Dual-pathway energy-inhibiting enzyme-based nano-therapeutics and preparation method and application thereof

Abstract

The present invention relates to the technical field of biomedical nanomaterials, and particularly relates to a dual-pathway energy inhibition enzyme-based nano-therapeutic agent, a preparation method thereof, and an application thereof. The dual-pathway energy inhibition enzyme-based nano-therapeutic agent comprises a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor. During transportation, due to the hydrophobic interaction between the hydrophobic oxidative phosphorylation inhibitor and the functional protein, the biosafety is improved; at the same time, the bacterial outer membrane vesicle encapsulates the functional protein and the oxidative phosphorylation inhibitor, improving the tumor accumulation of the nano-therapeutic agent. When the nano-therapeutic agent is in an acidic tumor microenvironment, the nano-therapeutic agent dissociates, the activity of the functional protein is restored, and the oxidative phosphorylation inhibitor is released, realizing the selective control of the dual-pathway energy inhibition enzyme-based nano-therapeutic agent.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a dual-path energy-inhibiting enzyme-based nano-therapeutic agent, a preparation method thereof, and an application thereof. Background Art

[0002] Most tumor cells transport and metabolize nutrients much faster than normal cells, and mainly obtain energy through anaerobic glycolysis rather than respiratory metabolism to meet their energy requirements for growth, invasion, and migration. Therefore, the proliferation and growth of cancer cells highly depend on glucose.

[0003] Given the key role of glucose in providing energy for tumor growth and metabolism, glucose oxidase (GOx) catalysis can effectively consume glucose and oxygen in tumor cells for starvation therapy, and lead to an increase in the acidity, hypoxia level, and hydrogen peroxide content in the tumor microenvironment. Based on this, GOx-induced glucose consumption in tumors can effectively block the energy supply of tumor cells, which provides an alternative non-invasive starvation strategy for treating cancer.

[0004] However, the complexity, diversity, and heterogeneity of the tumor microenvironment make it difficult to obtain effective treatment effects only through glucose oxidase for starvation therapy. The hypoxic microenvironment of tumors also limits the oxygen-consuming catalytic process of glucose oxidase. Research shows that the energy metabolism of tumors not only originates from anaerobic glycolysis, but oxidative phosphorylation is also one of the energy sources of many tumors. Therefore, simply consuming glucose cannot completely inhibit the proliferation of tumors, and inhibiting tumor energy metabolism from multiple aspects can more effectively treat tumors. Moreover, the limitations of glucose oxidase starvation therapy are not only affected by the tumor microenvironment, but as a natural enzyme, its own bioavailability and biosafety in vivo also need to be further verified in research.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, the purpose of the present invention is to provide a dual-path energy-inhibiting enzyme-based nano-therapeutic agent, a preparation method thereof, and an application thereof, aiming to solve the problems of limited tumor accumulation and poor effect of existing functional proteins.

[0007] The technical solution of the present invention is as follows:

[0008] A dual-path energy-inhibiting enzyme-based nano-therapeutic agent, comprising a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor.

[0009] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the mass ratio of the oxidative phosphorylation inhibitor to the functional protein is 1:5 to 1:40; the mass ratio of the bacterial outer membrane vesicles to the functional protein is (1 to 2):(1 to 2).

[0010] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the particle size of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is 179.87 to 207.43 nm.

[0011] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the functional protein is glucose oxidase.

[0012] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the oxidative phosphorylation inhibitor is selected from one or more of oligomycin A, metformin, phenformin, IACS-010759, antimycin A, BAY 87-2243, fenofibrate, α-TOS, atovaquone, rotenone, piericidin A, and bafilomycin A1.

[0013] A preparation method of a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, comprising the steps of:

[0014] Providing a functional protein and an oxidative phosphorylation inhibitor;

[0015] Mixing the functional protein and the oxidative phosphorylation inhibitor to obtain a mixture;

[0016] Adding bacterial outer membrane vesicles to the mixture, and performing mixing and extrusion treatment to obtain a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent.

[0017] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the step of mixing the functional protein and the oxidative phosphorylation inhibitor comprises:

[0018] Mixing the oxidative phosphorylation inhibitor with ethanol to obtain a first solution;

[0019] Mixing the functional protein with a buffer solution containing β-mercaptoethanol to obtain a second solution;

[0020] Mixing and stirring the first solution and the second solution.

[0021] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the buffer solution is phosphate buffer or tris(hydroxymethyl)aminomethane buffer.

[0022] The described dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, wherein the step of adding bacterial outer membrane vesicles to the mixture and performing mixing and extrusion treatment comprises:

[0023] After adding bacterial outer membrane vesicles to the mixture and mixing, extrusion is carried out through a filter membrane with a pore size of 0.19 - 0.23 μm for 18 - 22 times.

[0024] Application of a dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent in the preparation of a tumor - treating preparation.

[0025] Beneficial effects: The present invention provides a dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent, its preparation method and application. The dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent includes a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor. During the transportation process of this nano - therapeutic agent, due to the hydrophobic interaction between the hydrophobic oxidative phosphorylation inhibitor and the functional protein, the biosafety is improved; at the same time, the bacterial outer membrane vesicle encapsulates the functional protein and the oxidative phosphorylation inhibitor, enhancing the tumor accumulation of the nano - therapeutic agent. When the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent accumulates at the tumor site, in the acidic tumor microenvironment, the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent dissociates, the activity of the functional protein is restored, and the oxidative phosphorylation inhibitor is released, realizing the selective control of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent. This nano - therapeutic agent significantly improves the drug - loading capacity and bioavailability of the functional protein, can selectively control the activity of its functional protein, and increases the drug accumulation amount at the tumor site through encapsulation by bacterial outer membrane vesicles, thereby improving the curative effect and reducing the toxic and side effects. Brief Description of the Drawings

[0026] Figure 1 It is the synthetic route diagram of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent in Example 1 of the present invention;

[0027] Figure 2 It is the transmission electron microscope image (TEM) of OAGO prepared in Example 1 of the present invention;

[0028] Figure 3 It is the comparison diagram of the hydrogen peroxide concentration generated by GOx and OAG in Example 2 of the present invention;

[0029] Figure 4 It is the killing effect diagram of OA, OAG and OAGO on triple - negative breast cancer (4T1) tumor cells in Example 3 of the present invention;

[0030] Figure 5 It is the inhibitory effect diagram of OAGO on the metabolic pathway of 4T1 tumor cells in Example 4 of the present invention;

[0031] Figure 6 It is the fluorescence imaging diagram of tumor accumulation in mice in different treatment groups in Example 5 of the present invention;

[0032] Figure 7 This is the graph of the influence of different treatment groups on tumor blood oxygen saturation in Example 6 of the present invention;

[0033] Figure 8 This is the graph of the inhibitory effect of starvation / metabolism / photothermal combination therapy on the growth of 4T1 tumors in Example 7 of the present invention;

[0034] Figure 9 This is the graph of the blood biochemical parameters of healthy mice on the 15th day after intravenous injection of normal saline and OAGO in Example 8 of the present invention. Detailed implementation manners

[0035] The present invention provides a dual-path energy-inhibiting enzyme-based nano-therapeutic agent and its preparation method and application. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Those skilled in the art of the present technology can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the field to which the present invention belongs. It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted with an idealized or overly formal meaning unless specifically defined as here.

[0037] During the tumor proliferation process, energy is generated through the anaerobic glycolysis pathway. The complexity, diversity and heterogeneity of the tumor microenvironment make it difficult to obtain effective therapeutic effects only by glucose oxidase for starvation therapy. The hypoxic microenvironment of tumors also limits the oxygen-consuming catalytic process of glucose oxidase. Research shows that the energy metabolism of tumors not only originates from anaerobic glycolysis, but oxidative phosphorylation is also one of the energy sources of many tumors. Therefore, simply consuming glucose cannot completely inhibit tumor proliferation, and inhibiting tumor energy metabolism from multiple aspects can more effectively treat tumors.

[0038] Based on this, the present invention provides a dual-path energy-inhibiting enzyme-based nano-therapeutic agent, which includes a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor.

[0039] In this embodiment, the oxidative phosphorylation inhibitor is combined in the hydrophobic cavity of the functional protein to form the first nanoparticle, and then the first nanoparticle is encapsulated by bacterial outer membrane vesicles to obtain a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent (OAGO). During the transportation of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, since the hydrophobic oxidative phosphorylation inhibitor and the functional protein are combined by hydrophobic interaction, the inhibitor is bound in the hydrophobic cavity of the functional protein, and the activity of the functional protein is inhibited internally, improving the biosafety. At the same time, the bacterial outer membrane vesicles are used to encapsulate the functional protein and the oxidative phosphorylation inhibitor, increasing the tumor accumulation of OAGO.

[0040] Specifically, when the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent accumulates at the tumor site, the activity of the functional protein is restored and the oxidative phosphorylation inhibitor is released, realizing the selective control of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent. In addition, the oxidative phosphorylation inhibitor can effectively inhibit mitochondrial respiration, thereby reducing tumor oxygen consumption; on the other hand, the vascular rupture induced by bacterial outer membrane vesicles increases the tumor blood oxygen saturation, further enhancing the oxidative catalytic performance of the functional protein.

[0041] The dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent provided by the present invention significantly improves the drug loading and bioavailability of the functional protein, can selectively control the activity of its functional protein, and the encapsulation by bacterial outer membrane vesicles increases the accumulation amount of the drug at the tumor site, improving the curative effect and reducing the toxic and side effects. The dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent truly realizes high drug loading, high tumor accumulation, and protects the protein activity of the tumor microenvironment, and will have good application prospects in the field of tumor treatment.

[0042] In some embodiments, the mass ratio of the oxidative phosphorylation inhibitor to the functional protein is 1:5 to 1:40; within this ratio range, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent can have a suitable particle size and the maximum encapsulation efficiency. The encapsulation efficiency of the oxidative phosphorylation inhibitor reaches 66.59%, and the encapsulation efficiency of the functional protein is as high as 19.03%. At the same time, this ratio range can better realize the selective control of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, so that the activity of the functional protein is inhibited during transportation, and after accumulating at the tumor site, the functional protein is released and the activity of the functional protein is restored.

[0043] In some embodiments, the mass ratio of the bacterial outer membrane vesicles to the functional protein is (1-2):(1-2).

[0044] In a preferred embodiment, the mass ratio of the bacterial outer membrane vesicles to the functional protein is 1:1. At this ratio, the bacterial outer membrane vesicles can well encapsulate the first nanoparticles, better achieve the biosafety during the in vivo transportation of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, increase the stability of the nano-formulation, and effectively accumulate deep in the tumor.

[0045] In some embodiments, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is a second nanoparticle, and the particle size of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is 179.87 - 207.43 nm. That is, when the particle size of the second nanoparticle is between 179.87 - 207.43 nm, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent can better achieve the accumulation in the tumor and the metabolic treatment of the tumor.

[0046] Furthermore, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is a spherical nanoparticle.

[0047] In some embodiments, the functional protein is glucose oxidase (GOx).

[0048] Specifically, the protein not only has the role of a carrier, but also can achieve the treatment of tumors. GOx has glucose-specific catalytic properties and can produce gluconic acid and hydrogen peroxide by oxidizing glucose, thereby consuming the nutrients of the tumor and achieving the purpose of starving the tumor. Therefore, it can be used for the starvation therapy of cancer. When the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is formed by encapsulating GOx and hydrophobic binding oxidative phosphorylation inhibitor with bacterial outer membrane vesicles, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent can be used for the synergistic treatment of cancer starvation, metabolism and photothermal therapy. Among them, the activity of GOx is inhibited. After the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent encapsulated by bacterial outer membrane vesicles accumulates at the tumor site, the acidic tumor microenvironment causes the GOx nanoparticles encapsulated by bacterial outer membrane vesicles to dissociate and restore the activity of GOx; while GOx consumes the nutrients in the tumor environment by oxidizing glucose at the tumor site, achieving the effect of starving the tumor and realizing the starvation therapy of cancer.

[0049] In some embodiments, the oxidative phosphorylation inhibitor is selected from, but not limited to, one or more of oligomycin A (OA), metformin, phenformin, IACS-010759, antimycin A, BAY87-2243, fenofibrate, α-TOS, atovaquone, rotenone, piericidin A, and bafilomycin.

[0050] In a preferred embodiment, the oxidative phosphorylation inhibitor is selected from oligomycin A; the effect of oligomycin A on mitochondria is a complex and multi-stage process. Oligomycin A is a classical oxidative phosphorylation inhibitor that interacts with the carboxyl side chain of the GLU59 residue in the c-loop of adenosine triphosphate synthase (ATPase), and can effectively induce apoptosis of cancer cells. In addition, in the case of tumor starvation, the anti-cancer effect of oligomycin A can be effectively enhanced; considering the high energy demand for tumor growth and metabolism and the oxygen consumption in the GOx catalytic process, constructing a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is expected to overcome tumor hypoxia from multiple perspectives, inhibit energy supply, and thus enhance the anti-tumor efficacy.

[0051] In the present invention, a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is provided, which is composed of an oxidative phosphorylation inhibitor, a functional protein, and a bacterial outer membrane vesicle. After the disulfide bonds of the functional protein are opened by a reducing agent, more hydrophobic domains will be exposed, and then the hydrophobic oxidative phosphorylation inhibitor can be embedded and self-assembled to form a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent. When the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent accumulates at the tumor site, in the acidic tumor microenvironment, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent dissociates, the activity of the functional protein is restored, and the oxidative phosphorylation inhibitor is released, realizing the selective control of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent. In addition, the oxidative phosphorylation inhibitor effectively inhibits mitochondrial respiration, thereby reducing tumor oxygen consumption. On the other hand, the vascular rupture induced by bacterial outer membrane vesicles increases tumor blood oxygen saturation, further enhancing the oxidative catalytic performance of the functional protein.

[0052] In addition, the present invention also provides a preparation method of a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, including the steps of:

[0053] Step S10: Provide a functional protein and an oxidative phosphorylation inhibitor;

[0054] Step S20: Mix the functional protein and the oxidative phosphorylation inhibitor to obtain a mixture;

[0055] Step S30: Add bacterial outer membrane vesicles to the mixture, mix and extrude to obtain a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent.

[0056] In this embodiment, the preparation method has a simple process and convenient operation, does not require complex and expensive equipment, and is easy to realize industrial production. Moreover, the dual-path energy-inhibiting enzyme-based nano-therapeutic agent prepared by this preparation method significantly improves the drug loading and bioavailability of functional proteins, can selectively control the activity of functional proteins, thereby increasing the accumulation amount of drugs at the tumor site, enhancing the curative effect, and reducing the toxic and side effects. Furthermore, the dual-path energy-inhibiting enzyme-based nano-therapeutic agent truly realizes high drug loading, high tumor accumulation, and activation of protein activity in the tumor microenvironment, and will have good application prospects in the field of tumor treatment.

[0057] In some embodiments, in step S20, the step of mixing the functional protein and the oxidative phosphorylation inhibitor includes:

[0058] Step S21: Mix the oxidative phosphorylation inhibitor with ethanol to obtain a first solution;

[0059] Step S22: Mix the functional protein with a buffer solution containing β-mercaptoethanol to obtain a second solution;

[0060] Step S23: Mix and stir the first solution and the second solution.

[0061] In another embodiment, the step of mixing the functional protein and the oxidative phosphorylation inhibitor can also be: Add β-mercaptoethanol and the functional protein to a buffer solution for mixing, and then add the oxidative phosphorylation inhibitor dissolved in ethanol while stirring to obtain first nanoparticles; the β-mercaptoethanol therein is used to break the disulfide bond of the functional protein, open the structure of the functional protein, facilitate the hydrophobic binding of the protein to the oxidative phosphorylation inhibitor, and enable the oxidative phosphorylation inhibitor to bind in the hydrophobic cavity of the functional protein.

[0062] In some embodiments, after step S20 and before step S30, it further includes: performing ultrafiltration and centrifugation on the mixture to remove free oxidative phosphorylation inhibitor and β-mercaptoethanol in the mixture, for the purpose of purifying the product.

[0063] In some embodiments, the buffer solution is selected from phosphate (PBS) buffer solution or tris (hydroxymethyl) aminomethane (Tris) buffer solution.

[0064] In a preferred embodiment, the solvent of the PBS buffer solution or Tris buffer solution is ultrapure water, and ultrapure water has almost no impurities, which is more conducive to forming a pure biomimetic nanoemulsion.

[0065] In some embodiments, in step S30, the steps of adding bacterial outer membrane vesicles to the mixture and performing mixing and extrusion treatment include: after adding bacterial outer membrane vesicles to the mixture and mixing, extruding through a filter membrane with a pore size of 0.19 - 0.23 μm for 18 - 22 times.

[0066] In a preferred embodiment, the pore size of the filter membrane for the extrusion treatment is 0.22 μm, and the number of extrusion times is 20 times.

[0067] In addition, the present invention also provides an application of a dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent in the preparation of a tumor - treating preparation. And / or, an application of a dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent prepared by the preparation method of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent in the preparation of a tumor - treating preparation.

[0068] Specifically, the above - mentioned treatment can be the simultaneous treatment of starvation therapy, metabolic therapy, and photothermal therapy. The dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent in this embodiment can achieve high drug - loading capacity, high tumor accumulation, and tumor microenvironment - responsive protein activity. The oxidative phosphorylation inhibitor can effectively inhibit mitochondrial respiration, thereby reducing tumor oxygen consumption. On the other hand, the vascular rupture induced by bacterial outer membrane vesicles increases tumor blood oxygen saturation, further enhancing the oxidative catalytic performance of functional proteins, so as to achieve the effects of starvation therapy, metabolic therapy, and photothermal therapy, and will have good application prospects in the field of tumor treatment.

[0069] The following further gives examples to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non - essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention.

[0070] Example 1

[0071] In this example, a dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent is prepared, and its synthetic route diagram Figure 1 is shown as follows:

[0072] Step S1: Dissolve 10 mg of GOx in 5 mL of Tris buffer solution, and add β - mercaptoethanol after stirring for 10 min to obtain a GOx solution.

[0073] Step S2: Slowly add 300 μL of OA (5 mg / mL, dissolved in ethanol) to the GOx solution to obtain a primary product of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent, denoted as OAG.

[0074] Step S3: Remove free GOx and β-mercaptoethanol by ultrafiltration centrifugation to purify the final product (molecular weight cut-off = 30 kDa). Finally, mix OAG with bacterial outer membrane vesicles (OMVs), and extrude the mixture through a 0.22-μm filter membrane 20 times to obtain a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent, denoted as OAGO.

[0075] When the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent prepared in this example is not transported to the tumor site, it is in the "off" state of catalytic activity; when it is in the acidic tumor microenvironment, it is in the "on" state of catalytic activity. At this time, the GOx nanoparticles encapsulated by the bacterial outer membrane vesicles dissociate, restoring the activity of GOx, and producing gluconic acid and hydrogen peroxide by oxidizing glucose, thereby consuming the nutrients of the tumor and achieving the purpose of starving the tumor; while OA is an inhibitor of adenosine triphosphatase, which can effectively inhibit oxidative phosphorylation and reduce mitochondrial energy supply, thereby leading to an increase in tumor starvation.

[0076] The dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent prepared in this example was observed using a transmission electron microscope, and the results are as Figure 2 shown.

[0077] Example 2

[0078] Evaluation of the GOx catalytic activity of GOx, OAG, and acid-treated OAG on 4T1 tumor cells:

[0079] Incubate GOx, OAG, and acid-treated OAG ([GOx] = 200 ng / mL) with different concentrations of glucose (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10 mM) for 2 h. Then, collect the mixture and detect it using a hydrogen peroxide assay kit. The results are as Figure 3 shown. The catalytic ability of OAG is significantly inhibited in the glucose solution, while the catalytic ability of OAG is enhanced after acid pretreatment. The above results indicate that OAG has acid-activated enzyme catalytic properties, ensuring its effective catalysis and biosafety for cancer treatment.

[0080] Example 3

[0081] Evaluation of the toxicity of different drug treatments on 4T1 tumor cells:

[0082] Using the standard MTT method, evaluate the effect of drugs in different groups on the viability of 4T1 cells. Under the culture conditions of 37 °C and 5% CO2, seed 4T1 cells at 5×10 3Inoculate at a density into a 96-well plate. After 24 h, aspirate the old medium in the 96-well plate and add Dulbecco's Modified Eagle Medium (DMEM) containing 0, 250, 500, 1000, 2500 ng / mL of OA in Example 1 respectively. After continuing to incubate the cells for 24 h, aspirate the old medium in the 96-well plate, add 100 μL of medium solution containing 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to each well, and continue to culture for 4 h. Finally, add 150 μL of dimethyl sulfoxide (DMSO) to each well to replace the medium, and detect the absorbance (OD value, detection wavelength is 490 nm) of each well, denoted as the OA group.

[0083] Under the culture conditions of 37 °C and 5% CO2, inoculate 4T1 cells into a 96-well plate at a density of 5×10 3 Inoculate at a density into a 96-well plate. After 24 h, aspirate the old medium in the 96-well plate and add DMEM medium containing different concentrations of OAG ([OA]=0, 250, 500, 1000, 2500 ng / mL) respectively. After continuing to incubate the cells for 24 h, aspirate the old medium in the 96-well plate, add 100 μL of medium solution containing 5 mg / mL MTT to each well, and continue to culture for 4 h. Finally, add 150 μL of DMSO to each well to replace the medium, and detect the OD value of each well (detection wavelength is 490 nm), denoted as the OAG group.

[0084] Under the culture conditions of 37 °C and 5% CO2, inoculate 4T1 cells into a 96-well plate at a density of 5×10 3 Inoculate at a density into a 96-well plate. After 24 h, aspirate the old medium in the 96-well plate and add DMEM medium containing different concentrations of OAGO ([OA]=0, 250, 500, 1000, 2500 ng / mL) respectively. After continuing to incubate the cells for 24 h, aspirate the old medium in the 96-well plate, add 100 μL of medium solution containing 5 mg / mL MTT to each well, and continue to culture for 4 h. Finally, add 150 μL of DMSO to each well to replace the medium, and detect the OD value of each well (detection wavelength is 490 nm), denoted as the OAGO group.

[0085] Calculate the cell survival rate using the following formula:

[0086] Cell survival rate (%) = (OD490 value of the sample / blank OD490 value) × 100%, and the results are as Figure 4 shown. When OA is 1000 ng / mL, due to the presence of GOx, OAGO has a higher cell killing rate, indicating that OA-mediated tumor cell metabolic therapy enhances GOx-mediated starvation therapy.

[0087] Example 4

[0088] Comparison of the effects of different drug treatments on glycolytic stress and mitochondrial stress in 4T1 tumor cells:

[0089] After inoculating 4T1 cells at a density of 5×10 3 per well into a Seahorse cell culture plate for 12 h, the cells were then incubated for another 24 h with DMEM medium containing different treatments (where 1, 2, 3, 4, and 5 represent the control group, GOx group, OA group, OAG group, and OAGO group, respectively). The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the cells after different treatments were measured using a Seahorse XFe24 extracellular flux analyzer. The measurement procedure was carried out strictly in accordance with the protocol of the equipment supplier.

[0090] The results are as Figure 5 shown, where (a) is the inhibitory effect diagram of the glycolytic pathway of 4T1 cells in different treatment groups; (b) is the quantitative diagram of Figure (a); (c) is the inhibitory effect diagram of the oxidative phosphorylation pathway of 4T1 cells in different treatment groups; (d) is the quantitative diagram of Figure (c).

[0091] As Figure 5 shown in (a) and (c) of, after administration, the combined use of GOx and OA (i.e., the OAGO or OAG group) effectively exacerbated the degree of glycolytic inhibition. In addition, the real-time ECAR at 90 minutes in the OAGO and OAG groups (37.66 and 37.42 mpH / min, respectively) was much lower than that in the control group, GOx group, and OA group (92.46, 64.85, and 112.46 mpH / min, respectively).

[0092] As Figure 5 shown in (b) and (d) of, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), antimycin A, and rotenone were used to measure ATP production, basal respiration, and maximal respiration. Compared with the control group and GOx group, the other groups significantly inhibited basal respiration and ATP production, verifying that OA can induce mitochondrial dysfunction. As Figure 5 shown in (b) of, the cellular OCR in the OAG group and OAGO group decreased due to OA. In contrast, the cellular OCR in the control group and GOx group remained almost unchanged.

[0093] These results confirmed that OAGO can severely block glycolysis, inhibit mitochondrial respiration, and maximally block the energy supply of cancer cells.

[0094] Example 5

[0095] Evaluation of the accumulation effect of OAGO in 4T1 subcutaneous tumors in mice:

[0096] Female Balb / c mice (4 - 5 weeks old, 15 - 20 g) were selected and 1×10 6 4T1 tumor cells were subcutaneously injected into the right hind leg of the mice to establish a subcutaneous tumor model in mice. When the subcutaneous tumor volume exceeded 100 mm 3 , in vivo fluorescence imaging experiments were respectively carried out on the mice injected with GOx, OAG, and OAGO in Example 1. The change of tumor accumulation was observed through a small animal fluorescence imaging system, and the results were as Figure 6 shown.

[0097] As Figure 6 can be seen, 1 hour after tail vein administration, the accumulation of GOx, OAG, and OAGO in tumors reached the maximum, and then began to gradually metabolize. And the in vivo accumulation of OAGO reached the maximum 1 hour after tail vein administration, and the metabolism was relatively slow, indicating that OAGO could accumulate in the tumor site, and the accumulation efficiency was higher than that of GOx and OAG. Therefore, it shows that OAGO has good and relatively high tumor accumulation.

[0098] Example 6

[0099] Evaluation of the effect of OAGO on improving the hypoxia ability of 4T1 subcutaneous tumors in mice:

[0100] Female Balb / c mice (4 - 5 weeks old, 15 - 20 g) were selected and 1×10 6 4T1 tumor cells were subcutaneously injected into the right hind leg of the mice to establish a subcutaneous tumor model in mice. When the subcutaneous tumor volume exceeded 100 mm 3 , photoacoustic imaging experiments were respectively carried out on the mice injected with GOx, OA, OAG, and OAGO in Example 1.

[0101] The influence diagram of different treatment groups on tumor blood oxygen saturation is as Figure 7 shown. Among them, the change of tumor blood oxygen was observed through a photoacoustic imaging system, and the results were as Figure 7 shown in (a).

[0102] At different time points (0, 1, 2, 4, 6, 12, 24 h) after tail vein administration, photoacoustic imaging (PAI) was carried out. As Figure 7 shown in (b), GOx consumed oxygen, resulting in a decrease in the average value of tumor blood oxygen saturation (from 45.56% to 29.29%). However, due to the ability of OA to inhibit oxygen consumption, the average value of blood oxygen saturation in the OAG group increased significantly (from 38.47% to 46.87%). And due to the outer layer being wrapped with bacterial outer membrane vesicles, the blood oxygen saturation in the OAGO group showed the most significant increase (from 40.99% to 60.18%), confirming that OAGO has an excellent function of relieving tumor hypoxia.

[0103] Example 7

[0104] Evaluation of the inhibitory effect of starvation / metabolism / photothermal synergistic therapy on 4T1 tumor growth:

[0105] Female Balb / c mice (4 - 5 weeks old, 15 - 20 g) were selected and 1×10 6 4T1 tumor cells were subcutaneously injected into the right hind leg of the mice to establish a subcutaneous tumor model in mice. When the tumor volume reached 60 mm 3 , a treatment experiment was carried out. In the tumor model, the tumor-bearing mice were randomly divided into seven groups: (1) control group; (2) control light-irradiated group; (3) GOx injection group; (4) OA injection group; (5) OAG injection group; (6) OAGO injection group; (7) OAGO light-irradiated group. The tumor volume was measured every other day with a vernier caliper, and the body weight of the mice was monitored at the same time. The tumor volume was calculated according to the formula V = AB 2 / 2, where A is the long diameter (mm) of the tumor and B is the short diameter (mm) of the tumor. Each measurement result was normalized by the initial tumor volume before treatment. The experimental results are shown in Figure 8 .

[0106] Figure 8 (a) in shows the change of tumor volume of Balb / c mice in different treatment groups in the tumor model over days, Figure 8 (b) in shows the weights of the tumors after dissection of different treatment groups after 14 days of treatment (the 1, 2, 3, 4, 5, 6, 7 on the abscissa represent the control group, control light-irradiated group, GOx group, OA group, OAG group, OAGO group, OAGO light-irradiated group respectively). As can be seen from Figure 8 (a), the OAGO light-irradiated group can significantly inhibit the growth of subcutaneous 4T1 breast cancer tumors and has the best tumor inhibitory effect compared with other treatment groups. As can be seen from Figure 8 (b), after 14 days of treatment with OAGO and light irradiation, the weight of the excised tumor is the lightest, further indicating that the OAGO light irradiation treatment almost completely inhibits the growth of the tumor.

[0107] Example 8

[0108] Biological safety evaluation of OAGO on mice:

[0109] Fourteen days after treatment, representative orbital sinus blood samples were taken from the female Balb / c mice in the saline injection group and OAGO group in Example 7 and analyzed by a blood analyzer. The blood biochemical parameter graphs of healthy mice on the 15th day after intravenous injection of saline and OAGO are shown in Figure 9 ; among them, Figure 9(a) is the comparison chart of alanine aminotransferase in the blood of different groups of balb / c mice, (b) is the comparison chart of aspartate aminotransferase in the blood of different groups of balb / c mice, (c) is the comparison chart of creatinine in the blood of different groups of balb / c mice, and (d) is the comparison chart of blood urea nitrogen in the blood of different groups of balb / c mice; from Figure 9 As can be seen from (a), (b), (c), and (d) in Figure 9 , the numerical levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and blood urea nitrogen (UREA) are basically the same, indicating that OAGO does not damage the physiological functions of the liver and kidneys, proving its good biosafety.

[0110] In summary, the present invention provides a dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent and its preparation method and application. The dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent includes a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor. During the transportation process of the nano-therapeutic agent, due to the hydrophobic interaction between the hydrophobic oxidative phosphorylation inhibitor and the functional protein, the biosafety is improved; at the same time, the bacterial outer membrane vesicle encapsulates the functional protein and the oxidative phosphorylation inhibitor, increasing the tumor accumulation of the nano-therapeutic agent. When the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent is transported to the tumor site, in the acidic tumor microenvironment, the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent dissociates, the activity of the functional protein is restored, and the oxidative phosphorylation inhibitor is released, realizing the selective control of the dual-pathway energy-inhibiting enzyme-based nano-therapeutic agent. The nano-therapeutic agent significantly improves the drug loading and bioavailability of the functional protein, can selectively control the activity of the functional protein, and increases the accumulation amount of the drug at the tumor site by encapsulation with bacterial outer membrane vesicles, improving the curative effect and reducing the toxic and side effects.

[0111] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or changes can be made according to the above description, and all such improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

1. A dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent, characterized in that, It consists of a functional protein, an oxidative phosphorylation inhibitor bound in the hydrophobic cavity of the functional protein, and a bacterial outer membrane vesicle encapsulating the functional protein and the oxidative phosphorylation inhibitor; The mass ratio of the oxidative phosphorylation inhibitor to the functional protein is 1:5 to 1:40; the mass ratio of the bacterial outer membrane vesicle to the functional protein is (1-2):(1-2); The particle size of the dual-pathway energy inhibition type enzyme-based nano-therapeutic agent is 179.87-207.43 nm; The functional protein is glucose oxidase; The oxidative phosphorylation inhibitor is oligomycin A.

2. A preparation method of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent according to claim 1, characterized in that, It includes the steps of: Providing a functional protein and an oxidative phosphorylation inhibitor; Mixing the functional protein and the oxidative phosphorylation inhibitor to obtain a mixture; Adding a bacterial outer membrane vesicle to the mixture, and performing mixing and extrusion treatment to obtain a dual-pathway energy inhibition type enzyme-based nano-therapeutic agent.

3. According to the preparation method of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent described in claim 2, characterized in that, The step of mixing the functional protein and the oxidative phosphorylation inhibitor includes: Mixing the oxidative phosphorylation inhibitor with ethanol to obtain a first solution; Mixing the functional protein with a buffer solution containing β-mercaptoethanol to obtain a second solution; Mixing and stirring the first solution and the second solution.

4. According to the preparation method of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent described in claim 3, characterized in that, The buffer solution is phosphate buffer or tris(hydroxymethyl)aminomethane buffer.

5. According to the preparation method of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent described in claim 2, characterized in that, The step of adding a bacterial outer membrane vesicle to the mixture and performing mixing and extrusion treatment includes: After adding a bacterial outer membrane vesicle to the mixture and mixing, extruding through a 0.19-0.23 μm filter membrane 18-22 times.

6. An application of the dual - pathway energy - inhibiting enzyme - based nano - therapeutic agent according to claim 1 in the preparation of a tumor - treating preparation, wherein the tumor is breast cancer.

Images