CN112773894A - Multifunctional black phosphorus nanoparticle-based nano complex for photoacoustic immunotherapy and preparation method thereof - Google Patents
Multifunctional black phosphorus nanoparticle-based nano complex for photoacoustic immunotherapy and preparation method thereof Download PDFInfo
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Abstract
本发明公开一种基于多功能的黑磷纳米粒子的光声免疫治疗的纳米复合体及其制备方法，本发明的BP/R848/OVA/HS‑PEG‑NH2/TPP复合体是将免疫佐剂(R848)和鸡卵清蛋白(OVA)静电吸附到黑磷（BP）上，得到BP/R848/OVA，实现激活树突状细胞(DC细胞)的作用。然后将一端修饰巯基一端修饰氨基的聚乙二醇(HS‑PEG‑NH2)和活化的（3‑丙羧基）三苯基溴化膦(TPP)在室温下组装成HS‑PEG‑NH2/TPP，实现线粒体靶向作用。将BP/R848/OVA和HS‑PEG‑NH2/TPP在室温下搅拌得到复合纳米粒子。本发明中BP具有生物相容性好，负载效率高的特点，能够高效运载R848/OVA/HS‑PEG‑NH2/TPP复合体进入细胞来执行其功能；并且R848/OVA/HS‑PEG‑NH2/TPP复合体能保护BP，使其稳定性得到提高。结合免疫检查点抑制剂,实现精确的光声减瘤并产生免疫效应达到延长光声治疗的时间和扩展光声治疗的空间的功能。
The invention discloses a nanocomposite for photoacoustic immunotherapy based on multifunctional black phosphorus nanoparticles and a preparation method thereof. The BP/R848/OVA/HS-PEG-NH2/TPP complex of the present invention is an immune adjuvant (R848) and chicken ovalbumin (OVA) were electrostatically adsorbed to black phosphorus (BP) to obtain BP/R848/OVA, which could activate dendritic cells (DC cells). Then, polyethylene glycol (HS-PEG-NH2) modified with thiol group at one end and amino group at the other end and activated (3-propylcarboxy) triphenylphosphine bromide (TPP) were assembled into HS-PEG-NH2/TPP at room temperature , to achieve mitochondrial targeting. The composite nanoparticles were obtained by stirring BP/R848/OVA and HS‑PEG‑NH2/TPP at room temperature. The BP in the present invention has the characteristics of good biocompatibility and high loading efficiency, and can efficiently carry the R848/OVA/HS-PEG-NH2/TPP complex into cells to perform its function; and R848/OVA/HS-PEG-NH2 The /TPP complex protects BP and improves its stability. Combined with immune checkpoint inhibitors, we can achieve precise photoacoustic debulking and produce immune effects to extend the time and space of photoacoustic therapy.
Description
Technical Field
The invention belongs to the technical field of drug-loaded materials, and particularly relates to a degradable black phosphorus quantum dot high-efficiency delivery immunologic adjuvant and application thereof in tumor treatment in combination with a checkpoint inhibitor.
Background
The main drawback of current photothermal and photodynamic therapy is the shallow depth of kill due to low light transmission and poor drug selectivity. When the surface irradiation is carried out by the laser with the wavelength of 630 nanometers, under the conditions of conventional medicine dosage and illumination dosage, the tumor killing with the depth of 7-15 millimeters can be realized by one treatment, and at the moment, normal tissues subjected to the same illumination are hardly damaged. This is usually sufficient for transitional cell carcinoma of the bladder, a tumor with a shallow invasion depth, but is often insufficient for breast tumors, bronchopulmonary carcinoma, which may exceed this limit, but can be compensated by means of interstitial irradiation. Another disadvantage is that the patient is likely to develop skin photosensitivity for a certain period of time after administration, and it is desirable to avoid intense light exposure.
Photoacoustic therapy is a tumor therapy technique that has emerged in recent years, and is based on the photoacoustic effect of nanomaterials, i.e., nanomaterials generate strong enough photoacoustic shock waves under the excitation of pulse laser to mechanically destroy tumor cell membranes, thereby achieving the purpose of tumor therapy. Photoacoustic therapy has numerous advantages over other methods: 1. the photoacoustic therapy is mechanical damage to tumor cells, so that toxic and side effects are not easily caused, and drug resistance is not generated. 2. The laser power used by the photoacoustic therapy of the tumor is reduced by 150-1500 times compared with the traditional photothermal therapy, but the treatment efficiency is improved. 3. The photoacoustic therapy can kill tumor cells accurately without affecting surrounding normal tissue cells.
The nano probe mediated photoacoustic therapy as a novel tumor treatment method has great potential in clinical application and popularization due to the non-invasiveness, no toxic or side effect and tumor selectivity. But has a disadvantage in that it is not very effective for tumors that are easy to metastasize and tumors that are easy to recur. Therefore, there is a need to develop a treatment technique that is non-invasive, non-toxic and non-toxic, and can inhibit tumor metastasis and recurrence.
Disclosure of Invention
To overcome the disadvantages and drawbacks of the prior art, it is a primary object of the present invention to provide a photoacoustic immunotherapeutic nanocomposite (BP/R848/OVA/HS-PEG-NH 2/TPP).
Another object of the present invention is to provide a method for photoacoustic immunotherapy by using degradable black phosphorus nano quantum dots to efficiently carry immune adjuvants and combining checkpoint inhibitors.
The purpose of the invention is realized by the following technical scheme
The invention provides a nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) for photoacoustic immunotherapy, which is characterized in that an immunologic adjuvant (R848) and chicken Ovalbumin (OVA) are electrostatically adsorbed on black phosphorus quantum dots to obtain black phosphorus BP/R848/OVA modified by the immunologic adjuvant; then, polyethylene glycol (HS-PEG-NH2) with one end modified with sulfhydryl and one end modified with amino and activated (3-propylcarboxyl) triphenyl phosphine bromide (TPP) are assembled into a (3-propylcarboxyl) triphenyl phosphine bromide modified sulfhydryl polyethylene glycol (abbreviated as HS-PEG-NH2/TPP) complex at room temperature; mixing black phosphorus modified by immune adjuvant (BP/R848/OVA) and sulfhydryl polyethylene glycol modified by (3-propyl carboxyl) triphenyl phosphine bromide (HS-PEG-NH2/TPP) at room temperature to obtain the nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) for photoacoustic immunotherapy.
The black phosphorus is black phosphorus quantum dots, and the concentration of the black phosphorus is 2 mg/mL.
The molecular weight of the immunological adjuvant (R848) is 314.38, and the concentration is preferably 0.5 mg/mL.
The molecular weight of the Ovalbumin (OVA) is 45kD, and the concentration is preferably 0.5 mg/mL.
The heterofunctional group disubstituted ethylene glycol derivative (HS-PEG-NH 2). One end is amino and the other end is carboxyl, the molecular weight is 2000, and the concentration is preferably 20mg/mL.
The molecular weight of the (3-propylcarboxyl) triphenyl phosphine bromide (TPP) is 429, and the concentration is preferably 0.02mmol.
The final concentration of BP in the preferred nanocomposite (BP/R848/OVA/HS-PEG-NH 2/TPP) was 1mg/mL, the final concentration of R848/OVA was 0.2mg/mL, and the final concentration of HS-PEG-NH2/TPP was 0.2mmol.
The black phosphorus loaded immunoadjuvant combined with the nanoprobe of the mitochondrion targeting molecule specifically comprises the following steps:
(1) preparation of black phosphorus quantum dots
The black phosphorus quantum dots are obtained by a liquid phase stripping method. 20mg of black phosphorus crystal powder (BP) was dispersed in 20ml of N-methylpyrrolidone (NMP) and sonicated for 4 hours (cycle of 4s interval, 2s treatment time) with a sonicator (sonication frequency 19-25KHz, power 1200W). The mixture was then treated further overnight in an ice bath under ultrasound at a power of 300W. The dispersion was centrifuged at 7000rpm for 20min and the supernatant containing BP was gently removed. The supernatant was centrifuged at 12000rpm for 20min, the BP precipitate was collected and washed three times with distilled water. And obtaining the black phosphorus quantum dots.
(2) Immunoadjuvant-modified black phosphorus BP/R848/OVA
Weighing 0.4mg of black phosphorus quantum dots (BP), adding 200 mu L of triple distilled water, uniformly mixing, and carrying out ultrasonic treatment for 30min to obtain 2mg/mL of black phosphorus quantum dot dispersion liquid. Adding 0.2mL of 0.5mg/mL immunoadjuvant (R848) and Ovalbumin (OVA) into the BP dispersion, mixing uniformly, performing ultrasonic treatment in water bath for 1h, and stirring at room temperature for 6 h. Obtaining the immunoadjuvant modified black phosphorus BP/R848/OVA.
(3) Synthesis of PEG modified by mitochondria targeting molecule
8mg of (3-propylcarboxyl) triphenylphosphonium bromide (TPP) was weighed out and pre-dissolved in 0.2mL of dimethyl sulfoxide (DMSO). 9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 6mg of N-hydroxysuccinimide (NHS) were added and stirred for 4 hours. 0.5mL of 20mg/mL polyethylene glycol (HS-PEG-NH2) with a thiol group modified at one end and an amino group modified at the other end was added to the solution. The reaction is carried out for 12 hours at room temperature to obtain the final concentration of 0.2mmol of (3-propyl carboxyl) triphenyl phosphonium bromide modified sulfhydryl polyethylene glycol (HS-PEG-NH2/TPP) complex.
(4) Synthesis of photoacoustic immunotherapy nanocomplexes (BP/R848/OVA/HS-PEG-NH 2/TPP)
1mg/mL of immunoadjuvant-modified black phosphorus (BP/R848/OVA) and 0.2mmol (3-propylcarboxyl) of triphenylphosphine-bromide-modified mercaptopolyethylene glycol (HS-PEG-NH2/TPP) were mixed and stirred at room temperature for 12 hours, and washed three times with distilled water. Centrifuging at 7000rpm for 10min to obtain final 1mg/mL photoacoustic immunotherapy nano-complex (BP/R848/OVA/HS-PEG-NH 2/TPP).
(5) In vitro verification of targeting effect of nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) on mitochondria
Breast cancer cells (EMT 6) were cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS) in a cell culture chamber under the following conditions: the CO2 concentration was 5%, the humidity was 90%, and the temperature was 37 ℃. 20 μ L of 0.2mg/mL of the fluorescent dye CY5.5 and 1mL of 1mg/mL of the nanocomposite (BP/R848/OVA/HS-PEG-NH 2/TPP) were stirred at room temperature for 4 hours. The fluorescent dye CY5.5 modified nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) is obtained, and the concentration is 1 mg/mL. 50 mu L of the complex is incubated with breast cancer cells (EMT 6) for 4 hours, then mitochondrial fluorescent probes are added for half an hour, and the co-localization of the nanoparticles and the mitochondrial probes is observed under a confocal microscope.
(6) In vitro validation of the immune activation of the Nanocolex (BP/R848/OVA/HS-PEG-NH 2/TPP) on dendritic cells (DC 2.4 cell line)
50 mu.L of 1mg/mL nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP) was incubated with the DC2.4 cell line for 12 hours, anti CD80-PE was used to label CD80, anti CD86-APC was used to label CD86, and DC cells were counted by flow cytometry to verify the immune activation of DC cells by the nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP).
(7) And (3) verifying the immune activation of the tumor-associated antigen generated after the tumor cells are killed by the photoacoustic on the DC cells in vitro.
EMT6 cells were cultured in the upper transwell chamber and incubated with the nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP), and DC cells were cultured in the lower chamber. The breast cancer cells (EMT 6) were apoptotic by irradiating the upper chamber with a pulsed laser at 808nm at 20mj for 10 min. Apoptotic cells produce tumor-associated antigens that further promote DC cell maturation. DC cells were detected by flow cytometry.
For flow cytometry analysis, DC2.4 cells were seeded into 6-well plates (approximately 100,000 cells per well) under the same processing conditions as before. After 12 hours, the cells were washed 3 times with Phosphate Buffered Saline (PBS) and then digested with 2.5% trypsin at 37 ℃ for 1-2 min. The cells were then gently blown down and resuspended in PBS and analyzed by flow cytometry.
When analyzed by the laser scanning confocal microscope in the step (5), the wavelength of the excitation light used by the mitochondrial fluorescent probe is 488nm, the wavelength of the emission light is 498nm, the wavelength of the excitation light used by the CY5.5 is 633nm, and the wavelength of the emission light is 690 nm.
And (4) performing statistical analysis by using a flow cytometer in the step (6), wherein the wavelength of the excitation light used by the PE is 488nm, the wavelength of the emission light is 578nm, the wavelength of the excitation light used by the APC is 633nm, and the wavelength of the emission light is 660 nm.
In the research, the black phosphorus quantum dots are used as a carrier to load immune adjuvants for the first time to develop a high-efficiency, stable and high-biocompatibility photoacoustic immunotherapy system. The black phosphorus quantum dots have been widely used as bio-carriers for drug delivery and photothermal therapy, etc. because of their advantages of excellent biocompatibility, high loading efficiency, high thermal conversion efficiency, low toxicity, etc. Since black phosphorus is easily degraded in air, we use immune adjuvant (R848) and chicken Ovalbumin (OVA) to coat black phosphorus to prolong the degradation time of black phosphorus and exert the immune adjuvant effect of OVA and R848 to promote the maturation of DC cells. Covalent modification with thiolated PEG-TPP to OVA further promotes black phosphorus biocompatibility and enables targeting of subcellular organelles (mitochondria). The obtained product can kill tumor cells better. Because the carrier has the advantages of simple structure, stability, low toxicity, good biocompatibility and capability of activating immune effect, the composite of the black phosphorus quantum dot loaded immune adjuvant provides hope for clinical treatment.
The invention has the following advantages and effects compared with the prior art
(1) The method utilizes the black phosphorus quantum dots (BP) as a transportation carrier, has simple construction and can achieve the aim of low toxicity.
(2) The chicken Ovalbumin (OVA) and polyethylene glycol (PEG) modified black phosphorus quantum dots (BP) have the characteristics of good biocompatibility and high loading efficiency, and can efficiently carry the immunologic adjuvant R848 into cells to execute the functions.
(3) The Ovalbumin (OVA) and polyethylene glycol (PEG) modified black phosphorus quantum dot (BP) can protect the BP to slow down the degradation speed of black phosphorus, can enable the BP to better execute the function, and has high stability. Drawings
FIG. 1 is a schematic diagram of multifunctional black phosphorus nanoparticles for near-infrared light-triggered photoacoustic-immunotherapy
FIG. 2 is a representation of the UV absorption of the photoacoustic immunotherapy nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP).
FIG. 3 is a graph showing in vitro verification of the targeting effect of the photoacoustic immunotherapy nanocomplexes (BP/R848/OVA/HS-PEG-NH 2/TPP) on mitochondria
FIG. 4 is a graph showing in vitro verification of the immune activation of DC cells by the photoacoustic immunotherapy nanocomplexes (BP/R848/OVA/HS-PEG-NH 2/TPP)
FIG. 5 is a graph showing in vitro immune activation of DC cells by tumor-associated antigens generated after photoacoustic killing of tumor cells.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The experimental procedures for the following examples, where specific experimental conditions are not noted, are generally performed according to conventional experimental conditions or according to experimental conditions recommended by the manufacturer.
The basic principle of the invention is shown in figure 1, and in the research, the black phosphorus quantum dot is used as a carrier to load an immunologic adjuvant for the first time to develop a high-efficiency, stable and high-biocompatibility photoacoustic immunotherapy system. The black phosphorus quantum dots have been widely used as bio-carriers for drug delivery and photothermal therapy, etc. because of their advantages of excellent biocompatibility, high loading efficiency, high thermal conversion efficiency, low toxicity, etc. Since black phosphorus is easily degraded in air, we use immune adjuvant (R848) and chicken Ovalbumin (OVA) to coat black phosphorus to prolong the degradation time of black phosphorus and exert the immune adjuvant effect of OVA and R848 to promote the maturation of DC cells. Covalent modification with thiolated PEG-TPP to OVA further promotes black phosphorus biocompatibility and enables targeting of subcellular organelles (mitochondria). The obtained product can kill tumor cells better. Because the carrier has the advantages of simple structure, stability, low toxicity, good biocompatibility and capability of activating immune effect, the composite of the black phosphorus quantum dot loaded immune adjuvant provides hope for clinical treatment. The preparation method comprises the following steps:
1. preparation of black phosphorus quantum dots
The black phosphorus crystal powder was purchased from Nanjing Xiancheng nanomaterial science and technology Co., Ltd and stored in a dark argon-filled ampoule. N-methylpyrrolidone (NMP) was purchased from Shanghai Allantin Biotech Co., Ltd.
The black phosphorus quantum dots are obtained by a liquid phase stripping method. 20mg of black phosphorus crystal powder (BP) was dispersed in 20ml of N-methylpyrrolidone (NMP) and sonicated for 4 hours (cycle of 4s interval, 2s treatment time) with a sonicator (sonication frequency 19-25KHz, power 1200W). The mixture was then treated further overnight in an ice bath under ultrasound at a power of 300W. The dispersion was centrifuged at 7000rpm for 20min and the supernatant containing BP was gently removed. The supernatant was centrifuged at 12000rpm for 20min, the BP precipitate was collected and washed three times with distilled water. And obtaining the black phosphorus quantum dots.
2. Immunoadjuvant-modified black phosphorus BP/R848/OVA
Egg white protein (OVA) was purchased from sigma aldrich trade ltd and R848 was purchased from MedChemexpress biotechnology, usa.
Weighing 0.4mg of black phosphorus quantum dots (BP), adding 200 mu L of triple distilled water, uniformly mixing, and carrying out ultrasonic treatment for 30min to obtain 2mg/mL of black phosphorus quantum dot dispersion liquid. Adding 0.2mL of 0.5mg/mL immunoadjuvant (R848) and Ovalbumin (OVA) into the BP dispersion, mixing uniformly, performing ultrasonic treatment in water bath for 1h, and stirring at room temperature for 6 h. Obtaining the immunoadjuvant modified black phosphorus BP/R848/OVA.
3. Synthesis of PEG modified by mitochondria targeting molecule
(3-Propylcarboxy) Triphenyl phosphine Bromide (TPP) from Sigma Aldrich trade, Inc. [ alpha ] -sulfanyl-omega ] -aminopolyethylene glycol (HS-PEG-NH)2) Available from Shanghai Yanyi Biotech Ltd
8mg of (3-propylcarboxyl) triphenyl phosphine bromide (TPP) is weighed out and pre-dissolved in 0.2mL of DMSO. 9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 6mg of N-hydroxysuccinimide (NHS) were added and stirred for 4 hours. To the solution was added 0.5mL of 20mg/mL HS-PEG-NH 2. The reaction is carried out for 12 hours at room temperature to obtain the final concentration of 0.2mmol of (3-propyl carboxyl) triphenyl phosphonium bromide modified sulfhydryl polyethylene glycol (HS-PEG-NH2/TPP) complex.
4. Synthesis of photoacoustic immunotherapy nanocomplexes (BP/R848/OVA/HS-PEG-NH 2/TPP)
1mg/mL of immunoadjuvant-modified black phosphorus (BP/R848/OVA) and 0.2mmol (3-propylcarboxyl) of triphenylphosphine-bromide-modified mercaptopolyethylene glycol (HS-PEG-NH2/TPP) were mixed and stirred at room temperature for 12 hours, and washed three times with distilled water. Centrifuging at 7000rpm for 10min to obtain final 1mg/mL photoacoustic immunotherapy nano-complex (BP/R848/OVA/HS-PEG-NH 2/TPP).
Ultraviolet characterization of the photoacoustic immunotherapy nanocomplexes (BP/R848/OVA/HS-PEG-NH 2/TPP) with results shown in FIG. 2. The UV absorption peaks of the immunoadjuvant alone (R848) and of 3-propylcarboxyl) triphenylphosphonium bromide (TPP) are shown in FIG. two. And the absorption peak of each substance can be reproduced in the absorption curve of the nano-complex for photoacoustic immunotherapy (BP/R848/OVA/HS-PEG-NH 2/TPP). It can be concluded that: and (4) assembling a nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) for photoacoustic immunotherapy.
5. The targeting effect of the nano complex (BP/R848/OVA/HS-PEG-NH 2/TPP) of the photoacoustic immunotherapy on mitochondria is verified in vitro.
Fetal Bovine Serum (FBS) and cell culture medium (DMEM) were purchased from seimer feishell science (china) ltd (shanghai, china). The fluorescent dye CY5.5 was purchased from Beijing bridge Biotechnology, Inc., and the mitochondrial fluorescent probe was purchased from Shanghai Bin Yuntian Biotechnology, Inc. The EMT6 cell line came from the biophotonic institute of university of south china.
EMT6 cells were cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS) in a cell culture chamber under the following conditions: CO22The concentration was 5%, the humidity 90% and the temperature 37 ℃. 20 μ L of 0.2mg/mL CY5.5 and 1mL 1mg/mL nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP) were stirred at room temperature for 4 hours. The CY5.5 modified nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP) was obtained at a concentration of 1 mg/mL. 50 mu L of the complex is incubated with EMT6 cells for 4 hours, then mitochondrial fluorescent probes are added for half an hour, and the co-localization condition of the nanoparticles and the mitochondrial probes is observed under a confocal microscope. The excitation wavelength used by the mitochondrial fluorescent probe is 488nm, the emission wavelength is 498nm, the excitation wavelength used by CY5.5 is 633nm, and the emission wavelength is 690 nm. As a result, as shown in FIG. 3, red is fluorescence of the nanoparticle, green is fluorescence of the mitochondrial probe, and the overlapping of the two shows fluorescence of yellow. Therefore, the fluorescence energy of the nanoparticles is coincided with that of the mitochondrial probe, and the nanoparticles can be proved to target mitochondria.
6. The immune activation effect of the nano-complex of photoacoustic immunotherapy (BP/R848/OVA/HS-PEG-NH 2/TPP) on DC cells was verified in vitro.
Phosphate Buffered Saline (PBS), trypsin was obtained from Biotechnology engineering (Shanghai) GmbH. anti CD80 and anti CD86 were purchased from David Biotech Inc. (Biolegend), and the DC2.4 cell line was from biophotonic research at university of south China.
DC2.4 cells were seeded into 6-well plates (approximately 100,000 cells per well) under the same conditions as before. After 12 hours, the cells were washed 3 times with Phosphate Buffered Saline (PBS) and then digested with 2.5% trypsin at 37 ℃ for 1-2 min. The cells were then gently blown down and resuspended in PBS and analyzed by flow cytometry. 50 mu.L of 1mg/mL nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP) was incubated with DC2.4 cell line for 12 hours, anti CD80-PE was used to label CD80, anti CD86-APC was used to label CD86, and counting of DC cells by flow cytometry verified the immune activation of DC cells by the nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP). The results are shown in FIG. 4, and DC cells treated with BP-TPP + R848+ OVA produced 15% more CD80 and 1.5% more CD86 than the control group. Thus, the nanoparticles can stimulate immature DC cells to mature the immature DC cells. And (3) performing statistical analysis by using a flow cytometer, wherein the wavelength of excitation light used by the PE is 488nm, the wavelength of emission light is 578nm, the wavelength of excitation light used by the APC is 633nm, and the wavelength of emission light is 660 nm.
7. And (3) verifying the immune activation of the tumor-associated antigen generated after the tumor cells are killed by the photoacoustic on the DC cells in vitro.
EMT6 cells were cultured in the upper transwell chamber and incubated with the nanocomplex (BP/R848/OVA/HS-PEG-NH 2/TPP), and DC cells were cultured in the lower chamber. EMT6 cells were apoptotic by irradiating the upper chamber with 808nm pulsed laser at 20mj for 10 min. Apoptotic cells produce tumor-associated antigens that further promote DC cell maturation. DC cells were detected by flow cytometry. Results as shown in fig. 5, DC2.4 cells co-incubated with PBS and BP/R848/OVA stimulated some DC cell maturation compared to the control group, whereas co-incubated DC2.4 cells significantly stimulated DC cell maturation after treatment with light sound.
The R848/OVA/HS-PEG-NH2/TPP complex can protect BP and improve the stability of the BP. In combination with immune checkpoint inhibitors, precise photoacoustic tumor reduction is achieved and immune effects are generated with the effect of extending the time of photoacoustic therapy and expanding the space of photoacoustic therapy.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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