CN112402605B - Bionic nanoemulsion and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a bionic nanoemulsion and a preparation method and application thereof, wherein the bionic nanoemulsion comprises: albumin-perfluoro compound nano emulsion with surface combined with sodium porphyrin and PD-1 high expression cell membrane coated on the albumin-perfluoro compound nano emulsion with surface combined with sodium porphyrin. The bionic nanoemulsion can simultaneously realize fluorescence imaging of tumors and cooperative treatment combining photodynamic treatment and immunotherapy of the tumors, so that the bionic nanoemulsion has good application prospect in the fields of diagnosis and treatment of the tumors. Meanwhile, the preparation process is simple, convenient to operate, does not need complex and expensive equipment, and is easy to realize industrial production.
Description
Technical Field
The invention relates to the field of medical nano materials, in particular to a bionic nano emulsion and a preparation method and application thereof.
Background
Photodynamic therapy of tumors activates photosensitive drugs through illumination, so that oxygen in tissues is converted into toxic singlet oxygen, and tumor cells are killed, thereby achieving the purpose of treatment. Photodynamic therapy consists of three main factors: photosensitizers, light and oxygen. Wherein oxygen acts as a reactive substrate for singlet oxygen production, and its concentration significantly affects the efficiency of photodynamic therapy. However, there are different degrees of hypoxia in the deep part of solid tumors, limiting the efficacy of photodynamic therapy. Various liquid perfluorocarbons (such as perfluorohexane, perfluorotributylamine, perfluoro-15-crown-5-ether and the like) have higher affinity to oxygen, and can realize slow release of oxygen in the hypoxia part of solid tumors by pre-filling pure oxygen and packaging the pure oxygen in nano-carriers, so as to provide raw materials for photodynamic therapy, thereby improving the therapeutic effect of the solid tumors.
Photodynamic therapy is a local tumor treatment mode, and is difficult to effectively inhibit the whole-body metastasis of tumors. Tumor cell lysates generated after photodynamic therapy contain a large amount of tumor specific antigens, so that the tumor cell lysates generate in-situ vaccine-like effects, trigger downstream tumor specific immune responses, and are expected to inhibit remote tumor metastasis, but the immune responses are inhibited by immunosuppressive tumor microenvironments. Currently, the combination of immune checkpoint inhibitors (e.g., PD-1/PD-L1 antibodies) with a photodynamic therapy has been demonstrated to enhance the immune response elicited by photodynamic therapy, thereby achieving effective inhibition of distant tumor metastases. However, in the current research, a mode of separately injecting a photosensitive drug and an immune checkpoint inhibitor is adopted, the free immune checkpoint inhibitor is scattered and distributed in the whole body, has limited targeting capability on tumor parts, and has higher systemic toxic and side effects.
Accordingly, the prior art is subject to improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a bionic nanoemulsion and a preparation method and application thereof, and aims to solve the problem that existing immune checkpoint inhibitors are scattered throughout the whole body and have limited targeting capability on tumor parts.
The technical scheme of the invention is as follows:
a preparation method of bionic nanoemulsion, which comprises the following steps:
providing an albumin-perfluoro compound nanoemulsion;
combining sodium porphyrin (DVDMS) on the surface of the albumin-perfluorinated compound nanoemulsion to obtain an albumin-perfluorinated compound nanoemulsion with the sodium porphyrin combined on the surface;
and wrapping the PD-1 high-expression cell membrane on the albumin-perfluorinated compound nanoemulsion with the surface combined with the sodium porphyrin, so as to obtain the bionic nanoemulsion (PHD@PM).
The preparation method of the bionic nanoemulsion comprises the following steps:
mixing Human Serum Albumin (HSA) with water, then adding a perfluorinated compound, stirring, and carrying out ultrasonic treatment on the mixed solution obtained after stirring by using a probe to obtain the albumin-perfluorinated compound nanoemulsion.
The preparation method of the bionic nanoemulsion comprises the steps of combining sodium porphyrin in the surface of the albumin-perfluoro compound nanoemulsion to obtain the albumin-perfluoro compound nanoemulsion with the sodium porphyrin combined on the surface, and specifically comprises the following steps:
adding sodium warfarin into the albumin-perfluoro compound nanoemulsion, and stirring to obtain the albumin-perfluoro compound nanoemulsion with the surface combined with sodium warfarin.
The preparation method of the bionic nanoemulsion comprises the following steps of:
and digesting HEK293T cells by pancreatin, homogenizing by a homogenizer under the condition of ice bath, centrifuging, and taking supernatant to obtain cell membrane fragments, and sequentially passing through filter membranes of 0.8 mu m, 0.45 mu m and 0.22 mu m to obtain the PD-1 high-expression cell membrane.
The preparation method of the bionic nanoemulsion comprises the steps of wrapping the PD-1 high-expression cell membrane on the albumin-perfluoro compound nanoemulsion with the surface combined with sodium porphyrin, and specifically comprises the following steps of:
and uniformly mixing the albumin-perfluorinated compound nanoemulsion with the surface combined with the sodium porphyrin and the PD-1 high-expression cell membrane through a filter membrane with the diameter of 0.22 mu m respectively to obtain a mixture, and then extruding the mixture back and forth for 20 times by using a liposome extruder with the filter membrane with the diameter of 0.2 mu m to obtain the bionic nanoemulsion.
The bionic nanoemulsion is prepared by a preparation method, wherein the perfluoro compound is selected from one of perfluoro tripropylamine, perfluoro tributylamine, perfluoro hexane and perfluoro-15-crown-5 ether.
A biomimetic nanoemulsion as described above, wherein the biomimetic nanoemulsion comprises: albumin-perfluoro compound nanoemulsion with surface combined with sodium porphyrin, PD-1 high-expression cell membrane coated on the albumin-perfluoro compound nanoemulsion with surface combined with sodium porphyrin;
a bionic nanoemulsion as described above is prepared by the preparation method of the invention.
A biomimetic nanoemulsion as described above, wherein the diameter of the biomimetic nanoemulsion is 100-160 nm.
Use of a biomimetic nanoemulsion as described above in the preparation of a formulation for the treatment of a tumour, wherein the treatment is simultaneous photodynamic therapy and immunotherapy.
The beneficial effects are that: the invention provides a bionic nanoemulsion, which can simultaneously realize fluorescence imaging of tumors and cooperative treatment of combination of photodynamic therapy and immunotherapy of the tumors, and has good application prospects in the fields of diagnosis and treatment of the tumors. Meanwhile, the preparation process is simple, convenient to operate, does not need complex and expensive equipment, and is easy to realize industrial production.
Drawings
FIG. 1 is a TEM image of the biomimetic nanoemulsion of example 1 of the present invention;
FIG. 2 is a graph showing the killing effect of photodynamic/immunocooperative therapy on 4T1 tumor cells in example 2 of the present invention;
FIG. 3 is a graph showing the effect of PHD@PM on the passive accumulation of tumor sites in example 3 of the present invention;
FIG. 4 is a graph showing the inhibition effect of photodynamic/immunocooperative therapy on the growth of 4T1 tumor in example 4 of the present invention;
FIG. 5 is a graph showing the effect of photodynamic/immunocompetent treatment on the infiltration of tumor cells in example 5 of the present invention.
Detailed Description
The invention provides a bionic nanoemulsion and a preparation method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The embodiment of the invention provides a preparation method of a bionic nanoemulsion, which comprises the following steps:
s10, providing albumin-perfluoro compound nanoemulsion;
s20, combining sodium porphyrin bloom on the surface of the albumin-perfluoro compound nanoemulsion to obtain the albumin-perfluoro compound nanoemulsion with sodium porphyrin bloom combined on the surface;
s30, wrapping the PD-1 high-expression cell membrane on the albumin-perfluorinated compound nanoemulsion with the surface combined with the sodium porphyrin, so as to obtain the bionic nanoemulsion.
In this embodiment, in the albumin-perfluoro compound nanoemulsion, albumin is a drug already used in clinic, and has good biocompatibility, and perfluoro compound also has good biocompatibility. The albumin loads the perfluorinated compounds into the nano-emulsion through emulsification, and the albumin is on the surface of the nano-emulsion. The perfluoro compound has good biocompatibility and higher affinity to oxygen, and can realize slow release of oxygen in the hypoxic part of solid tumor by pre-filling pure oxygen and packaging the perfluoro compound in a nano carrier, thereby providing raw materials for photodynamic therapy and improving the therapeutic effect of the perfluoro compound.
In the embodiment, the strong adsorption between albumin and sodium porphyrin is utilized, the albumin-perfluoro compound nanoemulsion is used as a carrier material, and the sodium porphyrin is used as a photosensitizer to be combined on the albumin of the carrier material. The Hua Bulin sodium is a new-generation photosensitizer, has good biocompatibility, stable property and high singlet oxygen yield, and has good photodynamic therapy effect.
In the embodiment, the PD-1 high expression cell membrane has good biocompatibility as well, and can be used as an immune check point inhibitor to be combined with a photodynamic therapy to enhance immune response caused by photodynamic therapy and realize effective inhibition of a remote tumor metastasis.
The bionic nanoemulsion can simultaneously realize fluorescence imaging of tumors and cooperative treatment combining photodynamic treatment and immunotherapy of the tumors, so that the bionic nanoemulsion has good application prospects in the fields of diagnosis and treatment of the tumors. Meanwhile, the preparation process is simple, convenient to operate, does not need complex and expensive equipment, and is easy to realize industrial production.
In step S10, in one embodiment, the method for preparing the albumin-perfluoro compound nanoemulsion includes the steps of:
mixing human serum albumin with water, adding a perfluorinated compound, stirring, and carrying out ultrasonic treatment on the mixed solution obtained after stirring by using a probe to obtain the albumin-perfluorinated compound nanoemulsion.
In this example, human serum albumin is a drug which has been used in clinic, and has excellent biocompatibility, and the perfluoro compound has excellent biocompatibility. Human serum albumin loads the perfluorinated compounds inside the nanoemulsion by emulsification.
In one embodiment, the preparation method of the albumin-perfluoro compound nanoemulsion specifically comprises the following steps:
human serum albumin and water were mixed to give a solution concentration of 40mg/mL, wherein 240mg of human serum albumin was added slowly to 200-400. Mu.L of the perfluoro compound and stirred. And (3) treating the mixed solution with probe ultrasonic (250-400W) for 8 minutes to obtain the albumin-perfluoro compound nanoemulsion.
In one embodiment, the perfluoro compound may be selected from one of perfluoro tripropylamine, perfluoro tributylamine, perfluoro hexane, perfluoro-15-crown-5 ether, etc., but is not limited thereto.
In one embodiment, the water is ultrapure water, which has little impurities, which is more advantageous for forming pure albumin-perfluoro compound nanoemulsions.
In one embodiment, step S20 specifically includes: adding 20mg/mL sodium Huazhihua to the albumin-perfluoro compound nanoemulsion and stirring for 1-24h, wherein the mass ratio of albumin to sodium Huazhihua is 1:400-60:400, obtaining the albumin-perfluoro compound nano emulsion with the surface combined with the sodium porphyrin.
In step S30, in one embodiment, the PD-1 high expression cell membrane is extracted by the following method:
and digesting HEK293T cells by pancreatin, homogenizing by a homogenizer under the condition of ice bath, centrifuging, and taking supernatant to obtain cell membrane fragments, and sequentially passing through filter membranes of 0.8 mu m, 0.45 mu m and 0.22 mu m to obtain the PD-1 high-expression cell membrane.
In one embodiment, the PD-1 high expression cell membrane is extracted by the following specific method:
HEK293T cells are digested by pancreatin, centrifuged at 1500rpm for 5min, washed 3 times with PBS, resuspended in HM buffer, homogenized under ice bath conditions with a homogenizer for 100 min, uncleaved cell pellet removed after centrifugation at 1500rpm for 5min, organelle pellet removed after centrifugation at 7500rpm for 20min, finally centrifuged at 20000rpm for 60min to obtain cell membrane fragments, and sequentially filtered with 0.8 μm, 0.45 μm, 0.22 μm filters to obtain PD-1 highly expressed cell membranes, which are lyophilized and stored in a refrigerator at-80 ℃.
In the embodiment, under the condition of ice bath, the homogenization process can be carried out in the environment of approximately 0 ℃, which is more favorable for keeping the activity of each part of the cells, the homogenization purpose is to separate the cell membrane, the pancreatic enzyme digestion HEK293T cells are lysed after being homogenized, and then the centrifugation is carried out to remove the cell organelle precipitate, so as to obtain the PD-1 high-expression cell membrane.
In one embodiment, step S30 specifically includes:
and uniformly mixing the albumin-perfluorinated compound nanoemulsion with the surface combined with the sodium porphyrin and the PD-1 high-expression cell membrane through a filter membrane with the diameter of 0.22 mu m respectively to obtain a mixture, and then extruding the mixture back and forth for 20-100 times by using a liposome extruder with the filter membrane with the diameter of 0.2 mu m to obtain the bionic nanoemulsion. Wherein the PD-1 high expression cell membrane is wrapped on the albumin-perfluoro compound nanoemulsion with the surface combined with the sodium porphyrin.
The embodiment of the invention provides a bionic nanoemulsion, which comprises the following components: albumin-perfluoro compound nanoemulsion with surface combined with sodium porphyrin, PD-1 high-expression cell membrane coated on the albumin-perfluoro compound nanoemulsion with surface combined with sodium porphyrin;
a bionic nanoemulsion as described above is prepared by the preparation method of the invention.
In this embodiment, the biomimetic nanoemulsion is a biomimetic nanoemulsion combining fluorescence imaging, photodynamic therapy and immunotherapy. The Hua Bulin sodium is taken as a photosensitizer, can realize fluorescence imaging, and the PD-1 high-expression cell membrane is taken as an immune check point inhibitor, can be used with photodynamic therapy to enhance immune response caused by photodynamic therapy, and can realize effective inhibition of a remote tumor metastasis.
In one embodiment, the diameter of the bionic nanoemulsion is 100-160 nm, and the bionic nanoemulsion can better realize fluorescence imaging of tumors and cooperative treatment of combination of photodynamic treatment and immunotherapy of the tumors within the particle size range.
The embodiment of the invention also provides application of the bionic nanoemulsion in preparation of a preparation for treating tumors. Wherein, the tumor treatment is to adopt photodynamic treatment and immunotherapy simultaneously.
The bionic nanoemulsion can simultaneously realize fluorescence imaging of tumors and cooperative treatment combining photodynamic treatment and immunotherapy of the tumors, so that the bionic nanoemulsion has good application prospects in the fields of diagnosis and treatment of the tumors. Meanwhile, the bionic nanoemulsion disclosed by the embodiment is simple in preparation process, convenient to operate, free of complex and expensive equipment, and easy to realize industrial production.
The invention is further illustrated by the following specific examples.
Example 1
The preparation steps of the bionic nanoemulsion (PD-1 high expression cell membrane coated albumin-perfluoro tributylamine-Hua Bulin sodium) of the embodiment are as follows:
human serum albumin and water were mixed to a concentration of 30mg/mL, wherein 240mg of human serum albumin was obtained, and then 300. Mu.L of a perfluoro compound was slowly added and stirred. And (3) treating the mixed solution with probe ultrasonic 260W for 7 minutes to obtain the albumin-perfluoro compound nanoemulsion.
20mg/mL sodium Huazhihua is added into the albumin-perfluoro compound nano emulsion and stirred for 12 hours, and the mass ratio of albumin to sodium Huazhihua is 30:400, obtaining the albumin-perfluoro compound nano emulsion with the surface combined with the sodium porphyrin.
Extracting cell membranes with high expression of PD-1: the HEK293T cells are digested by pancreatin, centrifuged for 5min at 1500rpm to obtain cells, washed 3 times with PBS, resuspended in HM buffer, homogenized under ice bath conditions with a homogenizer for 100 min, and centrifuged for 5min at 1500rpm to remove uncleaved cell pellet, and centrifuged for 20min at 7500rpm to remove organelle pellet, finally centrifuged for 60min at 20000rpm to obtain cell membrane fragments, and sequentially filtered with 0.8 μm, 0.45 μm, 0.22 μm filters to obtain PD-1 highly expressed cell membranes, which are lyophilized and stored in a refrigerator at-80 ℃.
Wrapping PD-1 high expression cell membrane on the surface of albumin-perfluoro tributylamine nano emulsion with surface combined with sodium porphyrin bloom: the albumin-perfluoro tributylamine nanoemulsion with the surface combined with the sodium porphyrin and the cell membrane with high PD-1 expression are uniformly mixed after passing through a filter membrane with the thickness of 0.22 mu m respectively to obtain a mixture, the mass ratio of the albumin-perfluoro tributylamine nanoemulsion to the cell membrane is 1:5, and the mixture is extruded for 20 times by a liposome extruder with the filter membrane with the thickness of 0.2 mu m.
The TEM image of the obtained albumin-perfluoro tributylamine nanoemulsion with the surface combined with the sodium porphyrin bloom coated by the PD-1 high expression cell membrane is shown in figure 1. As can be seen from FIG. 1, the PD-1 high expression cell membrane is wrapped on albumin-perfluoro tributylamine nanoemulsion with surface bound sodium porphyrin, and the thickness is about 10nm.
Example 2
Toxicity evaluation of photodynamic therapy and immunotherapy synergistic therapy on 4T1 tumor cells
The effect of photodynamic therapy and immunotherapy co-therapy on 4T1 cell survival was evaluated using standard MTT methods. 4T1 cells at 5X 10 per well 3 Density inoculation into 96 well plates and exposure to 5% co at 37 ℃ 2 Incubate under conditions for 24h. Next, the old medium in the 96-well plate was aspirated, and 200ng/mL of DVDMS, PFTBA@HSA-DVDMS (PHD), PFTBA@HSA-DV were added, respectivelyDMEM medium with DMS@PD-1NVs (PHD@PM). After further culturing for 2 hours, the old medium in the 96-well plate was aspirated, 100. Mu.L of DMEM medium was added to each well, and then the cells were irradiated with 635nm laser for 5 minutes at a power of 200mW/cm for each well of the irradiated wells 2 . After the irradiation, the old medium in the 96-well plate was aspirated, and 100. Mu.L of a medium solution containing 5mg/mL MTT was added to each well, and the culture was continued for 12 hours. The OD value of each well (detection wavelength: 490 nm) was then measured on a Bio-Tel EL X800 type microplate reader, and the cell viability was calculated using the following formula. Cell viability (%) = (OD 490 value of sample/blank OD490 value) ×100%, experimental results are shown in fig. 2.
As shown in FIG. 2a, each drug group had no significant toxicity to 4T1 cells at concentrations ranging from 0-200 ng/mL; while figure 2b shows that after 5 minutes of 635nm laser irradiation, 4T1 cell viability was significantly reduced, indicating that the irradiation produced active oxygen in the photosensitizer, killing a large number of tumor cells. And due to the oxygen carrying capacity of the perfluoro tributylamine and the affinity of the PD-1 cell membrane and the tumor cell PD-L1, the cell survival rate of the PHD and PHD@PM group laser treatment is reduced more remarkably.
Example 3
Evaluation of effect of low-frequency ultrasonic irradiation on promoting accumulation of bionic nanoemulsion in subcutaneous tumor
Female Balb/c mice (3 weeks, 15-20 g), 2X 10 subcutaneous injections were given to the right hind legs of the mice 6 4T1 tumor cells, and establishing a mouse subcutaneous tumor model. When the double-sided tumor volume exceeds 100mm 3 At this time, a fluorescence imaging experiment was performed. After the medicine is injected into the tail vein of the tumor-bearing mice for 1,4,8, 12, 24, 48 and 72 hours, the change of tumor accumulation is observed through fluorescence imaging of sodium porphyrin, and quantitative analysis is carried out. The results are shown in FIG. 3.
In fig. 3a, drug accumulation in the tumor gradually increased with time following administration, and fluorescence decay was slow at the tumor sites for 24-72h, indicating a short residence time. FIG. 3b shows the fluorescence intensity values in the organs of mice isolated 24h after injection, and shows that sodium Huaporphyrin in the liver is rapidly metabolized 24h after injection, and sodium Huaporphyrin at the tumor site has a retention period of up to 2 days.
Example 4
Evaluation of inhibition effect of photodynamic/immunotherapy cooperative therapy on 4T1 tumor growth
Female Balb/c mice (3 weeks, 15-20 g), 2X 10 subcutaneous injections were given to the hind legs of the mice 6 4T1 tumor cells, and establishing a mouse subcutaneous tumor model. When the tumor volume reaches 60mm 3 At that time, a treatment experiment was performed. In the unilateral tumor model, tumor-bearing mice were randomly divided into six groups: (1) saline group (blank control); (2) injecting PHD group; (3) injecting PHD@PM group; (4) injecting a dvdms+ laser group; (5) injecting phd+ laser group; (6) injecting PHD@PM+ laser group. In the bilateral tumor model, tumor-bearing mice were randomly divided into five groups: (1) saline group (blank control); (2) injecting PHD@HM group; (3) injecting PHD@HM+ laser group; (4) injecting phd+ laser group; (5) injecting PHD@PM+ laser group; tumor volume was measured every other day with vernier calipers while monitoring the body weight of mice and following the formula v=ab 2 Tumor volume (Tumor volume) was calculated, where A is the major diameter of the Tumor and B is the minor diameter (mm) of the Tumor. The results of each measurement were normalized by the initial tumor volume before treatment and the experimental results are shown in fig. 4.
Panels a and b of fig. 4 show the change in tumor volume (Normalized tumor volume) over time (Day) for different treatment groups in single and double tumor models, respectively. As shown in fig. 4a, the phd@pm+ laser group can significantly inhibit the growth of unilateral subcutaneous 4T1 breast cancer tumor, and the tumor inhibition effect of the dvdms+ laser group and the phd+ laser group is equivalent. As shown in fig. 4b, phd@pm injection significantly inhibited growth of contralateral tumors, but tumors still recurred, whereas laser treatment added almost completely inhibited growth and recurrence.
Example 5
The tumor tissues of each group of mice are dissected and separated after 14 days of treatment by different treatment groups, the tumor tissues are digested for 30min by collagenase I, collagenase IV, DNase and hyaluronidase after being sheared, then erythrocyte lysate is added, the cells are leached by a cell culture medium after centrifugal resuspension, and finally, 5% BSA is added into the obtained cell suspension, and then the ice bath is closed for 10min. Adding fluorescent-labeled antibodies (Anti-CD 3, anti-CD 4),Anti-CD 8), detection of CD of tumor tissue using a flow analyzer 4+ /CD 8+ T lymphocyte infiltration, the experimental results are shown in FIG. 5.
As shown in FIG. 5, CD of physiological saline group 4+ /CD 8+ T lymphocytes are low in content, and CD in tumor tissue of PHD@PM+ laser group is injected 4+ /CD 8+ The T lymphocyte content was increased by 2.7-fold and 9.59-fold, respectively. Other groups were also improved to varying degrees relative to the normal saline group, but below the phd@pm+ laser group.
In conclusion, the bionic nanoemulsion can simultaneously realize fluorescence imaging of tumors and cooperative treatment combining photodynamic treatment and immunotherapy of the tumors, so that the bionic nanoemulsion has good application prospects in the fields of diagnosis and treatment of the tumors. Meanwhile, the preparation process is simple, convenient to operate, does not need complex and expensive equipment, and is easy to realize industrial production.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (4)
1. The preparation method of the bionic nanoemulsion is characterized by comprising the following steps of:
mixing human serum albumin with water, adding a perfluorinated compound, stirring, and carrying out ultrasonic treatment on the mixed solution obtained after stirring by using a probe to obtain the albumin-perfluorinated compound nanoemulsion;
adding sodium warfarin into the albumin-perfluoro compound nanoemulsion, and stirring to obtain an albumin-perfluoro compound nanoemulsion with the surface combined with sodium warfarin;
wrapping PD-1 high expression cell membrane on the albumin-perfluoro compound nanoemulsion with the surface combined with sodium porphyrin bloom to obtain the bionic nanoemulsion;
the diameter of the bionic nano emulsion is 100-160 nm;
the mass ratio of the albumin to the sodium porphyrin bloom is 30:400;
the bionic nanoemulsion is a bionic nanoemulsion combining fluorescence imaging, photodynamic therapy and immunotherapy, wherein Hua Bulin sodium is taken as a photosensitizer to realize fluorescence imaging, and the PD-1 high-expression cell membrane is taken as an immune checkpoint inhibitor to enhance immune response caused by photodynamic therapy in combination;
the PD-1 high expression cell membrane is extracted by the following method:
digesting HEK293T cells by pancreatin, homogenizing by a homogenizer under the condition of ice bath, centrifuging, and taking supernatant to obtain cell membrane fragments, wherein the cell membrane fragments sequentially pass through filter membranes of 0.8 mu m, 0.45 mu m and 0.22 mu m to obtain the PD-1 high-expression cell membrane;
the step of wrapping PD-1 high expression cell membrane on the albumin-perfluoro compound nanoemulsion with the surface combined with sodium porphyrin bloom to obtain the bionic nanoemulsion comprises the following steps:
uniformly mixing the albumin-perfluorinated compound nanoemulsion with the surface combined with the sodium porphyrin and PD-1 high-expression cell membranes through 0.22 mu m filter membranes respectively to obtain a mixture, and then repeatedly extruding the mixture for 20 times by using a liposome extruder with the 0.2 mu m filter membranes to obtain the bionic nanoemulsion;
the perfluoro compound is one selected from perfluoro tripropylamine, perfluoro tributylamine, perfluoro hexane and perfluoro-15-crown-5 ether.
2. A bionic nanoemulsion, characterized in that the bionic nanoemulsion is prepared by the preparation method of the bionic nanoemulsion of claim 1.
3. Use of the biomimetic nanoemulsion of claim 2 in the preparation of a formulation for treating tumors.
4. The use according to claim 3, wherein the treatment is simultaneous photodynamic therapy and immunotherapy.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101329646B1 (en) * | 2013-05-02 | 2013-11-14 | 주식회사 지니스 | Targeting-enhancing anticancer nanoparticles and preparation method the same |
| CN110251672A (en) * | 2019-06-18 | 2019-09-20 | 深圳大学 | A kind of nanometer of diagnosis and treatment agent and the preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN110251672A (en) * | 2019-06-18 | 2019-09-20 | 深圳大学 | A kind of nanometer of diagnosis and treatment agent and the preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Oxygen self-enriched nanoparticles functionalized with erythrocyte membranes for long circulation and enhanced phototherapy;Hao Ren et al.;《Acta Biomaterialia》;20170627;第59卷(第1期);第269页右栏1. Introduction-第270页右栏2.2. Preparation of IPH@RBC * |
| PD-1 Blockade Cellular Vesicles for Cancer Immunotherapy;Xudong Zhang et al.;《Adv Mater》;20180414;第30卷(第22期);第1页右栏第1段-第7页左栏第1段 * |
| 血清白蛋白:用于肿瘤精准诊疗的多元化载体;施德太等;《化学通报》;20200318;第83卷(第03期);第195-208页 * |
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