Cobalt carbonate nanorod as well as construction method and application thereof

Abstract

The invention discloses a cobalt carbonate nanorod as well as a construction method and application thereof, and the surface of the cobalt carbonate nanorod is modified by a liposome two-step method, specifically, the cobalt carbonate nanorod is firstly modified by DOPA and then modified by DPPC, cholesterol and DSPE-PEG. The cobalt carbonate nanorod (CoCO3) is prepared by adopting a hydrothermal method. The material not only can be used as proton nano sponge to neutralize an acidic tumor microenvironment, but also can be used as an acid-responsive drug carrier to effectively load a hydrophobic alkalescent drug EVO (Ethylene Vinyl Oxide). And after the surface modification, the CoCO3-PEG-EVO shows excellent biocompatibility. After the CoCO3-PEG-EVO is intravenously injected, the CoCO3-PEG-EVO can be gathered at a tumor part, and meanwhile, the acid neutralization effect of CoCO3 is utilized to overcome the ion capture effect and enhance the EVO uptake of cancer cells, so that the apoptosis of the cancer cells is promoted. On the other hand, excessive HCO3 <-> and H2O2 in the tumor are subjected to Fenton-like reaction with Co < 2 + >, and then a large amount of CO3 <-> is generated to cause cancer cell necrosis. The CoCO3-PEG-EVO can provide a potential strategy for enhancing the tumor treatment effect of weakly alkaline drugs by adjusting the tumor acidic microenvironment.

Description

Technical Field

[0001] The invention relates to a cobalt carbonate nanorod, a construction method and application thereof, and belongs to the field of drug carriers. Background Art

[0002] Compared with normal tissues, where both intracellular and extracellular pH are alkaline (pHe 7.4, pHi 7.2), tumor cells have only alkaline intracellular pH (pHi 7.1-7.8) and weakly acidic extracellular pH (pHe 6.4-7.1). The formation of this weakly acidic tumor microenvironment is closely related to local hypoxia, but its fundamental reason lies in the vigorous glycolysis of tumor cells. The large amount of lactic acid produced during glycolysis leads to the depletion of intracellular protons (H + ) concentration significantly increases, and in order to resist this acidic stress, tumor cells activate and express H + (and lactate) transporters to increase the intracellular pH. The cell membrane acts as a semipermeable barrier between the intracellular and extracellular environments, allowing uncharged small molecules to enter the cell through the phospholipid layer, while charged substances are difficult to penetrate. Therefore, weakly acidic drug molecules are easily taken up by cancer cells, while weakly alkaline drugs are ionized in the weakly acidic tumor microenvironment, making them difficult to be taken up by cancer cells and reducing the therapeutic effect. This phenomenon is called "ion capture."

[0003] Studies have shown that oral sodium bicarbonate (NaHCO3) can effectively neutralize the acidic tumor microenvironment, but it is worth noting that NaHCO3 does not directly target tumors, but increases the overall pH value outside the cells. Although this effect helps to neutralize acidity, it may cause metabolic alkalosis and pose a potential risk to the body. Therefore, clinical trials using NaHCO3 have not been successful. Achilefu et al. found that the CO3 on the surface of calcium carbonate (CaCO3) nanoparticles 2- It can effectively neutralize H in the tumor microenvironment + Therefore, in recent years, the application research of CaCO3 nanoparticles as an acid-responsive dissociated biomaterial in the biomedical field has gradually increased. However, the effect of killing cancer cells by relying solely on the acid neutralization of CaCO3 is very limited, and the excessive HCO3 released by the system - The effects on tumor growth and whether they can be repurposed to further achieve anticancer effects have not yet been studied.

[0004] In the field of biology, the Fenton reaction between ferrous ions and hydrogen peroxide is of great significance as the main source of oxidative stress. Normally, the Fenton reaction produces hydroxyl radicals (·OH) in acidic solutions, but in neutral solutions containing HCO3 - Even at low concentrations, carbonate anion radicals (CO3· -) becomes the main active oxidation product. The study also found that transition metals such as Mn 2+ and Co 2+ It can trigger a Fenton-like reaction in a neutral solution to produce CO3· - However, the current research on CO3 - The anticancer effects and related mechanisms of β-catenin have not yet been explored. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a cobalt carbonate nanorod with the function of acid neutralization and inducing cancer cell necrosis, as well as a construction method and application thereof.

[0006] Technical solution: The present invention provides a cobalt carbonate nanorod, the surface of which is modified by a two-step liposome method, first by DOPA modification, and then by DPPC, cholesterol and DSPE-PEG modification.

[0007] Further, the DSPE-PEG is DSPE-PEG 5k .

[0008] The present invention also provides a method for constructing the above-mentioned cobalt carbonate nanorods, and the construction method comprises the following steps:

[0009] (1) Synthesis of cobalt carbonate nanorods;

[0010] (2) The cobalt carbonate nanorods were modified by a two-step liposome method, first by DOPA modification, and then by DPPC, cholesterol and DSPE-PEG modification to obtain CoCO3-PEG.

[0011] Furthermore, the specific steps of step (1) include: dripping the ammonium carbonate aqueous solution into the cobalt acetate under magnetic stirring, heating at 120° C. under high pressure for 30 minutes, collecting the cobalt carbonate nanorods, and washing with ethanol to obtain the nanorods.

[0012] Furthermore, the specific steps of step (2) include: dispersing the cobalt carbonate nanorods obtained in step (1) in ethanol, adding DOPA under ultrasound, collecting the precipitate by centrifugation after the reaction, dispersing it in chloroform after washing with ethanol, adding DPPC, cholesterol, and DSPE-PEG and stirring overnight, removing the chloroform by rotary evaporation, and adding ultrapure water for hydration to finally form CoCO3-PEG.

[0013] Furthermore, the mass ratio of the cobalt carbonate nanorods, DPPC, cholesterol and DSPE-PEG is 1:4:2:8.

[0014] The present invention also provides the use of the cobalt carbonate nanorods in preparing a hydrophobic weakly alkaline drug carrier.

[0015] The present invention also provides the use of the cobalt carbonate nanorods in the preparation of anti-tumor drugs.

[0016] The cobalt carbonate nanorods of the present invention improve the uptake of hydrophobic weakly alkaline drugs by cancer cells by regulating the acidic microenvironment of tumors, and produce CO3· - , realizing the application of combined treatment effect.

[0017] The present invention also provides an anti-tumor drug, wherein EVO is loaded on the hydrophobic region of the liposome bilayer of the above-mentioned cobalt carbonate nanorod.

[0018] Furthermore, the mass ratio of EVO to cobalt in cobalt carbonate nanorods is 1:0.25-2.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention adopts a hydrothermal method to prepare cobalt carbonate nanorods (CoCO3). This material can not only act as a proton nanosponge to neutralize the acidic tumor microenvironment, but also act as an acid-responsive drug carrier to effectively load the hydrophobic weak alkaline drug EVO. After surface modification, CoCO3-PEG-EVO exhibits excellent biocompatibility. After intravenous injection of CoCO3-PEG-EVO, it can accumulate at the tumor site. At the same time, the acid neutralization effect of CoCO3 is used to overcome the ion capture effect, enhance the absorption of EVO by cancer cells, and thus promote cancer cell apoptosis. On the other hand, excess HCO3 in the tumor - and H2O2 and Co 2+ A Fenton-like reaction occurs, which produces a large amount of CO3 - Therefore, CoCO3-PEG-EVO can provide a potential strategy for enhancing the tumor therapeutic effect of weakly alkaline drugs by regulating the tumor acidic microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the construction route of CoCO3-PEG-EVO of an embodiment of the present invention and its application mechanism in tumor treatment;

[0021] Figure 2 This is a characterization result diagram of CoCO3-PEG of an embodiment of the present invention;

[0022] Figure 3 pH adjustment and CO3· - The ability to produce;

[0023] Figure 4 The drug loading capacity and acid-responsive release capacity of CoCO3-PEG of the embodiment of the present invention;

[0024] Figure 5 The ability of CoCO3-PEG of the present invention to regulate intracellular pH;

[0025] Figure 6 The in vitro combined treatment of CoCO3-PEG-EVO of the embodiment of the present invention;

[0026] Figure 7 The in vivo behavior of CoCO3-PEG of the embodiments of the present invention;

[0027] Figure 8 This is an in vivo therapeutic effect evaluation of CoCO3-PEG-EVO according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0029] Example 1 Construction of cobalt carbonate nanorods

[0030] S1: Synthesis of cobalt carbonate nanorods

[0031] Under magnetic stirring, an aqueous solution of ammonium carbonate (46.5 mg, 0.483 mmol) (30 mL) was dropped into cobalt acetate (40 mg, 0.161 mmol) (10 mL) using a syringe pump. The mixture was then transferred to a 100 mL reactor, which was heated to 120° C. in an oven for 30 minutes. The reactor was removed, cooled to room temperature, and the purple cobalt carbonate nanorods were collected by centrifugation and washed three times with ethanol.

[0032] S2: Two-step surface modification of liposomes

[0033] First, 0.1 mL of DOPA (dioleoylphosphatidic acid sodium salt) in chloroform (1 mg mL -1 ) was added to 1 mL of ethanol solution (1 mg mL -1 ) and mixed with ultrasound (160W) for 20 minutes. The mixed solution was then centrifuged (14800rpm, 5min), washed three times with ethanol and dispersed in 5mL chloroform, DPPC (dipalmitoylphosphatidylcholine), cholesterol and DSPE-PEG were added. 5k (Distearoylphosphatidylethanolamine-methoxypolyethylene glycol 5000) (CoCO3, DPPC, cholesterol and DSPE-PEG 5k After stirring overnight, the chloroform was removed by rotary evaporation, and 5 mL of ultrapure water was added for ultrasonic hydration to obtain CoCO3-PEG (the chloroform solution contained CoCO3, DPPC, cholesterol, DSPE-PEG 5k, mass ratio of 1:4:2:8), and CoCO3-PEG-EVO was prepared by the same method (the chloroform solution also contained EVO, wherein the mass ratio of EVO to cobalt in cobalt carbonate nanorods was 1:1).

[0034] The preparation process of CoCO3-PEG is as follows Figure 1 a; the preparation process of CoCO3-PEG-EVO is as follows Figure 4 As shown in a, the application mechanism in tumor treatment is as follows Figure 1 As shown in b.

[0035] Example 2 Performance Verification of Cobalt Carbonate Nanorods

[0036] 1. Characterization of cobalt carbonate nanorods

[0037] Cobalt carbonate (CoCO3) synthesized by hydrothermal method has a rod-like structure ( Figure 2 a), and the morphology of CoCO3-PEG obtained after surface liposome modification by two-step method remained unchanged ( Figure 2 b), but the size increases from 15nm to 25nm ( Figure 2 c). After lactic acid (7 mM) was added to the normal 1640 cell culture medium to reduce its pH from 8 to 6.8, CoCO3-PEG (Co: 0.848 mM) and sodium bicarbonate (7 mM) could slowly adjust the pH of the solution back to 8 within 3 h ( Figure 2 d), indicating that CoCO3-PEG has stronger acid neutralization ability than sodium bicarbonate.

[0038] 2. pH regulation and ROS generation ability verification of CoCO3-PEG

[0039] In pure HCl and HCl-NaHCO3 (1:0.5) solutions (the initial pH of the two solutions was 1.83 and 2.13, respectively), the morphology of CoCO3-PEG was significantly destroyed ( Figure 3 ab). Meanwhile, the size of CoCO3-PEG in HCl and HCl-NaHCO3 (1:0.5) solutions also decreased ( Figure 3 e). However, in HCl-NaHCO3(1:1) and HCl-NaHCO3(1:2) solutions (the initial pH of the two solutions were 3.87 and 6.35, respectively), the structure of CoCO3-PEG was partially damaged ( Figure 3 cd), and the size did not change significantly. In addition, due to the structural change of CoCO3-PEG, Co released from CoCO3-PEG was detected in this process. 2+ The amount of Figure 3f). In addition, CoCO3-PEG can also increase the pH of the other three solutions to neutral except the free HCl solution ( Figure 3 g) Detection of Co by using ROS probe DCFH-DA 2+ With excess HCO3 - CO3· - , it was found that as HCO3 - The increase in CO3 - The production of , and the fluorescence enhancement of DCFH-DA was the most significant ( Figure 3 h).

[0040] 3. Verification of drug loading capacity and acid-responsive release ability of CoCO3-PEG

[0041] This example uses the characteristic UV-visible absorption peak of EVO at 268 nm to measure the loading efficiency of CoCO3-PEG on EVO ( Figure 4 b). By gradually increasing the mass ratio of EVO to cobalt in CoCO3-PEG (1:0.25 to 1:2), we successfully improved the loading efficiency until a saturated loading rate of 72.5% was reached (EVO: Co 1:2, Figure 4 c). Subsequently, we selected a relatively stable 60% loading condition (EVO: Co 1:1) and tested the release behavior of EVO in normal and acidic 1640 cell culture media. The EVO release rate of CoCO3-PEG-EVO in acidic cell culture media (containing 7mM lactic acid) was significantly faster than that in neutral cell culture media ( Figure 4 d).

[0042] 4. Verification of the ability of CoCO3-PEG to regulate intracellular pH

[0043] This example detects the acid-base environment in mouse breast cancer cells 4T1 after treatment with lactic acid (LA) and CoCO3-PEG. 2',7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) is a cell-permeable non-fluorescent dye that can be converted into the fluorescent substance BCECF under the action of intracellular esterase to indicate changes in the pH inside the cell. 5After 4T1 cells were inoculated in a 12-well plate at a concentration of 1 / well for 24 hours, they were incubated with 1640 medium containing LA (7mM), 1640 medium containing LA (7mM) and sodium bicarbonate (7mM), and 1640 medium containing LA (7mM) and CoCO3-PEG (Co: 0.848mM) for 8 hours (6 replicates per group). It was found that the fluorescence intensity of intracellular BCECF decreased after incubation with medium containing LA (7mM), suggesting that the pH value inside the cells decreased. The addition of sodium bicarbonate and CoCO3-PEG can restore the fluorescence intensity of intracellular BCECF ( Figure 5 a, b), indicating that both sodium bicarbonate and CoCO3-PEG have the ability to neutralize lactic acid.

[0044] 5. Verification of the in vitro combined therapeutic effect of CoCO3-PEG-EVO

[0045] In this example, we evaluated the chemotherapeutic activity of EVO under different pH environments. First, 4T1 cells were seeded in a 96-well plate at a concentration of 8000 / well for 24 hours, and then incubated for 24 hours with 1640 medium containing EVO, 1640 medium containing LA (7mM) and EVO, and 1640 medium containing LA (7mM), sodium bicarbonate (7mM) and EVO, respectively. The concentration of EVO was in the range of 0-100μM, and finally the MTT experiment was used to detect the survival rate of the cells. The results showed that compared with the standard cell culture medium, the anticancer effect of EVO in the acidic cell culture medium treated with LA was weakened due to the ion capture effect ( Figure 6 a) This effect was counteracted by the additional addition of NaHCO3.

[0046] 4T1 cells were seeded in 96-well plates at a concentration of 8000 / well for 24 hours, and then incubated for 24 hours with 1640 medium containing CoCO3-PEG and 1640 medium containing LA (7mM) and CoCO3-PEG, respectively, where the concentration of Co was in the range of 0-50mg / L, and finally the MTT assay was used to detect the cell survival rate. The results showed that CoCO3-PEG could effectively induce cancer cell death in both normal and acidic cell culture media, which was attributed to the fact that it not only neutralized the H in the culture medium, but also induced the death of cancer cells. + The pH was adjusted to neutral and at neutral pH, with the aid of Co 2+ With HCO3 - The Fenton-like reaction with H2O2 generates a large amount of CO3 in the cell. - ( Figure 6 b) In addition, CoCO3 not only acts as a carrier to deliver EVO directly into cells, but also promotes the penetration of hydrophobic EVO into cells through neutralization and produces CO3· -The combined therapeutic effect was further enhanced. Therefore, compared with EVO and CoCO3-PEG, CoCO3-PEG-EVO had a more significant damaging effect on cancer cells in normal and acidic cell culture media ( Figure 6 c).

[0047] In addition, after the cells were seeded in a 6-well plate at a concentration of 300,000 / well for 24 hours, they were incubated with 1640 medium containing CoCO3-PEG (25 mg / L), EVO (50 μM) and CoCO3-PEG-EVO (Co: 25 mg / L; EVO: 50 μM) for 12 hours, and then the cells in each group were analyzed by flow cytometry. It was found that CoCO3-PEG mainly caused cancer cell necrosis, while EVO promoted cancer cell apoptosis. CoCO3-PEG-EVO showed a dual effect, which could induce cancer cell apoptosis and necrosis at the same time ( Figure 6 d and e). Western blotting experiments confirmed that EVO induced apoptosis of cancer cells by downregulating the anti-apoptotic protein Bcl-2 and upregulating the pro-apoptotic protein Bax, while CoCO3-PEG induced mitochondrial dysfunction and led to cell necrosis by upregulating serine-threonine kinase receptor interacting protein 1 (RIP1) and RIP3 ( Figure 6 f). In addition, CoCO3-PEG-EVO can induce cancer cell death through both EVO-mediated apoptosis and CoCO3-PEG-mediated necrosis pathways ( Figure 6 g).

[0048] 7. Verification of the in vivo behavior of CoCO3-PEG

[0049] This example explores the biological behavior of CoCO3-PEG nanoparticles in animals. First, CoCO3-PEG was labeled with a fluorescent dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindole dicarbocyanine, 4-chlorobenzenesulfonate, DiD), and then CoCO3-PEG-DiD was tracked in vivo using a small animal in vivo fluorescence imaging system ( Figure 7 a). 4T1 cells (2*10 7 / mL) was used to inoculate subcutaneous tumors in female Balb / c mice (6 weeks old, 25 g) purchased from the Experimental Animal Center of Yangzhou University. Each mouse was inoculated with 50 μL. When the tumor grew to 200 mm 3Free DiD or CoCO3-PEG-DiD was injected into tumor-bearing mice. Compared with mice injected with free DiD (0.74 mg / kg), the signal intensity in the tumor area of ​​mice injected with CoCO3-PEG-DiD (Co: 10 mg / kg; DiD: 0.74 mg / kg) was significantly and stably enhanced and reached the highest level 18 hours after injection. This finding suggests that these nanoparticles can be effectively enriched in the tumor site ( Figure 7 b). In addition, we quantitatively analyzed the pharmacokinetics of free DiD and CoCO3-PEG-DiD in mice by detecting the fluorescence signal of DiD in the blood. The results showed that free DiD was rapidly cleared in the body, while CoCO3-PEG-DiD showed a longer blood circulation time ( Figure 7 c). After 24 hours, fluorescence imaging of the main organs and tumors of mice showed that free DiD mainly accumulated in the spleen and liver, with minimal accumulation in tumors. Although CoCO3-PEG-DiD showed significant accumulation in the spleen and liver, it also accumulated significantly in the tumor site ( Figure 7 d, e). In addition, this study used BCECF as a pH fluorescent probe and analyzed the fluorescence of BCECF in the tumor site by imaging ( Figure 7 f), compared with the tumors of mice injected with free DiD, the ratio of the fluorescence intensity of BCECF in the tumor under 480nm and 440nm excitation was significantly increased due to the regulation of intratumoral pH by CoCO3-PEG-DiD.

[0050] 8. Verification of the in vivo therapeutic effect of CoCO3-PEG-EVO

[0051] In this example, tumor-bearing mice were divided into four groups, including a control group (PBS), a CoCO3-PEG (Co: 17 mg / kg) group, an EVO (EVO: 10 mg / kg) group, and a CoCO3-PEG-EVO (Co: 17 mg / kg; EVO: 10 mg / kg) group. 3 At 14 days, the four groups of mice were injected with the response material through the tail vein, and the day was recorded as day 0. The administration was repeated on days 3, 6, and 9. After 14 days of treatment and observation, it was found that although EVO could induce apoptosis of cancer cells, its low bioavailability limited its inhibitory effect on tumor growth. Due to the tumor enrichment and acid neutralization ability of CoCO3-PEG, CoCO3-PEG produces CO3 while neutralizing the acidic tumor microenvironment. -, thereby achieving moderate inhibition of tumor growth by promoting cancer cell necrosis. It is worth noting that the therapeutic advantages of CoCO3 and its ability to improve intracellular EVO uptake enable CoCO3-PEG-EVO to significantly inhibit tumor growth ( Figure 8 a). The most severe cell damage was observed in the tumor tissues of mice treated with the CoCO3-PEG-EVO combination, while the tumor cells in the other groups mostly or partially retained their normal morphology, which was confirmed by the histological staining results of the tumors in each group ( Figure 8 b).

[0052] In summary, this example successfully constructed a simple and universal method to prepare acid-dissociable and acid-neutralized CoCO3 nanostructures by hydrothermal method. 2+ and HCO3 - The release of CO3· - The Fenton-like reaction triggered the necrosis of cancer cells. By loading the EVO drug, the CoCO3 nanocarrier not only enhanced the uptake of EVO by cancer cells through endocytosis, but also broke the ion capture effect during acid neutralization, enhancing the diffusion of the weakly alkaline drug EVO into cancer cells. Our study provides a potential strategy for enhancing the tumor therapeutic effect of weakly alkaline drugs by regulating the tumor acidic microenvironment.

Claims

1. A cobalt carbonate nanorod, characterized in that: The surface of the cobalt carbonate nanorod is modified by a two-step liposome method, firstly by DOPA modification, and then by DPPC, cholesterol and DSPE-PEG modification.

2. The cobalt carbonate nanorods according to claim 1, characterized in that The DSPE-PEG is DSPE-PEG 5k .

3. A method for constructing cobalt carbonate nanorods according to any one of claims 1 to 2, characterized in that: The construction method comprises the following steps: (1) Synthesis of cobalt carbonate nanorods; (2) The cobalt carbonate nanorods were modified by a two-step liposome method, first by DOPA modification, and then by DPPC, cholesterol and DSPE-PEG modification to obtain CoCO3-PEG.

4. The construction method according to claim 3, characterized in that: The specific steps of step (1) include: dripping an aqueous solution of ammonium carbonate into cobalt acetate under magnetic stirring, heating at 120° C. under high pressure for 30 minutes, collecting cobalt carbonate nanorods, and washing with ethanol to obtain the obtained nanorods.

5. The construction method according to claim 3, characterized in that: The specific steps of step (2) include: dispersing the cobalt carbonate nanorods obtained in step (1) into ethanol, adding DOPA under ultrasound, collecting the precipitate by centrifugation after the reaction, dispersing it in chloroform after washing with ethanol, adding DPPC, cholesterol, and DSPE-PEG and stirring overnight, removing the chloroform by rotary evaporation, and adding ultrapure water for hydration to finally form CoCO3-PEG.

6. The construction method according to claim 5, characterized in that: The mass ratio of the cobalt carbonate nanorods, DPPC, cholesterol and DSPE-PEG is 1:4:2:

8.

7. Use of the cobalt carbonate nanorods according to any one of claims 1 to 2 in the preparation of hydrophobic weakly basic drug carriers.

8. Use of the cobalt carbonate nanorods according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: EVO is loaded on the hydrophobic region of the liposome bilayer of the cobalt carbonate nanorods according to any one of claims 1 to 2.

10. The anti-tumor drug according to claim 9, characterized in that: The mass ratio of EVO to cobalt in cobalt carbonate nanorods is 1:0.25-2.

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