CN113599518B - Composite sound-sensitive agent and preparation method thereof - Google Patents
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Abstract
The invention discloses a composite sound-sensitive agent, which comprises sound-sensitive agent nano particles and oxygen-producing microorganisms, wherein the oxygen-producing microorganisms generate oxygen through continuous photosynthesis under the irradiation of near-infrared laser so as to enable an anoxic tumor area to generate photosynthetic oxygenation, and the sound-sensitive agent is ultrasonically excited to convert the oxygen into a large amount of singlet oxygen with cytotoxicity, so that tumor cells are effectively killed and tumor tissues are damaged. The improvement of tumor hypoxia environment and immunogenic cell death caused by the acoustic dynamic therapy realize the cooperative therapy of acoustic dynamic and immunity. Meanwhile, the composite acoustic sensitivity agent can be used for magnetic resonance imaging and electronic computed tomography contrast imaging, and real-time monitoring of the acoustic dynamic treatment process is realized. This work provides good theoretical and experimental support for the development of good biocompatibility and effective acoustic dynamics using hybrid microorganisms and shows important clinical transformation prospects in acoustic dynamics of microbial nanomedicine.
Description
Technical Field
The invention relates to the field of ultrasonic anti-tumor, in particular to a compound sound-sensitive agent and a preparation method thereof.
Background
With the increasing morbidity and mortality, malignant tumors are a serious threat to human health and life safety. In recent years, induction of apoptosis by using reactive oxygen species has been widely used for cancer treatment. Among them, sonodynamic therapy has become an effective strategy for cancer treatment and is receiving much attention. Ultrasound (US) mediated sonodynamic therapy is a non-invasive, high penetration depth treatment with great potential in the treatment of deep solid tumors. The ultrasonic wave is used for triggering the sound-sensitive agent, excessive active oxygen is generated to trigger oxidative stress, and cancer cells are killed, so that the ultrasonic wave is an intrinsic mechanism for treating tumors through acoustic power. Ideal sonodynamic therapy requires three elements: ultrasonic excitation, oxygen, and sonosensitizers. Among them, tumor hypoxia and insufficient oxygen supply have become one of the main factors limiting the therapeutic effect of sonodynamic therapy on tumors, and further consumption of oxygen during sonodynamic therapy exacerbates the hypoxic state of tumors.
Research shows that the lack of oxygen supply inside the solid tumor is caused by the rapid proliferation of tumor cells of the solid tumor, the incomplete blood vessel development and the uneven distribution, so that the oxygen deficiency inside the tumor is caused. Thus, hypoxia is a major feature of advanced solid tumors. Hypoxia further aggravates the instability of tumor genes and activates tumor growth factors, promotes the metastasis of tumors, and severely limits the treatment effect. Wherein, the high expression of the Hypoxia inducible factor-1 alpha (Hypoxia induced factor1 alpha, HIF-1 alpha) related signal channel further inhibits the expression of an epithelial marker E-cadherin, promotes the expression of mesenchymal markers N-cadherin and Vimentin, and increases the mobility and invasiveness of tumor cells. Therefore, hypoxia is closely related to the occurrence, development, prognosis and metastasis of tumors. The efficient treatment aiming at the tumor hypoxia is one of the important problems which must be overcome to realize the complete cure of the tumor.
With the extensive development of nanotechnology, in order to alleviate or reverse the hypoxic region of tumors, researchers developed novel oxygen-producing nanomaterials based on perfluorocarbons, hemoglobin, catalase and the like, and improved the oxygen concentration in hypoxic regions of tumors, thereby improving the curative effect of sonodynamic therapy on hypoxic tumors. However, these methods have several drawbacks, for example, the low efficiency of oxygen loading by perfluorocarbons, and their own toxicity, the high concentration of which poses serious threats to the body; free hemoglobin tetramer molecules and decomposition products thereof can permeate vascular endothelium to enter organs and interstitial tissues, and side effects can be caused; due to the low hydrogen peroxide concentration in the tumor microenvironment, the catalase-loaded nanoparticles lack the reaction substrate to generate enough oxygen, so that the ideal treatment effect is difficult to achieve. Therefore, the development of a novel oxygen carrier, the design and construction of a high-efficiency and high-biosecurity continuous oxygen supply system for relieving the tumor hypoxia microenvironment is one of the best means for improving the acoustic dynamic treatment effect.
Disclosure of Invention
The invention aims to provide a composite sound-sensitive agent and a preparation method thereof, the composite sound-sensitive agent has high efficiency and high biological safety, can continuously supply oxygen to relieve a tumor hypoxia microenvironment, and improves the effect of sound power treatment.
In order to achieve the purpose, the invention provides a composite sound-sensitive agent which comprises sound-sensitive agent nano particles and oxygen-producing microorganisms.
Further, the acoustic sensitizer nano particles are bimetallic manganese tungsten oxide (expressed as Mn) a W b O x Or M) nanoparticles. Used for tumor sonodynamic treatment under multi-modal imaging navigation, mn (II) ions and W element exist a W b O x The nanoparticles can be used for magnetic resonance imaging and computed tomography imaging.
Further, the aerobic microorganism is a cyanobacterium (expressed as cyanobacterium, or Cyan). In recent years, microorganisms have received much attention in the field of bio-nanomedics. The microorganism and the nanometer material are integrated, the functions are complementary, the combined functions of treatment, molecular imaging, biological detection and the like are included, and the method has higher advantages for improving the human health. Cyanobacteria, the earliest photoautotroph on the earth, uses water as an electron donor and solar energy to convert CO 2 Reducing to organic carbon compounds and releasing oxygen. The cyanobacteria with oxygen production capacity is used as a carrier and a propeller of a sound sensitive agent, can produce oxygen in situ in hypoxic tumors through photosynthesis, and promote chemical reaction to generate singlet oxygen with cytotoxicityThereby solving the problem of low effect of the sonodynamic therapy on the hypoxic tumor and providing a new scientific basis for the ultrasonic treatment of the tumor.
Specifically, photosynthetic oxygen supply is accompanied by a sonosensitizer Mn a W b O x Improving the acoustic power effect of the hypoxic tumor. Under the irradiation of laser with the wavelength of 660nm, cyanobacteria generates photosynthesis to generate oxygen, so that the hypoxic tumor area generates photosynthetic oxygenation, the sonosensitizer is further ultrasonically excited, the oxygen is converted into a large amount of singlet oxygen with cytotoxicity, and therefore hypoxic tumor tissues and cells are seriously injured to achieve the purpose of efficient treatment. The improvement of tumor hypoxia environment and the acoustic dynamic therapy result in immunogenic cell death, and the blue algae can be used as an immunologic adjuvant to activate an immune system, thereby realizing the cooperative therapy of the acoustic dynamic therapy and the immunity. In addition, a large amount of oxygen release inhibits the expression of HIF-1 alpha gene, further improves the efficiency of sonodynamic therapy, and finally achieves the aim of efficiently treating tumors. The invention provides theoretical basis and experimental support for the application of microbial nano-medicine in adjuvant synergistic sonodynamic tumor treatment.
The invention also provides a method for preparing the composite sound-sensitive agent, which comprises the following steps: the construction of the composite sound-sensitive agent is completed by loading the sound-sensitive agent nano particles on the surface of the oxygen-producing microorganism through electrostatic adsorption.
Further, the surface of the aerobic microorganism is negatively charged, and the aerobic microorganism is dispersed in a phosphate buffer solution; the surface of the sonosensitizer nano-particle is provided with positive charges, the sonosensitizer nano-particle is added into the phosphate buffer solution, the mixture is stirred, and the oxygen-producing microorganism and the sonosensitizer nano-particle are combined through the adsorption of the positive charges and the negative charges (represented as Mn) a W b O x An, or M @ C).
Further, the sonosensitizer nano-particles are bimetal manganese tungsten oxide (Mn) a W b O x ) Nanoparticles of said bimetallic manganese tungsten oxide (Mn) a W b O x ) The preparation method of the nano-particles comprises the following steps: bimetallic manganese tungsten oxide (Mn) prepared using high temperature organic phase synthesis a W b O x ) An initial particle; and the use of aminopolyethylene glycol stearic acid (C) 18 －polyethylene glycol－amine，C 18 －PEG－NH 2 ) For the bimetal manganese tungsten oxide (Mn) a W b O x ) Modifying the initial particles to obtain the bimetal manganese tungsten oxide (Mn) a W b O x ) Nanoparticles.
Further, the high temperature organic phase synthesis method comprises the following steps: placing 10-20mL of benzyl ether, 1-1.5g of 1,2-dodecane diol and 0.35-0.5g of tungsten hexacarbonyl into a three-neck flask, stirring and mixing, heating the mixed system for the first time under the protection of inert gas, respectively adding 1-3mL of oleic acid and 1-3mL of oleylamine, heating the oleylamine for the second time, adding 0.25g of manganese acetylacetonate, reacting for 30 minutes, cooling the system to room temperature, adding excessive ethanol, fully mixing, centrifuging, repeatedly washing by using a detergent, and collecting the generated initial particles of the bimetallic manganese tungsten oxide.
Further, the temperature of the first heating is 100-120 ℃, and the temperature of the second heating is 230-260 ℃.
Further, the detergent is a mixture of cyclohexane and ethanol.
Further, the bimetal manganese tungsten oxide (Mn) a W b O x ) The step of modifying the initial particle comprises: 10-12mg of said bimetallic manganese tungsten oxide (Mn) a W b O x ) Primary particles and 50mg aminopolyethylene glycol stearic acid (C) 18 －polyethylene glycol－amine，C 18 －PEG－NH 2 ) Mixing in 4-5mL chloroform, performing ultrasonic action for 15-30min, stirring the mixed solution at room temperature for 2-3h, drying with nitrogen, dissolving in deionized water, and storing at 4 deg.C for use.
The invention provides a novel ultrasonic-excited microorganism-enhanced composite acoustic sensitizer with high biological safety and self-supplied oxygen, so as to realize efficient acoustic dynamic tumor treatment. A novel efficient microorganism-enhanced composite micro-nano acoustic sensitivity system is constructed by modifying a functional acoustic sensitivity agent on the surface of blue-green algae for the acoustic dynamic treatment of tumors. The method researches the intratumoral photosynthetic oxygen production action mechanism of the blue algae, optimizes the diagnosis and treatment performance of the sound-sensitive agent, and defines the synergistic action mechanism among the blue algae, the sound-sensitive agent and the ultrasound. The composite sonosensitizer can realize continuous production of active oxygen free radicals under the action of ultrasound so as to improve the efficiency of sonodynamic tumor treatment. Meanwhile, the acoustic dynamic therapy with enhanced photosynthesis can effectively improve the tumor immunosuppression microenvironment, induce the anti-tumor immune response mediated by immunogenic cell death, and finally realize the efficient synergistic treatment of acoustic dynamic and immune of the tumor.
Compared with the prior art, the composite sound-sensitive agent disclosed by the invention has the advantages that under the irradiation of near-infrared laser, the oxygen-producing microorganisms generate oxygen through continuous photosynthesis, so that the oxygen-deficient tumor area is subjected to photosynthetic oxygenation, and the sound-sensitive agent is excited by ultrasound to convert the oxygen into a large amount of singlet oxygen with cytotoxicity, so that tumor cells are effectively killed and tumor tissues are damaged. The improvement of tumor hypoxia environment and immunogenic cell death caused by the acoustic dynamic therapy realize the cooperative therapy of acoustic dynamic and immunity. Meanwhile, the composite acoustic sensitivity agent can be used for magnetic resonance imaging and electronic computed tomography contrast imaging, and real-time monitoring of the acoustic dynamic treatment process is realized. This work provides good theoretical and experimental support for the development of good biocompatibility and effective acoustic dynamics using hybrid microorganisms and shows important clinical transformation prospects in acoustic dynamics of microbial nanomedicine.
More specifically, the beneficial effects of the invention include:
(1) The microorganism-enhanced composite sound-sensitive system constructed by the invention has the advantages of simple preparation process, wide raw material source, simple experimental device, short preparation time and mild conditions.
(2) In order to solve the problem of tumor hypoxia in the sonodynamic therapy, the invention designs a nano acoustic sensitizer which has good stability, uniform particle size, good dispersibility and good biocompatibility, can be used as an imaging contrast agent for magnetic resonance imaging and electronic computer tomography imaging, and realizes the real-time monitoring of the sonodynamic curative effect; meanwhile, cyanobacteria is used as a carrier to construct an ultrasonic-excited microorganism-enhanced composite hybrid micro-nano acoustic-sensitive system, so that the continuous output of oxygen and singlet oxygen is realized, and a new method is provided for the ultrasonic treatment of the hypoxic solid tumor.
(3) In the constructed ultrasonic-excited microorganism-enhanced composite hybrid acoustic power micro-nano acoustic sensing system, active oxygen is generated to kill tumor cells under the participation of ultrasonic radiation, acoustic sensing agents and oxygen. The oxygen production mechanism is examined through the data results of HIF-1 alpha expression experiment and the like by oxygen content measurement. Constructed ultra-small oxygen-deficient manganese-tungsten bimetallic oxide Mn a W b O x Shows high-efficiency ultrasonic triggering generation of active oxygen due to Mn a W b O x The oxygen-poor structure serves as an electron trap site, so that electron-hole recombination can be prevented, meanwhile, oxygen is continuously and efficiently supplied, the yield of active oxygen is increased, and the acoustic dynamic triggering cancer cell killing is enhanced. The development of a novel high-performance multifunctional sound-sensitive agent lays a solid foundation for non-invasive, convenient and economic and effective cancer treatment triggered by ultrasonic waves.
(4) A hypoxic microenvironment exists in most solid tumors, and tumor cells activate a series of related molecular signaling pathways to adapt to the hypoxic microenvironment. HIF-1 alpha is a key transcription regulatory factor mediating cells to perform adaptive response to hypoxic microenvironment, and plays an important role in maintaining energy metabolism, cardiovascular formation, cell proliferation and apoptosis of tumor cells, invasion and metastasis of tumor cells and the like. The invention constructs a micro-nano acoustic-sensitive system, so that the tumor area is reoxygenated, an HIF-1 alpha channel is reduced, the tumor hypoxia microenvironment is improved, and immunogenic cell death is caused after acoustic dynamic therapy.
(5) The invention provides an effective method for enhancing sonodynamic therapy, oxygen is generated through cyanobacteria photosynthesis to improve the anaerobic state in tumors, a large amount of active oxygen is generated through ultrasonic excitation to kill cancer cells, fragments of the tumor cells can be used as antigens, an immunosuppression microenvironment is relieved, and anti-tumor immune response of the tumor cells is activated, so that efficient active oxygen and immune synergistic treatment of the tumors is realized, and tumor recurrence and metastasis are fundamentally prevented.
Drawings
FIG. 1 is a flow chart showing the experiment of the method of using a compound sonosensitizer for the sonodynamic and immune cooperative therapy of tumor;
FIG. 2 shows the experimental results of the preparation of the composite sonosensitizer, wherein a is Mn a W b O x TEM images of the nanoparticles; b is Mn a W b O x EDS-mapping images of nanoparticles; c is a TEM image of cyanobacterial cells; d is a composite sonosensitizer Mn a W b O x TEM images of @ Cyan; e is a composite sonosensitizer Mn a W b O x EDS-mapping image of @ Cyan;
FIG. 3 shows the results of a preliminary experiment for evaluating the biological safety of a sonosensitizer;
FIG. 4 shows the experimental results of the mechanism of oxygen production by cyanobacteria, wherein a is the intratumoral injection of cyanobacteria (1.1X 10) 9 Cells/ml, 100. Mu.L) and under laser irradiation (50 mW/cm) 2 ) Performing photoacoustic imaging of the tumor at a specified time point; b is the corresponding time course of the mean oxygen level as imaged by the PA; c is intratumoral injection of cyanobacteria (1.1X 10) 9 Cells/ml, 100. Mu.L) at various times&E, dyeing; d is intratumoral injection of cyanobacteria (1.1X 10) 9 Cell/ml, 100 μ L) post-HIF-1 α western blot analysis; e. f is the result of quantification of HIF-1 α and CD31 expression based on c, respectively.
FIG. 5 is a graph showing the viability of 4T1 cells assayed under hypoxic and normoxic conditions.
FIG. 6 shows the staining results of viable cells (green fluorescence) and dead or late apoptotic cells (red fluorescence) after different treatments, wherein the first row of control, the second row of US, the third row of M @ C + laser, the fourth row of MnWOxNTPs + US, and the fifth row of M @ C + laser + US.
FIG. 7 is an in vivo evaluation of cascaded photosynthetic oxidation enhanced sonodynamic therapy by intratumoral injection in 4T1 tumor-bearing mice.
FIGS. 8 to 10 are graphs showing the effects of the embodiment 5 of the present invention.
Detailed Description
The "ranges" disclosed herein are in the form of lower and upper limits. There may be one or more lower limits, and one or more upper limits, respectively. The given range is defined by the selection of a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range. For example, ranges of 60-120 and 80-110 are listed for particular parameters, with the understanding that ranges of 60-110 and 80-120 are also contemplated. Further, if the minimum range values of 1 and 2 are listed, and if the maximum range values of 3,4 and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present invention, all embodiments and preferred embodiments mentioned herein may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the steps mentioned herein may be performed sequentially or randomly, if not specifically stated, but preferably sequentially.
Example 1
The preparation method of the compound sound sensitive agent comprises the following steps:
(1) Preparation of Mn by high-temperature organic phase synthesis method a W b O x And (4) initial particles. Placing 20mL of benzyl ether, 1.5g of 1,2-dodecane diol and 0.35g of tungsten hexacarbonyl into a three-neck flask, and mixing by magnetic stirring; heating the mixed system to 120 ℃ under the protection of nitrogen, respectively adding 1ml of oleic acid and 1ml of oleylamine, heating the mixed system to 260 ℃, adding 0.25g of manganese acetylacetonate, reacting for 30 minutes, cooling the system to room temperature, adding excessive ethanol, fully mixing, centrifuging, repeatedly washing by cyclohexane and ethanol, and collectingMn produced by aggregation a W b O x And (4) initial particles.
(2) Using C 18 －PEG－NH 2 To modify the synthesized Mn a W b O x Primary particles, briefly, 10mgMn a W b O x Starting particles and 50mg C 18 －PEG－NH 2 Mixing in 4mL chloroform, and sonicating for 15min. Then stirring the mixed solution for 2 hours at room temperature, and drying by nitrogen to obtain Mn a W b O x Nanoparticles. Mn to be obtained a W b O x －PEG－NH 2 The samples were dissolved in deionized water and stored at 4 ℃ until use.
(3) Mn due to the negative charge on the surface of cyanobacteria a W b O x The surface of the nanoparticle is positively charged, so that the material and the cyanobacteria can be combined through the adsorption effect of the positive and negative charges. The method comprises the following specific steps: dispersing a certain amount of cyanobacteria cells in a phosphate buffer at room temperature, and adding Mn in a proper proportion a W b O x Adding the nanoparticle solution into the solution, mixing and stirring, and centrifuging and washing for later use.
Example 2
The in vivo biocompatibility of different groups of cells was systematically studied before the in vivo treatment evaluation was performed. Furthermore, hematological indicators such as White Blood Cells (WBC), mean Platelet Volume (MPV), red Blood Cells (RBC), mean Corpuscular Hemoglobin (MCH), lymphocytes, hemoglobin (HGB), and neutrophilic granulocytes (NEUT) are detected, and biochemical indicators such as serum glutamic pyruvic transaminase (ALT), aspartate Aminotransferase (AST), creatinine (CREA), and urea are detected.
Example 3
(1) Reoxygenation and related protein expression was tested in a tumor-bearing mouse model. First, by basing on the oxygen saturation pattern (sO) 2 ) Photoacoustic (PA) imaging of (a) evaluated the effect of tumor oxygenation in vivo under laser irradiation. Cyanobacteria (1.1X 10) 9 Cells/ml, 100 μ L) were injected into Balb/C nude mice bearing 4T1 tumors. PA imaging monitoring sO 2 Generation of (2);
(2) Sustained tumor oxygenation in vivo may lead to down-regulation of HIF-1 α expression. Expression of HIF-1 α was then monitored by Western blot;
(3) HE staining and immunohistochemical staining kinetic monitoring monitored HIF-1 α and CD31 expression.
Example 4
In vitro SDT performance was further evaluated using Calcein/propidium iodide (Calcein-AM/PI) staining. 4T1 cancer cells were seeded into a confocal culture dish to attach. And then placing the adherent 4T1 cells into a sealed anaerobic gas generating bag or a humidified incubator to obtain anoxic or normoxic cells. Mixing the M @ C with sound-sensitive agent or Mn after 12h 1.4 WO x The NP was incubated with 4T1 cells under hypoxic or normoxic conditions for 12h. Subsequently, 4T1 cells co-incubated with the M @ C biological mixed sound-sensitive agent were pre-irradiated for 1 minute with or without US irradiation. Will react with Mn 1.4 WO x NP-incubated 4T1 cells were irradiated with US. Then incubated at 37 ℃ for 15min with Calcein-AM/PI, washed with PBS, and then observed with a confocal laser microscope.
Example 5
20 mice implanted with 4T1 tumors were randomly divided into 5 groups, and the tumor-bearing mice were injected with different drugs intratumorally with or without laser irradiation for a 2-week test period. Tumor volume and mouse body weight were measured and calculated every two days to obtain a time-varying curve. At the end of the observation period, all mice were sacrificed and the major organs (heart, liver, spleen, lung, kidney) and tumor tissues were dissected out. Tumor tissues were weighed, photographed, H & E, TUNEL kit, and Ki67 antibody stained.
Fig. 1 is a flow chart of the experiment of the compound acoustic sensitizer in the scheme for the acoustic dynamic and immune synergistic treatment of tumors. Under the irradiation of near infrared laser, the composite sound-sensitive agent of the invention generates oxygen through the continuous photosynthesis of the aerobic microorganism cyanobacteria, so that the oxygen-deficient tumor area generates photosynthetic oxygenation, and the sound-sensitive agent Mn is ultrasonically excited a W b O x The nanoparticles convert oxygen into a large amount of cytotoxic singlet oxygen, thereby effectively killing tumor cells and destroying tumor tissues. Improvement of tumor hypoxic environment and immunogenic cell death following sonodynamic therapyAnd (4) death, realizing the cooperative treatment of the acoustic power and the immunity.
As shown in FIG. 2, it can be seen that Mn was preliminarily prepared at the early stage a W b O x 、Mn a W b O x －PEG、Mn a W b O x －NH 2 、Mn a W b O x @ Cyan nanomaterial, mn evidenced by TEM image a W b O x The nanoparticles exhibited a uniform spherical morphology with a narrow diameter distribution of about 2-4nm, and the elemental mapping showed coexistence of Mn, W, O elements in a uniform distribution (FIG. 2 b), while the TEM mapping showed cyanobacterial cells in a typical rod-like structure with a length of 2-4 μm (FIG. 2 c), in addition to which the elemental mapping of FIG. 2e further showed Mn a W b O x Nanoparticles can be successfully loaded on the cyans.
As shown in fig. 3, it can be seen that there was no death, behavior was significantly abnormal, and the difference in body weight was significant in both the experimental group and the control group in the observation of 30 days (fig. 3 a). The results of hematology indicators such as White Blood Cells (WBC), mean Platelet Volume (MPV), red Blood Cells (RBC), mean red blood cell hemoglobin (MCH), lymphocytes, hemoglobin (HGB), neutrophils (NEUT) indicate that there were no significant differences between the different groups (fig. 3 b-l). Meanwhile, the results of biochemical indexes such as serum glutamic-pyruvic transaminase (ALT), aspartate Aminotransferase (AST), creatinine (CREA) and urea show that M@C has no obvious hepatorenal toxicity to all mice.
As can be seen from FIG. 4, the oxygenation of tumors in the lower body irradiated with laser was evaluated by photoacoustic imaging technique, averaging the sO before injection of cyanobacteria 2 A value of 10.6% indicates the presence of a relative hypoxia level in the tumor region. When blue algae is injected and receives laser irradiation (50 mW/cm) for 10 minutes 2 ) When this is done, a significant oxygen tension is observed. As shown in FIG. 4a, saturation of sO can be observed at the end of irradiation 2 The level increases explosively. After 10 minutes of irradiation, the average saturation sO 2 The level increased from 8.2% to 28.0%. 60 min after injection, mean sO 2 The value was 16.2% (FIG. 4 b). Even after irradiation of sO 2 The level gradually decreases, and the high oxygen tension level can still be stably maintained for 75 minutes after injection,indicating that cyanobacteria continuously produce sO in vivo 2 The ability of the cell to perform. Continued tumor oxygenation in vivo may lead to down-regulation of HIF-1 α expression. HIF-1 α expression was then monitored dynamically by Western blotting and immunohistochemical staining. As shown in FIG. 4c, the combination of cyanobacteria and laser irradiation significantly down-regulated HIF-1 α expression, particularly 2 hours after administration. It then returned to the initial level at 24 hours (fig. 4 f). Furthermore, there was no significant change in CD31 expression (FIG. 4 e). Further WB analysis found HIF-1 α expression levels to be lowest 2 hours post injection, consistent with immunohistochemical staining (fig. 4 d). These results indicate that tumor reoxygenation by cyanobacteria photosynthesis can further down-regulate HIF-1 alpha expression. It is well known that HIF-1 α has been shown to play an important role in tumor progression as a steering mediator of hypoxia signaling. To some extent, down-regulation of HIF-1 α may increase therapeutic efficacy.
As shown in fig. 5, the viability of 4T1 cells was determined under hypoxic and normoxic conditions, respectively. In the absence of oxygen, with Mn 1.4 WO x Compared with the nanometer sound-sensitive agent and the US group (71.0 percent of cell viability), the cell viability is obviously reduced to 48.0 percent under the normal oxygen condition, which indicates that oxygen plays an indispensable role in high-efficiency SDT. Notably, the illumination M @ C showed stronger cytotoxicity (29.0% cell viability) under hypoxic conditions, indicating that the illumination M @ C had a stronger tumor cell killing function. Subsequently, the different treated live cells (green fluorescence) and dead or late apoptotic cells (red fluorescence) were further observed using calflavin-acetoxymethyl ester (calflavin-AM)/Propidium Iodide (PI) co-staining. The control, US and M @ C plus laser gave stronger green fluorescence, respectively (figure 6), indicating that the M @ C low intensity US irradiation had higher biocompatibility and lower cytotoxicity. But Mn is not limited to 1.4 WO x The + US group and the M @ C + laser + US group showed significant red fluorescence signals under the normoxic condition, indicating that Mn 1.4 WO x And M @ C are both useful as US-activated sonosensitizers.
As shown in fig. 7, 4T1 tumor-bearing mice were evaluated in vivo for cascade photosynthetic oxidation enhanced sonodynamic therapy by intratumoral injection. Tumor volumes and body weights were measured for each group of individual mice every two days after the different treatments. No significant changes in body weight were observed throughout the treatment period. After 14 days of treatment, as shown in fig. 8 and 9, the tumor growth of the M @ C + Laser + US group was significantly inhibited, and the tumor inhibition rate reached 91.3%, indicating that the hypoxia-resistant sonodynamic therapy had a higher therapeutic effect with the assistance of photo-oxidative and sonodynamic ROS generation supported by nano-acoustic sensitizer. H & E and immunohistochemical staining of the tumor sections are further collected, and the high inhibition effect of the tumor sections on the tumors is observed. Compared with the normal tumor tissue status of the control group, the US group and the M @ C + Laser group, significant tissue damage and cell necrosis, and nucleus shrinkage rupture were observed in the M @ C + Laser + US group (fig. 10). In Ki67 antibody staining (fig. 10), dark brown cells of the M @ C + Laser + US group represent significant inhibition of tumor cell proliferation. Also, TUNEL staining showed the greatest dark brown cell area that was apoptotic after the same group of different treatments. The results indicate that cyanobacteria-mediated improvement of the tumor microenvironment can fully exploit the advantages of sonodynamic therapy. In vitro and in vivo experimental results prove that the sonosensitizing M @ C biological hybridization can improve the generation rate of active oxygen, enhance the efficacy of the sonodynamic therapy and obviously inhibit the growth of tumors under the irradiation of laser.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Claims (8)
1. The composite sound-sensitive agent is characterized by comprising sound-sensitive agent nanoparticles and an oxygen-producing microorganism, wherein the sound-sensitive agent nanoparticles are double-metal manganese tungsten oxide nanoparticles, and the oxygen-producing microorganism is cyanobacteria.
2. A method for preparing the composite sound-sensitive agent of claim 1, wherein the construction of the composite sound-sensitive agent is completed by loading the sound-sensitive agent nanoparticles on the surface of the aerobic microorganism through electrostatic adsorption.
3. The method of claim 2, wherein said aerobic microorganisms have a negative charge on their surface and are dispersed in a phosphate buffer; the surface of the sound-sensitive agent nano particle is provided with positive charges, the sound-sensitive agent nano particle is added into the phosphate buffer solution, the mixture is stirred, and the oxygen-generating microorganism and the sound-sensitive agent nano particle are combined through the adsorption effect of the positive charges and the negative charges.
4. The method according to claim 2, wherein the sonosensitizer nanoparticles are bimetallic manganese tungsten oxide nanoparticles, and the preparation method of the bimetallic manganese tungsten oxide nanoparticles comprises the following steps:
preparing bimetal manganese tungsten oxide initial particles by using a high-temperature organic phase synthesis method; and
modifying the initial particles of the bimetal manganese tungsten oxide by using amino polyethylene glycol stearic acid to obtain the bimetal manganese tungsten oxide nano particles.
5. The method of claim 4, wherein the step of the high temperature organic phase synthesis comprises: placing 10-20mL of benzyl ether, 1-1.5g of 1,2-dodecane diol and 0.35-0.5g of tungsten hexacarbonyl into a three-neck flask, stirring and mixing, heating the mixed system for the first time under the protection of inert gas, respectively adding 1-3mL of oleic acid and 1-3mL of oleylamine, heating the oleic acid and the oleylamine for the second time, adding 0.25g of manganese acetylacetonate, reacting for 30 minutes, cooling the system to room temperature, adding excessive ethanol, fully mixing, centrifuging, repeatedly washing by using a detergent, and collecting the generated initial particles of the bimetallic manganese-tungsten oxide.
6. The method of claim 5, wherein the first heating is at a temperature of 100 ℃ to 120 ℃ and the second heating is at a temperature of 230 ℃ to 260 ℃.
7. The method of claim 5, wherein the detergent is a mixture of cyclohexane and ethanol.
8. The method of claim 4, wherein the step of modifying the primary particles of bimetallic manganese tungsten oxide comprises: mixing 10-12mg of the bimetal manganese tungsten oxide initial particles and 50mg of aminopolyglycol stearic acid in 4-5mL of chloroform, performing ultrasonic action for 15-30min, stirring the mixed solution at room temperature for 2-3h, and drying by nitrogen.
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