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CN118993962A - Zn-penicillamine nano chelating agent and preparation method and application thereof - Google Patents

Zn-penicillamine nano chelating agent and preparation method and application thereof Download PDF

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CN118993962A
CN118993962A CN202411065291.2A CN202411065291A CN118993962A CN 118993962 A CN118993962 A CN 118993962A CN 202411065291 A CN202411065291 A CN 202411065291A CN 118993962 A CN118993962 A CN 118993962A
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penicillamine
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李春霞
解玉林
高铭鸿
孙倩倩
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Shandong University
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Abstract

本发明属于抗肿瘤药物技术领域，涉及一种Zn‑青霉胺纳米螯合剂及其制备方法与应用。所述纳米螯合剂是由锌离子和D‑青霉胺通过鳌合配位形成非水溶性的纳米材料。其制备方法为：将D‑青霉胺和二价锌盐在溶液中混合均匀，调节pH至中性或碱性，持续搅拌反应，即得。本发明提供的Zn‑青霉胺纳米螯合剂能够干扰细胞离子稳态，在乳腺癌中不仅实现了氧化磷酸化(OXPHOS)和糖酵解代谢的双重抑制，而且有效激活了抗肿瘤免疫反应。

The present invention belongs to the technical field of anti-tumor drugs, and relates to a Zn-penicillamine nano-chelator, a preparation method and an application thereof. The nano-chelator is a water-insoluble nano-material formed by chelation coordination of zinc ions and D-penicillamine. The preparation method is as follows: D-penicillamine and a divalent zinc salt are mixed evenly in a solution, the pH is adjusted to neutral or alkaline, and the reaction is stirred continuously to obtain the product. The Zn-penicillamine nano-chelator provided by the present invention can interfere with cell ion homeostasis, and not only achieves dual inhibition of oxidative phosphorylation (OXPHOS) and glycolysis metabolism in breast cancer, but also effectively activates anti-tumor immune response.

Description

Zn-penicillamine nano chelating agent and preparation method and application thereof
Technical Field
The invention belongs to the technical field of antitumor drugs, and relates to a Zn-penicillamine nano chelating agent, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Copper is a common metal element, also a transition element, with redox activity. Under conventional chemical reactions and physiological conditions, reduced Cu + can be converted to oxidized Cu 2+. Copper ions participate in a variety of biochemical reactions by providing or accepting electrons. Copper ions can be combined with various proteins or enzymes to be used as auxiliary factors or structural components to participate in regulating and controlling various physiological processes such as energy metabolism, mitochondrial respiration, antioxidation and the like. The content of copper ions maintains dynamic balance, and unbalance can lead to oxidative stress, abnormal autophagy and the like, thereby inducing the occurrence of various copper or copper ion related diseases.
Copper death is a novel death mode distinguished from other programmed cell death (such as apoptosis, pyrodeath, necrosis and iron death), and its regulation process is closely related to mitochondrial metabolism. Excessive copper, through direct binding to lipoylated proteins in the mitochondrial tricarboxylic acid (TCA) cycle, leads to abnormal aggregation of the lipoylated proteins and loss of iron-sulfur cluster proteins in the respiratory chain complex, leading to protein toxic stress reactions, ultimately leading to cell death. However, the treatment mode of the metal ion overload is often uncontrollable, and the normal tissues are potentially influenced while the tumor cells are killed. Studies show that the content of Cu 2+ in serum of breast cancer patients is higher than that of normal people. Furthermore, mitochondrial copper chaperone and chaperone COX17 and SCO2 upregulation was shown in breast cancer patients, suggesting that the copper transport to the mitochondria is highly required by breast cancer cells compared to normal cells. If mitochondrial copper is consumed, this will shift metabolism from respiration to glycolysis and reduce energy production, which can be effective against oxidative phosphorylation (OXPHOS) dependent cancer types with less impact on normal tissues. However, due to metabolic reprogramming of tumor cells, when OXPHOS is inhibited, tumor cells maintain the energy required for their vital activities by enhancing glycolytic metabolism.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a Zn-penicillamine nano chelating agent and a preparation method and application thereof, the Zn-penicillamine nano chelating agent provided by the invention can interfere with the steady state of cell ions, in breast cancer, not only the double inhibition of oxidative phosphorylation (OXPHOS) and glycolytic metabolism is realized, but also the anti-tumor immune response is effectively activated.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
In a first aspect, a Zn-penicillamine nanocarrier is a nanomaterial that is water insoluble by chelation coordination of zinc ions and D-penicillamine.
The Zn-penicillamine nano chelating agent provided by the invention can chelate copper elements in breast cancer cells and simultaneously release Zn 2+. Copper depletion results in inhibition of oxidative phosphorylation (OXPHOS) metabolism by inhibiting mitochondrial cuprase cytochrome c oxidase complex IV (coxiv) activity. While elevated intracellular Zn 2+ levels activate the cGAS-STING pathway, which interferes with breast cancer glycolytic metabolism by inhibiting hexokinase II (HK 2) activity and triggers an immune response through IFN- β. Therefore, the Zn-penicillamine nano chelating agent not only realizes double inhibition of OXPHOS and glycolytic metabolism in the treatment of breast cancer, but also effectively activates anti-tumor immune response.
Meanwhile, the reagent capable of chelating copper ions not only comprises D-penicillamine, but also comprises other copper ion chelating agents such as trientine, tetrathiomolybdic acid and the like. Compared with other copper ion chelating agents (such as trientine, tetrathiomolybdic acid and the like), the D-penicillamine has a simple structure and has amino, carboxyl and sulfhydryl groups. These groups facilitate the chelating coordination of D-penicillamine with metal ions (Zn 2+), thereby forming a water-insoluble nanomaterial which is beneficial to long-term accumulation at tumor sites. However, trientine and tetrathiomolybdic acid form water-soluble complex with metal ion (Zn 2+) easily, and are rapidly discharged by the body, so that the therapeutic effect cannot be achieved. In addition, the price of D-penicillamine is cheaper and is 1/8 of that of trientine and 1/4 of that of tetrathiomolybdic acid, which greatly reduces the treatment cost of diseases. Therefore, compared with other copper ion chelating agents, the Zn-penicillamine nano chelating agent can play a role in a tumor part for a long time, thereby achieving better treatment effect.
In addition, the breast cancer inhibition experiment shows that the combination of Zn and penicillamine has a synergistic promotion effect, and the tumor killing effect of the nano chelating agent is enhanced.
The nanomaterial described in the present invention may be a nanoparticle, a nanofiber, or the like. In some embodiments, the nanomaterial is a nanofiber. Compared with other morphologies of nano materials, the nano particles have smaller size (especially radial size), have deeper tumor penetration capability, and are beneficial to exerting better anti-tumor effect.
In some embodiments, the coordination ratio of zinc ions to D-penicillamine is 1:1.
On the other hand, the preparation method of the Zn-penicillamine nano chelating agent comprises the steps of uniformly mixing D-penicillamine and divalent zinc salt in a solution, regulating pH to be neutral or alkaline, and continuously stirring for reaction to obtain the Zn-penicillamine nano chelating agent.
The divalent zinc salt is a compound of which the cations are divalent zinc ions, such as zinc chloride, zinc sulfate, zinc nitrate and the like. According to the invention, researches on zinc chloride and zinc nitrate show that the anions of the divalent zinc salt have little influence on the formation and morphology of the nano material.
The molar ratio of D-penicillamine to bivalent zinc salt is 1:0.5-2.0, and researches show that different molar ratios have little influence on the morphology of the Zn-penicillamine nano chelating agent. In some embodiments, the molar ratio of D-penicillamine to divalent zinc salt is from 1:0.9 to 1.1.
The pH value is regulated to 7-14, especially the pH value is regulated to 7-12, and under different pH conditions, the complexing constants of D-penicillamine and zinc ions are different, so that Zn-penicillamine nano chelating agents with different morphologies can be formed. In some embodiments, the pH is adjusted to 7.3 to 7.7. Studies have shown that this condition enables the preparation of nanofibrous Zn-penicillamine nanocarriers.
The reaction time of continuous stirring is 2-14 h. In some embodiments, the reaction is continued with stirring for a period of 11 to 13 hours.
In some embodiments, solid-liquid separation is performed after continuous stirring of the reaction. Specifically, the solid-liquid separation mode is centrifugal separation. More specifically, the rotational speed of centrifugal separation is 5000-10000 rpm, and the centrifugal time is 5-15 min; preferably, the rotational speed of centrifugation is 9900-10000 rpm, and the centrifugation time is 9-11 min.
Specifically, the solid-liquid separation is followed by washing. The number of times of washing is generally 2 to 3.
In a third aspect, a pharmaceutical composition comprises an active ingredient and a pharmaceutical excipient, wherein the active ingredient is the Zn-penicillamine nano-chelating agent according to the first aspect of the invention or the Zn-penicillamine nano-chelating agent obtained by the preparation method according to the second method of the invention.
In some embodiments, the pharmaceutical adjuvant is water, physiological saline, a buffer solution, an immunoadjuvant, an emulsifier, a suspending agent, a filler, a disintegrant, a preservative, or the like.
In some embodiments, the pharmaceutical composition is administered by subcutaneous injection or intravenous injection, or the like.
In a fourth aspect, the Zn-penicillamine nano-chelating agent according to the first aspect of the present invention or the Zn-penicillamine nano-chelating agent obtained by the preparation method according to the second aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention is used in the preparation of an anti-breast cancer drug.
The positive surface charge of Zn-penicillamine favors internalization of breast cancer cells (4T 1) and accumulation at mitochondria. At the mitochondria, zn-penicillamine binds to copper, resulting in release of Zn 2+, interfering with mitochondrial function, triggering apoptosis of 4T1 cells. Second, released Zn 2+ stimulates cGAS-STING signaling pathway, induces release of interferon- β (IFN- β), further promotes maturation of DCs, enhancing immune response. In addition, copper depletion and Zn 2+ proliferation prevented OXPHOS and glycolytic metabolism in 4T1 cells.
In some embodiments, the anti-breast cancer drug has the effect of inhibiting oxidative phosphorylation and glycolytic metabolism and/or activating an anti-tumor immune response.
The beneficial effects of the invention are as follows:
(1) Traditional chelation therapy is to consume the metal elements of host body overload by intravenous injection of chelators, which are prone to systemic toxicity due to lack of targeting. The Zn-penicillamine nano chelating agent provided by the invention can be accumulated at a tumor part for a long time, so that systemic toxicity is avoided. In addition, zn-penicillamine can consume copper elements necessary for the growth and development of breast cancer and also release Zn 2+ with anti-tumor properties.
(2) The Zn-penicillamine nano chelating agent provided by the invention is prepared by chelating coordination of Zn 2+ and D-penicillamine, and has extremely high penicillamine chelating agent loading compared with a chelating agent loaded by a nano material.
(3) The Zn-penicillamine nano chelating agent provided by the invention not only realizes double inhibition of OXPHOS and glycolysis metabolism in breast cancer, but also activates anti-tumor immune response and effectively blocks metastasis.
(4) The preparation method is simple, has strong practicability and is easy to popularize.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a Transmission Electron Microscope (TEM) image of the Zn-penicillamine nanoshelpers prepared in example 1.
FIG. 2 is a TEM image of the Zn-penicillamine nanoshelpers prepared in example 2.
FIG. 3 is a TEM image of the Zn-penicillamine nanoshelpers prepared in example 3.
FIG. 4 is a TEM image of the Zn-penicillamine nanoshelpers prepared in example 4.
FIG. 5 is a graph of X-ray photoelectron spectroscopy (XPS) characterization of the Zn-penicillamine nanoshelter prepared in example 1.
FIG. 6 is an XPS characterization of the nanomaterial after the Zn-penicillamine nanoshelter and Cu chelate prepared in example 1.
FIG. 7 is a characterization of Zn 2+ release after preparation of Zn-penicillamine nanoshelpers and Cu chelation in example 1.
FIG. 8 is a flow cytometer characterization graph of the Zn-penicillamine nanoshelpers prepared in example 1 to sequester Cu in 4T1 cells.
FIG. 9 is a flow cytometer characterization diagram of Zn 2+ released in 4T1 cells by preparing Zn-penicillamine nanocarriers in example 1.
FIG. 10 is a graph showing the results of toxicity test of the Zn-penicillamine nanocarrier prepared in example 1 on tumor cells (4T 1) and normal cells (HUVECs); a is 4T1 cells and b is HUVECs cells.
FIG. 11 is a graph showing the comparison of the toxicity test results of the Zn-penicillamine nanoshelating agent with zinc ions and D-penicillamine on tumor cells (4T 1) prepared in example 1.
FIG. 12 is a graph showing the results of the test for the induction of apoptosis of tumor cells (4T 1) by the Zn-penicillamine nanocarrier prepared in example 1.
FIG. 13 is a graph showing the results of the activity test of the Zn-penicillamine nanocarrier prepared in example 1 in inhibiting COX IV of tumor cells (4T 1).
FIG. 14 is a graph showing the results of the activity test of the Zn-penicillamine nanocarrier prepared in example 1 against HK2 of tumor cells (4T 1).
FIG. 15 is a graph showing the results of the metabolic test for tumor cell inhibition (4T 1) by the Zn-penicillamine nanocarrier prepared in example 1; a is glucose content, b is lactic acid content, and c is ATP content.
FIG. 16 is a graph showing the results of the test for the Zn-penicillamine nanoshelating agent treated tumor cells (4T 1) prepared in example 1 to promote maturation of DCs.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
1. The preparation method of the Zn-penicillamine nano chelating agent comprises the following steps:
1) 0.5mmol of ZnCl 2 and D-penicillamine were weighed separately into 50mL beakers.
2) 20ML of deionized water was added and dissolved by stirring with a magnet.
3) The pH of the solution was adjusted to 7.5 using NaOH solution.
4) Stirring for 12h at room temperature, and centrifuging for 10min at 10000rpm to obtain silvery white solid Zn-penicillamine nano chelating agent. And then washing for 3 times by using deionized water, and centrifuging again to finally obtain the Zn-penicillamine nano chelating agent.
The Zn-penicillamine nanocarrier prepared in example 1 was characterized by a transmission electron microscope, as shown in fig. 1, from which it can be seen that the prepared Zn-penicillamine nanocarrier was a fibrous nanomaterial.
2. XPS characterization of Zn-penicillamine nanoshelpers
1) And drying the Zn-penicillamine nano-chelating agent prepared in the steps at 70 ℃.
2) The solid Zn-penicillamine nanocarrier was ground into powder using an agate mortar.
3) And finally, preparing a sample to test XPS (XPS) full spectrum of the Zn-penicillamine nano chelating agent.
As shown in fig. 5, XPS characterization confirmed the presence of Zn, S, N elements in the prepared Zn-penicillamine nanoshelating agent.
3. XPS characterization of Cu-chelated Zn-penicillamine nanoshelpers
1) The prepared Zn-penicillamine nanoshelpers were dispersed in 20mL deionized water.
2) 50MM CuCl 2 solution was added and sonicated for 10min.
3) Centrifuging the solution at 10000rpm for 10min to obtain the Cu-chelated Zn-penicillamine nanometer chelating agent.
4) The subsequent testing steps are consistent with the two steps.
As shown in fig. 6, XPS full spectrum characterization confirmed the presence of Cu, zn, S, N element in the Cu-chelated Zn-penicillamine nanoshelpers.
4. Characterization of Zn-penicillamine nanoshelpers chelating Cu 2+ to release Zn 2+
1) The prepared Zn-penicillamine nano-chelating agent was dispersed in 10mL deionized water, and the average was divided into 5 parts, with 2mL of solution in each tube.
2) Then, cuCl 2 solutions with different concentrations of 0mM, 6.25 mM, 12.5 mM, 25 mM and 50mM are respectively added into each tube, and the ultrasound is continued for 10min.
3) Centrifuging the solution at 10000rpm for 10min, and taking supernatant to test Zn 2+ concentration by inductively coupled plasma-mass spectrometry.
As shown in fig. 7, the content of Zn 2+ in the supernatant solution gradually increased with increasing concentration of Cu 2+, which demonstrates that Zn 2+ can be released after the Zn-penicillamine nanoshelter chelates Cu 2+.
5. Characterization of Zn-penicillamine nano-chelator chelated Cu in breast cancer
1) 4T1 cells were seeded in 6-well plates (10 5 cells/well) and incubated in an incubator at 37℃and 5% CO 2 for 24h.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(10μg mL-1); (3) D-penicillamine (23 μg mL -1); (4) Zn-penicillamine (33. Mu.g mL -1).
3) After 4h of different treatments, the prepared copper ion fluorescent probe-RHB is added, incubated for 30min and washed 3 times by PBS.
4) Changes in intracellular copper ions were detected using flow cytometry.
As shown in fig. 8, the copper content in the breast cancer cells of the control group and Zn 2+ group were similar, indicating that Zn 2+ did not affect the copper level in the breast cancer cells. The intracellular copper content of D-penicillamine and Zn-penicillamine treatment is obviously reduced, and the capacity of Zn-penicillamine to chelate copper in breast cancer cells is proved.
6. Characterization of Zn 2+ released by Zn-penicillamine nanoshelpers in breast cancer
1) 4T1 cells were seeded in 6-well plates (10 5 cells/well) and incubated in an incubator at 37℃and 5% CO 2 for 24h.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(10μg mL-1); (3) D-penicillamine (23 μg mL -1); (4) Zn-penicillamine (33. Mu.g mL -1).
3) After 4h of different treatments, the prepared zinc ion fluorescent probe-TSQ is added, incubated for 30min and washed 3 times with PBS.
4) Changes in intracellular zinc ions were detected using flow cytometry.
As shown in fig. 9, the D-penicillamine group had reduced intracellular zinc content in breast cancer cells compared to the control group, which is probably caused by the fact that D-penicillamine also sequesters intracellular Zn. The significantly increased intracellular zinc content of Zn 2+ and Zn-penicillamine treated cells demonstrated the ability of Zn-penicillamine to release Zn 2+ in breast cancer cells.
7. Cytotoxicity of Zn-penicillamine nanoshelpers against breast cancer cells (4T 1) and normal cells (HUVECs)
1) HUVECs and 4T1 cells were seeded in 96-well plates (8X 10 3 cells/well), respectively.
2) Cells in 96-well plates were treated with different concentrations (0, 20, 40, 60, 80, 100 μg mL -1) of Zn-penicillamine nanocarriers and cultured for 24h.
3) Cell viability was measured using the standard 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) method.
As shown in FIG. 10, the inhibition rate of Zn-PEN on 4T1 cells reached 71.3% even at a low concentration (40. Mu.g mL -1). At high concentrations (100 μg mL -1), the survival of 4T1 cells was below 20%, while the survival of HUVECs remained above 75%, confirming that Zn-penicillamine nanocarriers were highly cytotoxic to tumor cells with less impact on normal cells.
8. Cytotoxicity of different treatments on breast cancer cells (4T 1).
1) 4T1 cells were seeded in 96-well plates (8X 10 3 cells/well) and incubated in an incubator at 37℃and 5% CO 2 for 24h.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(30μg mL-1); (3) D-penicillamine (70 μg mL -1); (4) Zn-penicillamine (100 μg mL -1); cells in 96-well plates were treated according to experimental groups and cultured for 24h.
3) Cell viability was measured using the standard 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) method.
As shown in FIG. 11, 4T1 cell viability was 46.2% after zinc ion treatment alone, while 4T1 cell viability was 84.2% after D-penicillamine treatment. The inhibition rate of Zn-PEN to 4T1 cells can reach 79.5 percent, which is higher than the sum (70.6 percent) of single zinc ions and D-penicillamine, thus showing that the combination of Zn and penicillamine has synergistic promotion effect and enhances the tumor killing effect of the nano chelating agent.
9. Zn-penicillamine nano chelating agent induced breast cancer cell (4T 1) apoptosis detection
1) 4T1 cells were seeded in 6-well plates (10 5 cells/well) and incubated in an incubator at 37℃and 5% CO 2 for 24h.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(30μg mL-1); (3) D-penicillamine (70 μg mL -1); (4) Zn-penicillamine (100. Mu.g mL -1).
3) After 4h of different treatments, the old medium in the 6-well plate was removed and washed 3 times with PBS.
4) Cells in 6-well plates were digested with trypsin, centrifuged at 1000rpm for 5min, and the pellet was taken to give differently treated cells.
5) And adding the prepared apoptosis detection kit into the obtained cells, lightly blowing the cells to resuspend the cells, continuously incubating for 30min, and detecting the apoptosis effect of the cells by using a flow cytometry.
As shown in FIG. 12, the highest apoptosis rate of tumor cells in the Zn-penicillamine treated group was 62.3%.
10. OxiPHOS metabolism assay for inhibition of 4T1 by Zn-penicillamine nanoshelpers
1) 4T1 cells were seeded on a petri dish (4X 10 6 cells/dish) and cultured in an incubator at 37℃and 5% CO 2 for 24 hours.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(10μg mL-1); (3) D-penicillamine (23 μg mL -1); (4) Zn-penicillamine (33. Mu.g mL -1).
3) After 4h of different treatments, the old medium in the dishes was removed and washed 3 times with PBS.
4) COX IV activity of 4T1 cells after different treatments was measured according to the procedure of COX IV assay kit.
As shown in FIG. 13, the activity of COX IV in 4T1 cells was reduced by 44.6% and 55.4% respectively in the control group after treatment with copper chelators D-penicillamine and Zn-penicillamine, indicating that Zn-penicillamine inhibited COX IV activity by copper depletion, thereby inhibiting OXPHOS metabolism in breast cancer cells.
11. Glycolytic metabolism detection of inhibition of 4T1 by Zn-penicillamine nano chelating agent
1) 4T1 cells were seeded on a petri dish (4X 10 6 cells/dish) and cultured in an incubator at 37℃and 5% CO 2 for 24 hours.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(10μg mL-1); (3) D-penicillamine (23 μg mL -1); (4) Zn-penicillamine (33. Mu.g mL -1).
3) After 4h of different treatments, the old medium in the dishes was removed and washed 3 times with PBS.
4) HK2 activity of 4T1 cells after different treatments was tested according to the procedure of HK2 test kit.
As shown in FIG. 14, zn 2+ and Zn-penicillamine treatments resulted in 77.1% and 79.2% decrease in HK2 enzyme activity in 4T1 cells, respectively, as compared to the control group. This suggests that Zn-penicillamine inhibits HK2 activity by activating STING, thereby inhibiting glycolytic metabolism of breast cancer cells.
12. Metabolic assay for inhibition of 4T1 by Zn-penicillamine nanoshelpers
1) 4T1 cells were seeded in 6-well plates (10 5 cells/well) and incubated in an incubator at 37℃and 5% CO 2 for 24h.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(10μg mL-1); (3) D-penicillamine (23 μg mL -1); (4) Zn-penicillamine (33. Mu.g mL -1).
3) After 4h of different treatments, the old medium in the 6-well plate was removed and washed 3 times with PBS.
4) Cells in 6-well plates were digested with trypsin, centrifuged at 1000rpm for 5min, and the pellet was taken to give differently treated cells.
5) And detecting the change of the contents of glucose, lactic acid and ATP in the breast cancer cells after different treatments by using a glucose detection kit, a lactic acid detection kit and an ATP detection kit respectively.
As shown in fig. 15, the glucose content of the Zn 2+ -treated 4T1 cells was increased and the lactic acid and ATP levels were decreased compared to the control group. This phenomenon is due to the inhibition of glycolytic metabolism caused by the increase in Zn 2+. D-penicillamine treated 4T1 cells had elevated glucose and lactate levels, while ATP levels were slightly reduced. This phenomenon may be due to the inhibition of OXPHOS metabolism in breast cancer cells, leading to an increase in glycolytic metabolism. Finally, a significant decrease in ATP levels was found in Zn-penicillamine treated 4T1 cells, indicating that inhibition of OXPHOS and glycolysis by copper depletion and elevated Zn 2+ effectively disrupts the energy supply to breast cancer cells.
13. Zn-penicillamine nanoshelating agent treated 4T 1-induced DCs maturation assay
1) 4T1 cells were seeded in 6-well plates (10 5 cells/well) while DCs were seeded in the lower chamber of Transwell 6-well plates (10 5 cells/well) and incubated for 24h at 37℃in an incubator with 5% CO 2.
2) 4 Sets of experiments were set up, respectively: (1) a control group; (2) Zn 2+(30μg mL-1); (3) D-penicillamine (70 μg mL -1); (4) Zn-penicillamine (100. Mu.g mL -1).
3) After 4h of different treatments of 4T1 cells, old medium in 6-well plates was removed and washed 3 times with PBS.
4) 4T1 cells in 6-well plates were digested with trypsin, centrifuged at 1000rpm for 5min, and the pellet was taken to give 4T1 cells of different treatments.
5) The different treated 4T1 cells were seeded into the upper chamber of the Transwell 6 well plate described above and cultured in an incubator at 37℃and 5% CO 2 for 24 hours.
6) DCs cells in the lower chamber of the Transwell 6 well plate were digested with trypsin and centrifuged at 1000rpm for 5min to obtain the precipitate.
7) The DCs were stained with FITC anti-CD 11c, PE anti-CD 86 and APC anti-CD 80 antibodies for 1h and finally analyzed for DCs maturation by flow cytometry.
As shown in FIG. 16, the proportion of mature DCs in the control group was only 9.8%. However, zn 2+ and Zn-penicillamine treatments significantly enhanced maturation of DCs at ratios of 21.8% and 27.9%, respectively. These results confirm the immune activation of Zn-penicillamine.
Example 2
The preparation method of the Zn-penicillamine nano chelating agent comprises the following steps:
1) 0.5mmol of ZnCl 2/Zn(NO3)2 and D-penicillamine were weighed separately into 50mL beakers.
2) 20ML of deionized water was added and dissolved by stirring with a magnet.
3) The pH of the solution was adjusted to 12 using NaOH solution.
4) Stirring for 12h at room temperature, and centrifuging for 10min at 10000rpm to obtain silvery white solid Zn-penicillamine nano chelating agent. And then washing for 3 times by using deionized water, and centrifuging again to finally obtain the Zn-penicillamine nano chelating agent.
The Zn-penicillamine nano-chelating agent prepared in the example 2 is characterized by a transmission electron microscope, as shown in fig. 2, the prepared Zn-penicillamine nano-chelating agent is a larger diamond nano-material at the pH of 12, and the appearance influence of zinc salt is smaller.
Example 3
The preparation method of the Zn-penicillamine nano chelating agent comprises the following steps:
1) The ZnCl 2 and the D-penicillamine with different molar ratios are respectively weighed into a 50mL beaker, and the molar ratios are respectively 0.5:1, 1:1 and 1:2.
2) 20ML of deionized water was added and dissolved by stirring with a magnet.
3) The pH of the solution was adjusted to 12 using NaOH solution.
4) Stirring for 12h at room temperature, and centrifuging for 10min at 10000rpm to obtain silvery white solid Zn-penicillamine nano chelating agent. And then washing for 3 times by using deionized water, and centrifuging again to finally obtain the Zn-penicillamine nano chelating agent.
Characterization of the Zn-penicillamine nanoshelpers prepared in example 3 was performed by transmission electron microscopy, as shown in FIG. 3, it can be seen from the graph that the Zn-penicillamine nanoshelpers prepared by the reactants with different molar ratios are also larger diamond-shaped nanomaterials at pH 12, and the different molar ratios of the reactants have less influence on the morphology.
Example 4
The preparation method of the Zn-penicillamine nano chelating agent is as follows:
1) 0.5mmol of ZnCl 2 and D-penicillamine were weighed separately into 50mL beakers.
2) 20ML of deionized water was added and dissolved by stirring with a magnet.
3) The pH of the above solutions was adjusted to 7.5, 8, 9, 10, 11, 12, respectively, using NaOH solution.
4) Stirring for 12h at room temperature, and centrifuging for 10min at 10000rpm to obtain silvery white solid Zn-penicillamine nano chelating agent. And then washing for 2-3 times by using deionized water, and centrifuging again to finally obtain the Zn-penicillamine nano chelating agent.
Characterization of the Zn-penicillamine nano-chelator prepared in example 4 was performed by transmission electron microscopy, as shown in FIG. 4, it can be seen from the graph that the morphology of the prepared Zn-penicillamine nano-chelator has a large change when the pH is different. At pH 7.5, the prepared Zn-penicillamine nano-chelating agent is nanofiber-shaped. The difference in morphology of Zn-penicillamine nanoshelpers may be due to the different complexing constants of D-penicillamine and Zn 2+ at different pH values. Studies have shown that nanoparticle size plays a critical role in cellular uptake, smaller nanomaterials exhibiting deeper tumor penetration. Finally, smaller fibrous Zn-penicillamine produced under weakly alkaline conditions (ph=7.5) was selected for subsequent application studies.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1.一种Zn-青霉胺纳米螯合剂，其特征是，所述纳米螯合剂是由锌离子和D-青霉胺通过鳌合配位形成非水溶性的纳米材料。1. A Zn-penicillamine nano-chelate, characterized in that the nano-chelate is a water-insoluble nano-material formed by chelation coordination of zinc ions and D-penicillamine. 2.如权利要求1所述的Zn-青霉胺纳米螯合剂，其特征是，所述的纳米材料为纳米纤维。2. The Zn-penicillamine nano-chelating agent according to claim 1, wherein the nano-material is nano-fiber. 3.一种Zn-青霉胺纳米螯合剂的制备方法，其特征是，将D-青霉胺和二价锌盐在溶液中混合均匀，调节pH至中性或碱性，持续搅拌反应后即得Zn-青霉胺纳米螯合剂。3. A method for preparing a Zn-penicillamine nano-chelating agent, characterized in that D-penicillamine and a divalent zinc salt are uniformly mixed in a solution, the pH is adjusted to neutral or alkaline, and the Zn-penicillamine nano-chelating agent is obtained after continuous stirring and reaction. 4.如权利要求3所述的Zn-青霉胺纳米螯合剂的制备方法，其特征是，D-青霉胺和二价锌盐的摩尔比为1:0.9～1.1。4. The method for preparing a Zn-penicillamine nano-chelating agent as claimed in claim 3, wherein the molar ratio of D-penicillamine to divalent zinc salt is 1:0.9-1.1. 5.如权利要求3所述的Zn-青霉胺纳米螯合剂的制备方法，其特征是，调节pH值至7.3～7.7。5. The method for preparing the Zn-penicillamine nano-chelating agent according to claim 3, characterized in that the pH value is adjusted to 7.3-7.7. 6.如权利要求3所述的Zn-青霉胺纳米螯合剂的制备方法，其特征是，持续搅拌反应的时间为11～13h。6. The method for preparing a Zn-penicillamine nano-chelating agent according to claim 3, wherein the stirring reaction time is 11 to 13 hours. 7.一种药物组合物，包括活性成分和药用辅料，其特征是，所述活性成分为权利要求1或2所述的Zn-青霉胺纳米螯合剂或权利要求3～6任一所述的制备方法获得的Zn-青霉胺纳米螯合剂。7. A pharmaceutical composition comprising an active ingredient and a pharmaceutical excipient, wherein the active ingredient is the Zn-penicillamine nano-chelate according to claim 1 or 2 or the Zn-penicillamine nano-chelate obtained by the preparation method according to any one of claims 3 to 6. 8.如去哪里要求7所述的药物组合物，其特征是，所述药用辅料为水、生理盐水、缓冲溶液、免疫佐剂、乳化剂、悬浮剂、填充剂、崩解剂或防腐剂；8. The pharmaceutical composition as described in claim 7, characterized in that the pharmaceutical excipient is water, physiological saline, buffer solution, immune adjuvant, emulsifier, suspending agent, filler, disintegrant or preservative; 或，所述药物组合物的施用方式为皮下注射、静脉注射等。Alternatively, the pharmaceutical composition is administered by subcutaneous injection, intravenous injection, etc. 9.一种权利要求1或2所述的Zn-青霉胺纳米螯合剂或权利要求3～6任一所述的制备方法获得的Zn-青霉胺纳米螯合剂或权利要求7或8所述的药物组合物在制备抗乳腺癌药物中的应用。9. Use of the Zn-penicillamine nanochelate according to claim 1 or 2, or the Zn-penicillamine nanochelate obtained by the preparation method according to any one of claims 3 to 6, or the pharmaceutical composition according to claim 7 or 8 in the preparation of anti-breast cancer drugs. 10.如权利要求9所述的应用，其特征是，所述抗乳腺癌药物具有抑制氧化磷酸化和糖酵解代谢和/或激活抗肿瘤免疫反应的作用。10. The use according to claim 9, characterized in that the anti-breast cancer drug has the effect of inhibiting oxidative phosphorylation and glycolysis metabolism and/or activating anti-tumor immune response.
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