Preparation method and application of nanoparticles for treating oral squamous cell carcinoma

Abstract

The invention discloses a preparation method and application of nano particles for treating oral squamous cell carcinoma, the multifunctional nano particles take dendritic macroporous mesoporous silica as a template, titanium dioxide continues to grow on the surface of the template, and then the obtained mesoporous titanium dioxide is subjected to quaternary ammonium functionalization to obtain the nano particles for treating oral squamous cell carcinoma. And finally, carrying porphyromonas gingivalis cell walls with multiple functions through electrostatic interaction. The nano particles have sonodynamic and chemical dynamic therapy performance, hydrogen peroxide can be catalyzed to generate oxygen, the oxygen and glutathione are subjected to an oxidation-reduction reaction, and carried porphyromonas gingivalis cell walls can activate anti-tumor immune response. The nanoparticles can be used for treating oral squamous cell carcinoma in combination with sonodynamic, chemical dynamic and bacterial immunity under ultrasonic excitation, and recurrence and metastasis of tumors are effectively inhibited through combination of multiple strategies. The method is reasonable in design and simple to operate, can improve the treatment effect of oral squamous cell carcinoma, and has important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of nanoparticles for treating oral squamous cell carcinoma. Background Art

[0002] Oral squamous cell carcinoma is the most common pathological type of oral cancer, with the characteristics of strong invasiveness, high malignancy, easy recurrence and metastasis, and poor prognosis. At present, the main clinical treatment methods are surgical resection, radiotherapy and chemotherapy, targeted therapy and immunotherapy. Among them, surgical resection of superficial and easily resectable tumors can control the growth of tumors, but it will affect the beauty of the patient's face and cause the loss of biological functions; radiotherapy and chemotherapy have strong toxic side effects and are easy to produce tolerance; targeted therapy and immunotherapy have gradually become more promising treatment methods for oral squamous cell carcinoma in recent years. Among them, immunotherapy kills tumors by inducing immune response and can prevent tumor metastasis and recurrence. Compared with traditional treatment methods such as surgery and radiotherapy and chemotherapy, immunotherapy has the advantages of strong specificity and few side effects. However, because many tumors have an immunosuppressive microenvironment and little inflammatory cell infiltration, only a small number of patients have a good effect on this single mechanism of immunotherapy, and the immune response rate during the treatment process is very low, less than 20%.

[0003] Combining bacterial immunotherapy with other non-invasive physical therapy methods can effectively kill tumor cells and effectively treat oral squamous cell carcinoma. Sonodynamic therapy has become a new treatment method developed in recent years due to its strong penetration. By exciting sonosensitizers with ultrasound, hydroxyl radicals (·OH) and singlet oxygen ( 1 O2), superoxide anion (·O 2- ) and other cytotoxic reactive oxygen species to kill tumor cells, and only local ultrasound is performed on the tumor site, which has high accuracy and can reduce damage to surrounding tissues. Chemodynamic therapy is also a new tumor treatment method in recent years. It mainly converts excess hydrogen peroxide in the tumor site into ·OH through the Fenton / Fenton-like reaction, that is, transition metal ions (such as Fe, Co, Ni, Cu, Mn, etc.) undergo redox reactions with hydrogen peroxide under the slightly acidic conditions of the tumor microenvironment to produce highly toxic ·OH, leading to oxidative stress, destroying biological signal molecules such as DNA, proteins, lipids or other substances that maintain life activities in tumor cells, thereby inducing cell apoptosis and necrosis.

[0004] Whether it is sonodynamic therapy produced by ultrasound or chemodynamic therapy relying on endogenous stimulation, reactive oxygen species will be generated to damage tumor cells and cause them to undergo immunogenic cell death. The damage-associated molecular patterns subsequently released can serve as endogenous auxiliary signals to stimulate inflammatory cell infiltration and drive the maturation of antigen-presenting cells. These endogenous auxiliary signals include adenosine triphosphate, calreticulin, and high-mobility protein 1. In addition, bacterial structures such as flagella and lipopolysaccharides can serve as pathogen-associated molecular patterns to stimulate immune responses, further enhancing the immune response caused by the immunogenic death of tumor cells, thereby producing a powerful immune activation response.

[0005] Porphyromonas gingivalis, the main pathogen of periodontitis, is widely present in the oral cavity. It maintains its life activities and virulence by taking in foreign heme, and the excess is stored in its cell wall in the form of heme dimers ([Fe(Ⅲ)PPIX]2O), which are converted from heme and oxygen or other oxygen-containing substances. The porphyrin ring structure of the heme dimer can be used as an organic sonosensitizer to produce reactive oxygen under ultrasound to achieve sonodynamic therapy; heme can catalyze hydrogen peroxide to generate oxygen, reduce itself to heme, and undergo redox reactions with glutathione, consume glutathione, and generate heme; heme can generate ·OH in the presence of hydrogen peroxide to achieve chemodynamic therapy; lipopolysaccharide on its cell wall can be used as an immune adjuvant to achieve immune activation. Therefore, the bacterial wall of Porphyromonas gingivalis has the above multiple functions and can be used in the treatment of oral squamous cell carcinoma by combining sonodynamics / chemodynamics / bacterial immunity. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and application of nanoparticles for treating oral squamous cell carcinoma, so as to solve the technical problem that the immunotherapy in the prior art produces a very low immune response rate during the treatment process. The present invention utilizes the cell wall of Porphyromonas gingivalis with multiple functions, loads it on mesoporous titanium dioxide nanoparticles, and achieves the purpose of efficiently treating oral squamous cell carcinoma by combining sonodynamic / chemodynamic / bacterial immunotherapy. The preparation method is to use dendritic mesoporous macroporous silica as a template, continue to grow titanium dioxide on it, and further quaternary ammonium functionalize it, and use electrostatic adsorption to load the cell wall of Porphyromonas gingivalis to obtain multifunctional nanoparticles carrying the cell wall of Porphyromonas gingivalis.

[0007] The present invention makes full use of the multiple functions of the cell wall of Porphyromonas gingivalis and nanosizes it through nanotechnology to achieve a highly efficient combination of catalytic oxygen production, glutathione consumption, sonodynamics, chemodynamics and bacterial immunotherapy for the treatment of oral squamous cell carcinoma.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing nanoparticles for treating oral squamous cell carcinoma.

[0010] (1) triethanolamine and deionized water are fully mixed, and hexadecyltrimethylammonium bromide is added under 80-85°C oil bath conditions. After stirring for 1-2 hours, deferasirox is added, and after stirring for 2-3 hours, tetraethyl orthosilicate is added. After reacting for 15-20 minutes, an ethanol solution is added to terminate the reaction; the white product is collected by centrifugation, washed with methanol and ethanol, and then dispersed in a mixed solution of anhydrous methanol / concentrated hydrochloric acid (10 / 1, v / v), and condensed and refluxed for 5-6 hours. The reaction is repeated three times, and the template is removed to obtain dendritic macroporous silica nanoparticles (defined as DLMSNs);

[0011] (2) ultrasonically dispersing the DLMSNs obtained in step (1) in an ethanol / acetonitrile mixed solution (3 / 1, v / v), slowly adding 25 wt% ammonia water under stirring, separately dispersing butyl titanate in an ethanol / acetonitrile mixed solution (3 / 1, v / v), then dropwise adding it into the DLMSNs mixed solution, stirring and reacting for 0.5-1 h, centrifuging the product; washing with acetonitrile, dispersing it in a mixed solution of acetonitrile and deionized water (15 / 1, v / v), placing it in a polytetrafluoroethylene reactor in a muffle furnace at 150-170° C. for reaction for 5-6 h, collecting the obtained product by centrifugation, and washing it with acetonitrile to obtain mesoporous titanium dioxide nanoparticles (defined as DT);

[0012] (3) dispersing the DT obtained in step (2) into an ethanol solution, adding deionized water and 25 wt% ammonia water, stirring at 30-40° C. for 2-3 min, then adding 50 wt% N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride solution, and continuing to stir for 24-36 h. The product is collected by centrifugation and washed with ethanol to obtain quaternary ammonium functionalized DT;

[0013] (4) collecting Porphyromonas gingivalis grown on Columbia blood agar plates, treating them with lysozyme, shaking them in a shaker at 37° C. for 3-4 hours in a dark environment, centrifuging them for the first time to discard the supernatant, washing them with deionized water and centrifuging them 4-6 times, collecting and merging the supernatants to obtain Porphyromonas gingivalis bacterial cell walls (defined as PgCW); adding the extracted cell wall solution to the quaternary ammonium functionalized DT solution obtained in step (3), stirring for 10-15 minutes, and centrifuging to obtain a multifunctional nanoparticle product (defined as DT-PgCW).

[0014] Furthermore, in step (1), the obtained DLMSNs have a particle size of 60-150 nm.

[0015] Furthermore, in step (2), the mass ratio of the DLMSNs to DT is 1:0.5-2.

[0016] Furthermore, in step (4), the mass ratio of DT to PgCW is 1:0.01-1.

[0017] Furthermore, the particle size of the nanoparticle DT-PgCW is 80-200nm.

[0018] The present invention also provides the use of multifunctional nanoparticles in treating oral squamous cell carcinoma. The multifunctional nanoparticles treat oral squamous cell carcinoma under the action of ultrasound.

[0019] Furthermore, the multifunctional nanoparticles are prepared by the preparation method of nanoparticles for treating oral squamous cell carcinoma. The multifunctional nanoparticles for treating oral squamous cell carcinoma based on combined sonodynamic / chemodynamic / bacterial immunotherapy have good sonodynamic performance, chemodynamic performance, catalytic oxygen production, glutathione consumption and activation of anti-tumor immune response, and can be used for the efficient treatment of oral squamous cell carcinoma.

[0020] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects:

[0021] (1) The present invention studies the cell wall of Porphyromonas gingivalis and innovatively discovers that it has multiple functions such as sonodynamic properties, chemodynamic properties, catalytic oxygen production, glutathione consumption, and activation of anti-tumor immunity, and successfully constructs nanoparticles that integrate multiple functions.

[0022] (2) The present invention adopts nano-drug delivery technology. By using dendritic macroporous mesoporous silica with good biocompatibility as a template, mesoporous titanium dioxide (DT) with a large specific surface area is prepared. It not only combines inorganic nano-sonosensitizers, but also can better deliver the bacterial cell wall of Porphyromonas gingivalis into tumor cells.

[0023] (3) The constructed DT-PgCW nanoparticles have good biosafety and can combine sonodynamic, chemodynamic, and bacterial immunotherapy under ultrasound stimulation to achieve efficient treatment of oral squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0025] Figure 1 is a TEM image of the nanoparticles prepared in Example 1 of the present invention,

[0026] Where A: DLMSNs; B: DT; C: DT-PgCW;

[0027] Figure 2 It is the element scanning and energy spectrum analysis of DT-PgCW prepared in Example 1 of the present invention;

[0028] Figure 3 is the loading condition of the nanoparticles prepared in Example 1 of the present invention on PgCW,

[0029] A: protein bands of DT, PgCW and DT-PgCW after gel electrophoresis and Coomassie Brilliant Blue staining; B: detection results of lipopolysaccharide of DT-PgCW nanoparticles;

[0030] Figure 4 is the performance of the nanoparticle DT-PgCW prepared in Example 1 of the present invention,

[0031] Where A: DT-PgCW catalytic oxygen production performance; B: DT-PgCW glutathione consumption performance;

[0032] Figure 5 The nanoparticles DLMSNs, DT and DT-PgCW prepared in Example 1 of the present invention were subjected to the conditions of hydrogen peroxide (1 mM) or ultrasound (1 W / cm 2 , 5min) after the action of the active oxygen level,

[0033] Where A: total reactive oxygen level; B: ·OH level; C: 1 O2 levels;

[0034] Figure 6 The nanoparticles DLMSNs, DT and DT-PgCW prepared in Example 1 of the present invention were subjected to ultrasound (1 W / cm 2 , 5min) against oral squamous cell carcinoma;

[0035] Figure 7 The primary tumor inhibition effect of the nanoparticle DT-PgCW prepared in Example 1 of the present invention,

[0036] A: Changes in tumor volume during tumor treatment; B: In vitro tumors of mice 14 days after treatment; C: In vitro tumor mass of mice 14 days after treatment;

[0037] Figure 8 The distal tumor inhibition effect of the nanoparticle DT-PgCW prepared in Example 1 of the present invention,

[0038] A: Changes in distal tumor volume during tumor treatment; B: In vitro images of distal tumors in mice 14 days after treatment. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1: Preparation and characterization of nanoparticles DT-PgCW, and anti-tumor properties in vitro and in vivo

[0045] 1) Preparation of DLMSNs

[0046] Add 400.0 mg of triethanolamine to a 50 mL two-necked bottle and mix thoroughly with 20 mL of deionized water. Continue to add 304.0 mg of hexadecyltrimethylammonium bromide in an 80 ° C oil bath. After stirring for 1 hour, weigh 91.2 mg of deferasirox and add it to the above reaction. After continuing to stir the reaction for 3 hours, add 3.2 mL of tetraethyl orthosilicate solution. Continue stirring for 20 minutes and then add ethanol solution to terminate the reaction. The white product is obtained and collected by centrifugation and washed with methanol. It is redispersed in a mixed solution of anhydrous methanol / concentrated hydrochloric acid (10 / 1, v / v), condensed and refluxed for 6 hours, and the reflux operation is repeated three times. Finally, the product is washed with water and ethanol respectively, then dispersed in ethanol and stored at 4 ° C to obtain DLMSNs. The transmission electron microscopy results of DLMSNs are shown in Figure 1 A.

[0047] 2) Preparation of DT

[0048] The ethanol and acetonitrile solution were mixed evenly at a volume ratio of 3 / 1 (v / v), and the total system was 48 mL. 60 mg of DLMSNs were ultrasonically dispersed in the above mixed solution. 100 μL of concentrated ammonia (25 wt%) was slowly added under stirring conditions of 30°C and 800 rpm. 0.4 g of butyl phthalate was dispersed in a total system of 8 mL of ethanol and acetonitrile (3 / 1, v / v) mixed solution and continued to be added dropwise to the above DLMSNs solution. After stirring for 1 hour, the product was centrifuged, washed with acetonitrile, dispersed in a mixed solution of acetonitrile and water of 15 / 1 (v / v), placed in a polytetrafluoroethylene reactor and reacted in a muffle furnace at 170°C for 5 hours. The obtained product was collected by centrifugation, washed with acetonitrile, and then dispersed in anhydrous ethanol for use to obtain DT. The transmission electron microscopy results of DT are shown in Figure 1 B.

[0049] 3) Preparation of quaternized DT

[0050] Take 10 mL of 2 mg / mL DT ethanol solution, add 200 μL of ultrapure water and 100 μL of ammonia water (25wt%) in sequence, stir for 2 min, then add 200 μL of N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride methanol solution (50wt%), stir at 40 ° C for 24 h, collect the product by centrifugation and wash with ethanol, then disperse in ethanol solution and store at 4 ° C to obtain quaternary ammonium DT.

[0051] 4) Preparation of DT-PgCW

[0052] Porphyromonas gingivalis grown on Columbia blood agar was transferred to a lysozyme solution with a concentration of 2 mg / mL, mixed evenly, and shaken in a 37°C shaker for 3 hours in a dark environment; then centrifuged at 5000 rpm at 4°C for 10 minutes, discarded the light yellow supernatant after the first centrifugation, and then added ultrapure water and repeatedly blown to disperse, and the brown supernatant after 4 centrifugations was collected to obtain a PgCW solution.

[0053] Take 2 mL of the quaternized DT solution with a concentration of 2 mg / mL obtained in step 3) and add it dropwise to the 8.6×10 7 The PgCW solution of 1000 bacteria was stirred for 10 minutes and collected by centrifugation to obtain DT-PgCW. The transmission electron microscopy results of DT-PgCW are shown in Figure 1 C.

[0054] 5) Coomassie Brilliant Blue staining to investigate PgCW-related proteins

[0055] Gel electrophoresis-Coomassie brilliant blue staining was used to detect the types of proteins on DT, DT-PgCW and PgCW. Figure 3 A.

[0056] 6) Investigation of lipopolysaccharide content on DT-PgCW

[0057] The content of lipopolysaccharide on PgCW and DT-PgCW treated with equal concentrations of lysozyme was detected using a horseshoe crab kit. Figure 3 B.

[0058] 7) Investigation of DT-PgCW catalytic oxygen production performance

[0059] The JPBJ-608 portable dissolved oxygen meter was used to measure the catalytic oxygen production ability of 100 μg / mL DT in 1 mM hydrogen peroxide solution and the same concentration of DT-PgCW in 0, 1 and 5 mM hydrogen peroxide solutions. Figure 4 A.

[0060] 8) Detection of the ability of DT-PgCW to consume GSH

[0061] The Ellman's reagent was used to detect the consumption capacity of different concentrations of DT-PgCW on the same concentration of GSH in the solution, where the concentration gradient of the DT-PgCW solution was set to 5μg / mL, 20μg / mL, and 30μg / mL. Figure 4 B.

[0062] 9) Investigation of the performance of active oxygen generation stimulated by ultrasound

[0063] The fluorescent probe 2',7'-dichlorofluorescein diacetate was used to detect the level of reactive oxygen production in DT-PgCW solution under different conditions to evaluate its sonodynamic and chemodynamic properties. The experimental groups were Control, DLMSNs, DT, DT-PgCW, DT-PgCW+H2O2, Control+US, DLMSNs+US, DT+US, DT-PgCW+US, and DT-PgCW+H2O2+US. Among them, the final concentrations of 2',7'-dichlorofluorescein diacetate and samples in each group were 20μM and 100μg / mL, respectively, the H2O2 concentration was 100μM, and the ultrasonic parameters were set to 1W / cm2, 5min, 1MHz, and 50% duty cycle. Finally, the fluorescence intensity of each sample solution was detected by a fluorescence spectrophotometer, and the detection conditions were an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0064] The fluorescent probe terephthalic acid was used to detect the level of ·OH produced by DT-PgCW under different conditions. The experimental groups were the same as above. The final concentrations of terephthalic acid and samples in each group were 2.5mM and 100μg / mL, respectively, and the H2O2 concentration was 100μM. The ultrasonic parameters were set as above. Finally, the fluorescence intensity of each sample solution was detected by a fluorescence spectrophotometer, with an excitation wavelength of 315nm and an emission wavelength of 435nm.

[0065] The singlet oxygen fluorescence detection probe (SOSG) was used to detect the generation of DT-PgCW under different conditions. 1 O2 level. The experimental groups were the same as above, and the final concentrations of the probe and sample in each group were 5μM and 100μg / mL, respectively. The ultrasound parameters were set as above. The fluorescence value of each sample at 504nm was detected by a fluorescence spectrophotometer. The results are shown in Figure 5 A, B, C.

[0066] 10) Investigate the anti-tumor effect of nanoparticles DT-PgCW in vitro

[0067] The MTT method was used to evaluate the effects of different concentrations of each sample with or without ultrasound (1.5 W / cm 2 , 3min) on the cytotoxicity of SCC-7 cells. Figure 6 .

[0068] 11) Investigate the anti-tumor effect of nanoparticles DT-PgCW in vivo

[0069] Step 1: Evaluation of the in vivo therapeutic efficacy of nanoparticle DT-PgCW-mediated combined therapy on primary oral squamous cell carcinoma. The specific operation is as follows: construct a mouse subcutaneous oral squamous cell carcinoma model, wait for the tumor to grow to a volume of about 100mm 3Treatment was started at 14:00. C57 mice were randomly divided into 8 groups (5 mice / group), and the group treatment scheme was as follows: saline control group, DLMSNs treatment group, DT treatment group, DT-PgCW treatment group, saline + US treatment group, DLMSNs + US treatment group, DT + US treatment group, DT-PgCW + US treatment group. Drugs were administered by intratumoral injection. After 6 hours, ultrasound treatment was given at the tumor lesion in the ultrasound group. The ultrasound condition was 2W / cm2 for 5 minutes. Intratumoral injection and ultrasound treatment were performed on the first and fourth days of treatment, respectively. From the first day of treatment, the tumor volume of mice was measured every 2 days, and the weight of mice was measured at the same time, and the measurement was continued until the end of the 14th day of treatment. After the end of treatment, all mice were euthanized, and the tumor sites were collected, photographed, and weighed.

[0070] Step 2: Evaluation of the in vivo inhibitory effect of nanoparticles DT-PgCW on distal tumors. The specific operation is as follows: After the above primary tumor construction is completed, the SCC-7 cell suspension is injected into the other side of the mouse back in the same way one day before treatment to construct a distal tumor model. Subsequently, during the treatment period, the tumor volume of the mice was measured every 2 days, and after the treatment, the distal tumors of all mice were photographed in vitro.

[0071] The transmission electron microscopy results of the nanoparticles obtained in each step of the embodiment are shown in Figure 1 A, B, C, the results show that the average particle size of DT-PgCW nanoparticles is 109±6nm, and it is clearly observed that the contrast of the nanoparticles increases significantly after loading PgCW. Compared with DT, the pores of the nanoparticles are no longer clear, and it can be seen that PgCW is successfully loaded on DT.

[0072] Figure 2 Figure 2 is the element scanning and energy spectrum of DT-PgCW nanoparticles. The results show that DT-PgCW contains elements such as Si, Ti, O, C and a small amount of Fe, among which Fe comes from the porphyrin dimer ([Fe(Ⅲ)PPIX]2O) of PgCW, indicating the successful synthesis of DT and the successful loading of PgCW.

[0073] Figure 3 A is the protein bands of DT-PgCW and PgCW after gel electrophoresis-Coomassie Brilliant Blue staining. The results show that the protein types of the two are basically the same, indicating that PgCW is effectively loaded on the surface of DT-PgCW and the effective ingredients of PgCW are retained. Figure 3 B is the detection of lipopolysaccharide content on the surface of DT-PgCW. The results show that the lipopolysaccharide content on DT-PgCW is basically similar to that of the invested PgCW. From this result, it can be calculated that 87.6% of the invested PgCW is loaded on DT, indicating that the surface of DT-PgCW contains lipopolysaccharide, which is expected to activate the body's immune response.

[0074] Figure 4 A is the investigation of the catalytic oxygen production performance of the nanoparticles. It can be seen that the DT nanoparticles do not produce O2 even in the presence of H2O2, while the DT-PgCW nanoparticles loaded with PgCW can catalyze the production of O2 in the presence of H2O2, and the level of O2 produced gradually increases with the increase in the concentration of H2O2. This fully demonstrates that the DT-PgCW nanoparticles have the ability to catalyze H2O2 to produce O2, and can be used to catalyze the abundant H2O2 in tumor tissues to produce O2, thereby improving the hypoxic state of tumor tissues, thereby further enhancing the therapeutic effect of sonodynamic therapy. Figure 4 B is the investigation of the ability of DT-PgCW nanoparticles to consume glutathione. It can be seen that the higher the concentration of DT-PgCW nanoparticles, the stronger the glutathione consumption ability.

[0075] Figure 5 The levels of reactive oxygen species generated by different nanoparticles under different treatment conditions. Figure 5 A shows the use of DCFH-DA probe to detect the level of total reactive oxygen species produced by different nanoparticles in the presence or absence of H2O2. Under non-ultrasound conditions, compared with the water control group, the DLMSNs and DT groups showed fluorescence intensities similar to those of water, but DT-PgCW showed a certain fluorescence signal, which may be because PgCW itself reacted with DCFH-DA. According to literature reports, DCFH can be oxidized by oxidizing substances, thereby enhancing fluorescence. PgCW contains oxidizing Fe 2+ , which can enhance the fluorescence signal of DCFH. When H2O2 was added to the DT-PgCW group, the fluorescence intensity was significantly enhanced. This is due to the fact that Fe 2+ The Fenton reaction with H2O2 promoted the production of reactive oxygen species, indicating that DT-PgCW has a chemical dynamic effect. After ultrasound treatment, the fluorescence intensity of each experimental group increased to varying degrees, among which the DT-PgCW+H2O2 group combined with ultrasound showed the strongest fluorescence signal enhancement effect, indicating its excellent ability to produce reactive oxygen species. Figure 5B is a terephthalic acid fluorescent probe to detect the ability of different nanoparticles to produce ·OH. Since only a small amount of ·OH is produced during the sonodynamic process, this test is mainly used to evaluate the chemical kinetic properties of nanoparticles. From the results, it can be seen that when H2O2 is not added, the fluorescence intensity of the DLMSNs, DT, and DT-PgCW groups is close to that of the Control group, indicating that there is basically no ·OH produced; when H2O2 is added, the fluorescence signal of the DT-PgCW group is significantly enhanced, proving its chemical kinetic properties. After ultrasonic treatment, the DLMSNs, DT, and DT-PgCW groups all showed enhanced levels of reactive oxygen generation; when H2O2 was added to the DT-PgCW group at the same time, under the combined action of H2O2 and ultrasound, its ·OH production efficiency was greatly enhanced. Figure 5 C is detected using SOSG fluorescent probe 1 O2 results. The active oxygen generated by the sonodynamic process is mainly 1 O2, so this test is mainly used to evaluate the sonodynamic properties of nanoparticles. From the results, it can be seen that the fluorescence signals of each nanoparticle did not show obvious differences when not acted on by ultrasound; after ultrasonic treatment, the fluorescence signals of each group were enhanced to varying degrees compared with the control group, and were enhanced successively with each step of modification of the nanoparticles. When H2O2 was added to the DT-PgCW group, enhanced fluorescence intensity was shown. This is because DT-PgCW can catalyze H2O2 to produce O2, which, as an oxidizing substance, can also react with SOSG, thereby enhancing its fluorescence signal; when combined with ultrasound, the DT-PgCW+H2O2+US group showed the strongest fluorescence intensity, indicating that the O2 produced by PgCW catalysis can further enhance 1 Production of O2.

[0076] Figure 6 The killing performance of DLMSNs, DT, DT-PgCW and their ultrasonic treatment on mouse oral squamous cell carcinoma cells SCC-7 was evaluated. The results showed that when the concentration was 0-400μg / mL, the DLMSNs and DT groups did not produce obvious cytotoxicity, while DT-PgCW had a 60% killing effect when the concentration reached 400μg / mL. Compared with the non-ultrasound group, the ultrasound group showed certain sonodynamic therapeutic performance in the DLMSNs and DT groups of nanoparticles, causing a certain degree of cell death. When the DT-PgCW nanoparticles were at a concentration of 100μg / mL, the cell survival rate was only 50% under ultrasonic excitation, and at 400μg / mL combined with ultrasonic action, the cell survival rate was less than 20%.

[0077] Figure 7 Evaluation of the inhibitory effect of DT-PgCW nanoparticles on primary tumor growth. Figure 7A's tumor growth curve shows that without ultrasound treatment, the tumor growth rates of the saline group, DLMSNs group and DT group were relatively fast, with no obvious tumor inhibition effect, and the tumor growth rates were relatively close. However, the tumor growth rate of the DT-PgCW group was significantly inhibited, proving that DT-PgCW can play a chemokinetic therapeutic role, produce certain reactive oxygen species to kill tumor cells, and the immunogenic cell death effect induced by killing cells can effectively combine with the lipopolysaccharide of PgCW to stimulate immune response, thereby inhibiting tumor growth. After ultrasound treatment was applied on the basis of drug treatment, the saline group and DLMSNs group had no obvious tumor growth inhibition effect. The DT combined with ultrasound group showed a significant tumor inhibition effect, indicating that DT has good sonodynamic therapeutic performance. DT-PgCW combined with ultrasound treatment greatly inhibited tumor growth, proving that in addition to its chemokinetic properties, DT-PgCW can also achieve better sonodynamic therapeutic performance on the basis of alleviating tumor tissue deficiency. Moreover, the sonodynamic properties of the combination of inorganic sonosensitizer DT and organic sonosensitizer PgCW can further enhance the production of reactive oxygen species, thereby achieving the purpose of inhibiting tumor growth. At the same time, the effective combination of immunogenic cell death effects and PgCW immune adjuvants can exert a more effective anti-tumor immune activation effect under the action of ultrasound. It is fully proved that DT-PgCW-mediated combined therapy can effectively treat OSCC. Figure 7 The in vitro photos and weight results of the tumors after treatment in B and C also showed the same trend.

[0078] Figure 8 To evaluate the inhibitory effect of DT-PgCW-mediated combined therapy on tumor metastasis. A bilateral tumor model was established to simulate tumor metastasis and verify the distal effect of the therapy. The results showed that the growth trend of distal tumors in the DLMSNs and DT groups was not much different from that in the saline group without ultrasound treatment, indicating that these two nanoparticles had no effect on the growth of distal tumors, but the tumor growth in the DT-PgCW group was relatively slow, indicating that the chemical power generated by the loaded PgCW induced immunogenic cell death and activated the immune response in mice with its immune adjuvant effect, reducing the tumor growth rate to a certain extent; under ultrasound conditions, the DT group also had a certain distal tumor inhibition effect compared with the saline group and the DLMSNs treatment group, indicating that the immunogenic cell death produced by DT under ultrasound can promote the recognition of tumor cell fragments by immune cells, thereby transmitting signals to the body, thereby promoting the infiltration of immune cells into tumor tissues and alleviating the immunosuppression of tumor tissues. On this basis, DT-PgCW as an immune adjuvant further stimulates the body's immune response, proving that DT-PgCW combined with ultrasound can not only kill primary tumors, but also effectively inhibit the growth of distal tumors.

[0079] Finally, it should be noted that:

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing nanoparticles for treating oral squamous cell carcinoma, characterized in that: The following steps are involved: (1) Under 80-85 ℃ oil bath conditions, hexadecyltrimethylammonium bromide, deferasirox, and tetraethyl orthosilicate are added to a mixture of triethanolamine and water in sequence, and after the reaction is completed, an ethanol solution is added to terminate the reaction; the white product is collected by centrifugation and washed, and then dispersed in a mixture of anhydrous methanol / concentrated hydrochloric acid, and condensed and refluxed to remove the template to obtain dendritic macroporous silica nanoparticles DLMSNs; (2) DLMSNs were ultrasonically dispersed in an ethanol / acetonitrile mixed solution, 25 wt% ammonia water was slowly added under stirring, butyl titanate was dispersed in an ethanol / acetonitrile mixed solution, and then it was added dropwise to the DLMSNs mixed solution. After the stirring reaction was completed, the product was centrifuged; washed with acetonitrile, dispersed in a mixed solution of acetonitrile and deionized water, reacted at 150-170 °C, the obtained product was collected by centrifugation, and washed with acetonitrile to obtain mesoporous titanium dioxide nanoparticles DT; (3) Dispersing DT in an ethanol solution, adding deionized water and 25 wt% ammonia water, stirring at 30-40 °C, adding 50 wt% N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride solution, and continuing stirring for 24-36 h. The product was collected by centrifugation and washed with ethanol to obtain quaternary ammonium functionalized DT. (4) Add the Porphyromonas gingivalis cell wall solution PgCW to the quaternary ammonium functionalized DT solution obtained in (3), stir for 10-15 min, and centrifuge to obtain the multifunctional nanoparticle product DT-PgCW.

2. The method for preparing nanoparticles for treating oral squamous cell carcinoma according to claim 1, characterized in that: The particle size of DLMSNs in (1) is 60-150 nm, and the volume ratio of anhydrous methanol / concentrated hydrochloric acid is 10:

1.

3. The method for preparing nanoparticles for treating oral squamous cell carcinoma according to claim 1, characterized in that: The mass ratio of DLMSNs to DT in (2) is 1:0.5-2, the volume ratio of ethanol / acetonitrile is 3:1, and the volume ratio of acetonitrile to deionized water is 15:

1.

4. The method for preparing nanoparticles for treating oral squamous cell carcinoma according to claim 1, characterized in that: The mass ratio of DT to PgCW in (4) is 1:0.01-1.

5. The method for preparing nanoparticles for treating oral squamous cell carcinoma according to claim 1, characterized in that: The particle size of DT-PgCW in (4) is 80-200 nm.

6. The method for preparing nanoparticles for treating oral squamous cell carcinoma according to claim 1, characterized in that: The extraction of the Porphyromonas gingivalis cell wall solution in (4) is as follows: Porphyromonas gingivalis is treated with lysozyme, shaken in a shaker at 37°C for 3-4 hours in a dark environment, then centrifuged for the first time to discard the supernatant, then washed with deionized water and centrifuged 4-6 times, and the supernatant was collected and combined to obtain the Porphyromonas gingivalis cell wall solution PgCW.

7. Use of multifunctional nanoparticles in the treatment of oral squamous cell carcinoma, characterized in that: The multifunctional nanoparticles are used to treat oral squamous cell carcinoma under the action of ultrasound.

8. Use of the multifunctional nanoparticles according to claim 7 in treating oral squamous cell carcinoma, characterized in that: The multifunctional nanoparticles are prepared by a preparation method of nanoparticles for treating oral squamous cell carcinoma.

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