCT to cancer cells is only about 2 times that of normal cells, so the therapeutic window is too small and there is a high safety risk in clinical application. 50 The concentration of SCT is as high as several millimolar, which is hundreds of times that of current first-line anticancer drugs (such as cisplatin), greatly increasing the difficulty of delivering sufficient drugs to tumor tissues. Therefore, it is necessary to develop composite nanomedicines that can efficiently load and controllably release SCT, increase the window concentration of SCT in treating tumors, and reduce the IC value of SCT in treating tumors. 50 , which is of great significance. Summary of the Invention

[0004] Purpose of the invention: To address the problem of too small therapeutic window and IC in the treatment of tumors using SCT. 50 The present invention provides a method for synthesizing a composite nanoparticle drug (SSLINP) that efficiently loads and temperature-controlled releases sodium citrate (SCT). The present invention first synthesizes SiO2 loaded with SCT, then uses DMPC and DPPC, the raw materials of thermosensitive liposomes, to coat SiO2, and finally adsorbs the photothermal agent ICG on its surface to finally obtain SSLINP. The composite nanoparticle drug synthesized by the present invention greatly increases the window concentration of sodium citrate for treating tumors and significantly reduces the IC value of sodium citrate for treating tumors. 50, has excellent safety and anti-tumor activity, and solves the bottleneck problem faced by using SCT to treat tumors.

[0005] The invention also provides synthesized composite nano medicine and application thereof.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a method for synthesizing a composite nanoparticle drug (SSLINP) with efficient loading and temperature-controlled release of sodium citrate (SCT), comprising the following steps:

[0007] (1) Dissolving DMPC and DPPC, the raw materials for synthesizing liposomes, and rotary evaporating to obtain a thermosensitive liposome film (lip);

[0008] (2) Anhydrous ethanol was added to the formed liposome film, and then a sodium citrate aqueous solution containing ammonia was added. After the solution turned milky white, tetraethyl orthosilicate was added dropwise. After stirring, the reaction was carried out to obtain the product SiO2@SCT@lip. Indocyanine green (ICG) was added at a ratio of SiO2@SCT@lip:ICG = 1:0.5. After stirring, the mixture was centrifuged and dried to finally obtain the SSLINP nanomedicine.

[0009] Wherein, the mass ratio of DMPC to DPPC in step (1) is 1:1-2.

[0010] Preferably, the mass ratio of DMPC to DPPC in step (1) is 1:1.

[0011] Wherein, in step (1), DMPC and DPPC are dissolved in chloroform, and the ratio of DMPC and DPPC to chloroform is 6-10:1 mg / mL.

[0012] Preferably, in step (1), DMPC and DPPC are dissolved in chloroform, and the ratio of DMPC to DPPC and chloroform is 6:1 mg / mL.

[0013] Wherein, the sodium citrate aqueous solution containing ammonia water in step (2) is prepared by dissolving 36-40 mg of sodium citrate in 2-3 mL of purified water, and then adding 400-420 μL of ammonia water.

[0014] Preferably, the sodium citrate aqueous solution containing ammonia water in step (2) is prepared by dissolving 36 mg of sodium citrate in 2 mL of purified water, and then adding 420 μL of ammonia water.

[0015] Wherein, in step (2), 200-250 μL of tetraethyl orthosilicate is added dropwise; tetraethyl orthosilicate is added to the reaction in 5 batches.

[0016] Wherein, the reaction in step (2) is carried out at room temperature for 12-15 hours, and the stirring speed is 500-800 rpm.

[0017] Preferably, the reaction in step (2) is carried out at room temperature for 14 hours with a stirring speed of 600 rpm.

[0018] Wherein, in step (2), the mass ratio of indocyanine green ICG to the reaction product SCT@SiO2@lip is 1-2:0.5.

[0019] Preferably, in step (2), the mass ratio of indocyanine green ICG to the reaction product SCT@SiO2@lip is 1:0.5.

[0020] In step (2), indocyanine green (ICG) was added and stirred at room temperature for 3-5 hours at a stirring speed of 500-600 rpm.

[0021] Preferably, indocyanine green (ICG) is added in step (2), and the mixture is stirred at room temperature for 4 h at a stirring speed of 600 rpm.

[0022] The composite nanomedicine (SSLINP) with efficient loading and temperature-controlled release of sodium citrate (SCT) synthesized by the synthesis method of the present invention.

[0023] The invention relates to the use of the composite nanomedicine (SSLINP) in the preparation of anti-tumor drugs.

[0024] Preferably, the synthesis method of the composite nanomedicine (SSLINP) comprises the following steps:

[0025] 30 mg of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) (1:1 mass ratio) were weighed and dissolved in 5 mL of chloroform. The mixture was placed in a 50 mL round-bottom flask and rotary evaporated to obtain thin-film liposomes, to which 50 mL of anhydrous ethanol was added. Next, 36 mg of sodium citrate was dissolved in 2 mL of purified water. 420 μL of ammonia was added to the sodium citrate aqueous solution, mixed thoroughly, and added all at once to the 50 mL of anhydrous ethanol in the flask. After a 3-minute reaction, 250 μL of tetraethyl silicate was added dropwise (50 μL was added to the reaction at intervals of 1 minute). The reaction was allowed to proceed for 14 hours, followed by centrifugation and washing with a water-ethanol mixture to obtain the product, SiO2@SCT@lip. Finally, the product was loaded with ICG at a mass ratio of 1:0.5. The product was stirred with a magnetic stirrer for 4 hours and then dried to obtain the SSLINP nanoparticle.

[0026] During the preparation of the invention, the active drug sodium citrate is encapsulated in the SiO2 template by the SiO2 salt template method, and the temperature-controlled release of the active drug sodium citrate is achieved by utilizing the combined action of thermosensitive liposomes and indocyanine green.

[0027] The present invention encapsulates salt drugs in SiO2 to form a nano drug with a particle size of only 200nm, thereby avoiding a large amount of loss. The prior art is to directly use sodium citrate for cancer treatment, and the active concentration required is very large, and the amount required for sodium citrate is very large, reaching more than 5mM (Adv.Sci.2021, 2101553). The designed nano drug of the present invention is released by precise temperature control, greatly reducing the amount of sodium citrate, and the amount of sodium citrate can be controlled within tens of micromoles (for SCT and SSLINP in terms of SCT quantification, the conversion formula is 1mM=0.294mg / mL). It solves the problem that the amount of citrate used is large, which easily causes drug resistance in tumor patients and the harm of the cardiac toxic side effects of citrate. In addition, the structure of the present invention uses hollow SiO2 as a template, the active drug sodium citrate is encapsulated in the SiO2, a layer of thermosensitive liposome film is wrapped outside the SiO2, and the photosensitizer indocyanine green ICG is loaded on the liposome film. Under the irradiation of 808nm laser, the thermosensitive liposome is cleaved, thereby destroying the SiO2 structure and finally releasing the active drug sodium citrate.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] 1. The SSLINP prepared in the present invention greatly increases the window concentration for treating tumors with sodium citrate.

[0030] 2. The preparation of SSLINP in the present invention significantly reduces the IC value of sodium citrate in treating tumors 50 , significantly improving the tumor therapeutic activity.

[0031] 3. The SSLINP of the present invention has excellent safety and anti-tumor activity, and has very good selectivity for normal cells and tumor cells.

[0032] 4. The preparation method of the present invention is simple, the raw materials are widely available, and it can be produced and utilized on a large scale and used as a tumor treatment drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Transmission electron microscopy image of SSLINP (scale = 500 nm);

[0034] Figure 2 This is the photothermal performance diagram of SSLINP;

[0035] Figure 3 This is a diagram of SCT release triggered by SSLINP photothermal stimulation;

[0036] Figure 4 The graph shows the activity of SCT and SSLINP on tumor cells (mouse breast cancer cells, 4T1) and normal cells (mouse fibroblasts, L929).

[0037] Figure 5 Schematic diagram of in vivo anticancer activity detection. DETAILED DESCRIPTION

[0038] The present invention can be better understood based on the following examples. Those skilled in the art will readily appreciate that the contents described in the examples are merely illustrative of the present invention and should not and will not limit the present invention described in detail in the claims. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions or under conditions recommended by the manufacturer.

[0039] The sodium citrate (SCT) used in the present invention is sodium citrate dihydrate, CAS No.: 6132-04-3.

[0040] Example 1

[0041] Synthesis of SSLINP

[0042] Dissolve 30 mg of equal weight of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) in 5 mL of chloroform and place in a round-bottom flask. Rotary evaporate for 10 minutes to obtain thin film liposomes. Add 50 mL of anhydrous ethanol to all the thin film liposomes. Then, 36 mg of sodium citrate was dissolved in 2 mL of pure water, and 420 μL of 25% ammonia water was added to the sodium citrate aqueous solution. After mixing evenly, the mixture was added to the above flask containing 50 mL of anhydrous ethanol at one time. After 3 minutes, 250 μL of tetraethyl silicate was added dropwise (added in 5 times, 50 μL was added to the reaction each time, with an interval of 1 minute). The mixture was stirred at room temperature (500 rpm) for 14 hours. The precipitate was washed by centrifugation with a water-ethanol (1:1) mixed solution and dried to obtain the product SiO2@SCT@lip. Finally, a DMSO solution containing ICG was added according to a mass ratio of 1:0.5 between the product SiO2@SCT@lip and ICG. After stirring at room temperature (600 rpm) for 4 hours, the precipitate was washed once with anhydrous ethanol and water respectively, and the solid was dried to obtain SSLINP nanomedicine (SiO2@lip@ICG). Figure 1 As shown in Figure 2, transmission electron microscopy observation of the size and morphology of SSLINP showed that SSLINP was uniform and single-distributed spherical particles. Chemical spectroscopy (ICP) analysis showed that SSLINP contained approximately 36% SCT by mass.

[0043] Example 2

[0044] SSLINP's light and thermal performance testing

[0045] 50 μg, 75 μg, and 100 μg of the SSLINP nanomaterial in Example 1 were respectively dispersed in 1 mL of water to prepare SSLINP solutions with concentrations of 50 μg / mL, 75 μg / mL, and 100 μg / mL. The power of the 808 nm laser was 1 W / cm 2 , irradiate the SSLINP solution for 5 minutes, and use a thermal imager to observe and record the temperature change of the SSLINP solution. Figure 2 As shown in the figure, with the extension of illumination time, the temperature of SSLINP solutions of various concentrations increased significantly, proving that SSLINP has photothermal conversion ability.

[0046] Example 3

[0047] SSLINP's light-triggered temperature-controlled release SCT assay

[0048] 5 mg of SSLINP in Example 1 was dispersed in 5 mL of water to obtain 1 mg / mL of nanomaterial. After standing at room temperature for 1 min, 2 min, 3 min, 4 min, and 5 min, the supernatant was centrifuged and the SCT concentration (Cu) was measured. 2+ Under 808 nm laser irradiation conditions, after 1 min, 2 min, 3 min, 4 min, and 5 min, the SSLINP supernatant was centrifuged and the SCT concentration (Cu 2+ Complex UV method). Figure 3 The results show that SCT release from SSLINPs under irradiation and non-irradiation conditions increased with increasing exposure time. However, SCT release from the non-irradiated group showed no significant increase over time. This demonstrates that SSLINPs can release the active drug SCT in a temperature-controlled manner under 808nm laser triggering.

[0049] Example 4

[0050] Dark toxicity and phototoxicity of SSLINP and SCT in normal cells and tumor cells

[0051] 4T1 and L929 cells were cultured at a pressure of (2x10 5The cells were seeded in 96-well plates with DMEM medium containing 10% fetal bovine serum and cultured overnight at 37°C in 5% CO2. The next day, different concentrations of the above-mentioned SSLINP (based on the SCT content in the SSLINP) (94, 5.88, 8.82, 14.7, and 20.58 mg / mL, and 2, 4, 6, 8, and 10 mg / mL for the 4T1 cell group) and the SSLINP synthesized in Example 1 (3.6, 10.8, 18, and 25.2 mg / mL for the L929 cell group and 18, 36, 57.6, and 86.4 μg / mL for the 4T1 cell group, based on the SCT content in the SSLINP) were added to the L929 and 4T1 cells, respectively. The cells were incubated in a 37°C incubator for 24 hours, and cell viability was determined by the MTT assay.

[0052] 4T1 and L929 cells were seeded in 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 overnight. The next day, the SSLINP synthesized in Example 1 (the concentrations of the 4T1 cell group were 3.6, 7.2, and 14.4 μg / mL; the concentrations of the L929 cell group were 72, 216, and 360 μg / mL, and the above concentrations were quantitatively determined based on the SCT content in SSLINP) were added to the cells. The cells were placed in an incubator and incubated at 37°C for 24 hours. The cells were then illuminated with an 808 nm laser (1 W / cm 2 ) irradiated cells for 5 min, then placed the cells in an incubator at 37°C for 24 h, and then the cell viability was determined by MTT assay. Figure 4 It was shown that under non-illumination conditions, the IC of SSLINP in L929 cells 50 375 times that of 4T1 cells; Under light conditions, the IC of SSLINP in L929 cells 50 The IC of SCT in L929 cells was 31.63 times that of 4T1 cells. 50 The IC value of SSLINP on 4T1 tumor cells was 1.87 times that of 4T1 cells under both illumination and non-illumination conditions. 50 The above results indicate that SSLINP significantly increases the window concentration of sodium citrate for tumor treatment and significantly reduces the IC value of sodium citrate for tumor treatment. 50 , its tumor therapeutic activity is significantly increased, and its selectivity for tumor cells and normal cells is stronger. It has excellent safety and anti-tumor activity, and solves the bottleneck problem faced by using SCT to treat tumors.

[0053] Example 5

[0054] In vivo anticancer activity assay

[0055] Female Balb / c mice were treated with 4T1 cells to bear tumors. When the tumors grew to 100 mm 3 The tumor-bearing mice were divided into 5 groups, and their body weight and tumor volume were measured every day after illumination. On the 1st, 3rd, 5th, 7th, 9th, 11th and 13th days, PBS (200uL, pH=7.4) and the SSLINP (compound drug) in Example 1 (15mg / kg mouse body weight), high concentration sodium citrate (30mg / kg mouse body weight) and low concentration sodium citrate (5.4mg / kg mouse body weight) were injected into the tail vein respectively. The content of sodium citrate in the SSLINP administration group was also 5.4mg / kg. Among them, the SSLINP (compound drug) sodium citrate nanomaterial synthesized in Example 1 was formulated into a solution with DMSO and injected at the above dose. After 4 hours, one of the two SSLINP administration groups was selected and laser-assisted with 808nm laser (0.3W / cm 2 ) The tumor tissues of mice were irradiated for 6 minutes. Fourteen days later, the mice were euthanized. The volume changes of mice in each group were recorded during the 14-day treatment period. Figure 5 As shown, CDT treatment in the SSLINP group treated with light completely inhibited tumor growth and had the best in vivo anti-tumor activity, followed by the high-dose sodium citrate group. The non-light-treated SSLINP group and the low-dose sodium citrate group had poor in vivo anti-tumor effects.

Claims

1. A method for synthesizing a composite nanoparticle drug (SSLINP) with efficient loading and temperature-controlled release of sodium citrate (SCT), characterized in that: The steps include: (1) Dissolve the raw materials DMPC and DPPC for synthesizing liposomes, and obtain a thermosensitive liposome film (lip) after rotary evaporation; (2) Anhydrous ethanol was added to the formed liposome film, followed by a sodium citrate aqueous solution containing ammonia, and then tetraethyl orthosilicate was added. After stirring, the reaction was performed to obtain the product SiO2@SCT@lip. Indocyanine green (ICG) was added, and after stirring, the mixture was centrifuged and dried to finally obtain the SSLINP nanomedicine. The mass ratio of DMPC to DPPC in step (1) is 1:1-2; The sodium citrate aqueous solution containing ammonia in step (2) is prepared by dissolving 36-40 mg of sodium citrate in 2-3 mL of purified water, and then adding 400-420 μL of ammonia; In step (2), add 200-250 μL of tetraethyl orthosilicate dropwise in multiple additions; The reaction in step (2) is carried out at room temperature for 12-15 hours with a stirring speed of 500-800 rpm; In step (2), the mass ratio of indocyanine green ICG to the reaction product SCT@SiO2@lip is 1:0.5-1; In step (2), indocyanine green (ICG) was added and stirred at room temperature for 3-5 h at a stirring speed of 500-600 rpm.

2. The synthesis method according to claim 1, wherein In step (1), DMPC and DPPC are dissolved in chloroform, and the ratio of DMPC and DPPC to chloroform is 6-10:1 mg / mL.

3. A composite nanoparticle drug (SSLINP) with efficient loading and temperature-controlled release of sodium citrate (SCT) synthesized by the synthesis method of claim 1.

4. Use of the composite nanomedicine (SSLINP) according to claim 3 in the preparation of anti-tumor drugs.

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