CN113058031B - Galgi-body and genetic-engineering-exosome hybrid-membrane-coated retinoic acid in-situ spray hydrogel vaccine, and preparation method and application thereof - Google Patents
Galgi-body and genetic-engineering-exosome hybrid-membrane-coated retinoic acid in-situ spray hydrogel vaccine, and preparation method and application thereof Download PDFInfo
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- CN113058031B CN113058031B CN202110289031.3A CN202110289031A CN113058031B CN 113058031 B CN113058031 B CN 113058031B CN 202110289031 A CN202110289031 A CN 202110289031A CN 113058031 B CN113058031 B CN 113058031B
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Abstract
A retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes, a preparation method and application thereof, belonging to the technical field of medicines; in particular to a retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi and genetic engineering exosomes for inhibiting secretion of exosomes PD-L1 and activating systemic immune response by re-vibrating lymph node immune cells and application thereof in inhibiting and/or treating postoperative recurrence of melanoma.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi body and genetic engineering exosome and used for inhibiting secretion of exosome PD-L1, triggering immune memory by re-vibrating lymph node immune cells and further activating systemic immune response, and application of the retinoic acid in inhibiting and/or treating and inhibiting postoperative recurrence of melanoma.
Background
Local residual tumors and microassays of Circulating Tumor Cells (CTCs) tend to induce tumor recurrence. Immunotherapy can effectively suppress the recurrence and metastasis of cancer, of which PD-L1 mab shows great promise in treatment. But the patient response rate to immunotherapy is still low and most patients experience relapse within months. This is because PD-L1 is also located on the extracellular vesicle surface, enhances the growth of immune-dependent tumors, inhibits T cell function, particularly in Draining Lymph Nodes (DLNs) as well as spleen sites, and is predisposed to resistance to treatment with PD-L1 mab.
The CRISPR/Cas9 gene editing technology has great treatment potential in treating tumors. Studies have shown that elimination of biogenesis of exosomes or immune checkpoint genes can actively remodel the immune microenvironment of a tumor. These edited cells or exosomes can act as vaccines, sensitizing immune cells and inducing systemic anti-tumor immunity and memory. PD-L1, a membrane protein, was transferred from the lumen of the Endoplasmic Reticulum (ER) to the Golgi apparatus (Golgi appaatus) to further modify the mannosidase sequence with many glycan modifications and then traverse the cell surface. It then enters and fuses with endosomes, fusing the products of the golgi apparatus in vesicles, and then secreting as exosomes in an Rab27 a-dependent manner. Deregulation of glycosylation results in misfolded or unassembled proteins, which are polyubiquitinated. These proteins are transported back to the cytoplasm by cytoplasmic proteases for endoplasmic reticulum-associated degradation (ERAD).
Cell membrane coating techniques enhance the biological interface properties of Nanoparticles (NPs). Research shows that the coating of the hybrid membrane of the platelet and the neutrophil can improve the targeting affinity of the tumor. It mimics the properties of the source cell and has the ability to fuse its function to that of the source cell. The Golgi Membrane (GM), an important intracellular membrane, is not clearly understood. However, when used as a biomimetic drug delivery method, it has the potential to deliver drugs to the golgi apparatus via the endosomal-golgi-endoplasmic reticulum pathway. The vitamin a derivative retinoic acid changes its structure in the golgi.
Polyvinyl alcohol (PVA) is a unique synthetic polymer with good biodegradability, which is widely used in hydrogel preparation because it is easy to modify hydroxyl groups to obtain functional derivatives for crosslinking, compared to other polymers.
Disclosure of Invention
The technical problem solved by the present invention is to overcome the drawbacks of the prior art, considering that melanoma cells (B16-F10 cells) express PD-L1 on both cell surface as well as exosomes, the present invention provides a new internal and external blocking strategy (IEB), i.e. spray hydrogel vaccine in situ. In addition, a preparation form of the vaccine, a preparation method of the vaccine and application of the vaccine in inhibiting and/or preventing postoperative recurrence of melanoma tumor are also provided.
The first purpose of the invention is to provide a retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes.
The second purpose of the invention is to provide a dosage form of the in-situ spray hydrogel vaccine, which is retinoic acid bionic nanoparticles (GENPs) coated by a hybrid membrane of Golgi bodies and genetically engineered exosomes of immune cells in a stress-induced draining lymph node capable of destroying the Golgi bodies of tumor cells, and is loaded by polyvinyl alcohol acetoacetate (PVAA)/Chitosan (CS) Gel (aPD-L1@ GENPs @ Gel).
The third purpose of the invention is to provide a preparation method of the tretinoin in-situ spray hydrogel vaccine coated by the loaded Golgi apparatus and the genetic engineering exosome hybrid membrane.
The fourth purpose of the invention is to provide the application of the retinoic acid in-situ hydrogel vaccine coated by the hybrid membrane of the Golgi apparatus and the genetic engineering exosome in inhibiting postoperative recurrence of melanoma.
In order to achieve the purpose, the invention adopts the technical scheme that:
the in-situ spray hydrogel vaccine provided by the invention aims to inhibit local tumor recurrence and distant tumor development after operation. The hydrogel was formed by mixing short-chain Chitosan (CS) with an aqueous solution of polyvinyl alcohol acetoacetate (PVAA) as a nanoparticle reservoir at room temperature. Short-chain CS is a natural polymer that exhibits high water solubility, and PVAA is non-cytotoxic and non-inflammatory. The in-situ spray hydrogel vaccine is prepared by placing vitamin A acid bionic nanoparticles (GENPs) coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes in CS, and placing a PD-L1 monoclonal antibody in PVAA. After mixing CS and PVAA by spraying, the acetoacetate groups of the PVAA react with the CS amino groups to form dynamic olefinic bonds. aPD-L1 in PVAA can mask PD-L1 on the cell surface of B16-F10, GENPs in CS can target cells through homologous targeting and remain on Golgi through the endosome-Golgi body-endoplasmic reticulum pathway to release retinoic acid. The in-situ spray hydrogel vaccine provided by the method can break the glycosylation process of PD-L1 and inhibit the release of exosome, thereby reducing the exosome PD-L1. In addition, GENPs have the ability to enter draining lymph nodes by mimicking the behavior of exosomes, which can revitalize potent immune cells, thereby persistently activating T cells.
The in situ spray hydrogel vaccine comprises: tretinoin, a hybrid membrane and a nano carrier, wherein the mass percentages of the components are as follows: 1% -3% of tretinoin, 47% -49% of nano carrier material and the balance of hybrid membrane, wherein the preferable proportion is as follows: 1.5:48.5:50.
The tretinoin can also be a tretinoin derivative.
The hybrid membrane is a hybrid membrane of Golgi apparatus and genetic engineering exosomes; the Golgi body and genetic engineering exosome hybrid membrane is of murine origin and is extracted from murine melanoma B16-F10 cells.
The nano carrier can be any carrier capable of encapsulating tretinoin or derivatives thereof, and comprises one or more of polylactic-co-glycolic acid (PLGA), mesoporous silicon, quantum dots and gold nanocages; preferably a polylactic acid-glycolic acid copolymer.
A preparation form of an in-situ spray hydrogel vaccine is specifically tretinoin bionic nanoparticles (GENPs) coated by a hybrid membrane of a Golgi apparatus and a genetic engineering exosome, and is loaded by polyvinyl alcohol acetoacetate (PVAA)/Chitosan (CS) Gel (aPD-L1@ GENPs @ Gel).
The PVAA/CS gel contains 5% by mass of PVAA and 1.5% by mass of CS, the balance being PBS (phosphate buffered saline, pH 7.4).
The tretinoin bionic nano-particles are a hybrid membrane with the surface coated with Golgi bodies and genetic engineering exosomes, and the interior is coated with tretinoin nano-carriers.
Specifically, the retinoic acid bionic nanoparticles comprise retinoic acid, a nano-carrier and a hybrid membrane of Golgi body and genetic engineering exosome; wherein the mass percent of each component is as follows: 1 to 3 percent of tretinoin, 47 to 49 percent of nano-carrier and the balance of a Golgi apparatus and genetic engineering exosome hybrid membrane. The preferable mass percentage is as follows: 1.5% of tretinoin, 48.5% of nano-carrier and 50% of hybrid membrane of Golgi body and genetic engineering exosome.
The tretinoin can also be a tretinoin derivative.
The nano carrier can be any carrier capable of encapsulating tretinoin or derivatives thereof, and comprises one or more of polylactic-co-glycolic acid (PLGA), mesoporous silicon, quantum dots and gold nanocages; preferably a polylactic acid-glycolic acid copolymer.
The particle size of the tretinoin bionic nano-particles is 112-142 nm.
The Golgi body and genetic engineering exosome hybrid membrane is of murine origin and is extracted from murine melanoma B16-F10 cells. The bionic nano-particles of the retinoic acid are coated on a Golgi body and genetic engineering exosome hybrid membrane extracted from a mouse melanoma B16-F10 cell according to the mass ratio of 1:1, for example, 1mg of the core of the retinoic acid nano-particles is coated on a 1mg of the Golgi body and genetic engineering exosome hybrid membrane.
The preparation method of the retinoic acid bionic nanoparticles coated by the hybrid membrane of the Golgi apparatus and the genetic engineering exosomes comprises the following steps:
preparing a retinoic acid nano-carrier; preparing tretinoin-loaded nanoparticles by volatilization of an oil-in-water (O/W) emulsion solvent; the method specifically comprises the following steps: dissolving a nano carrier material and tretinoin in an organic solvent, transferring an organic phase into a PVA solution, and performing ultrasonic oscillation in an ice bath to prepare the nano particles. The organic solvent is dichloromethane, and the mass volume concentration of the PVA solution is as follows: 0.5 to 1.5 percent.
Preparing a hybrid membrane of Golgi apparatus and genetic engineering exosomes; collecting B16-F10 cells, washing with buffer solution, centrifuging, collecting Golgi apparatus membrane, and storing at-80 deg.C; mixing B16-F10Pd-l1-/-Culturing the cells and a culture medium with 10% of fetal calf serum without exosomes at 37 ℃, and then extracting a genetic engineering exosome membrane by a differential centrifugation method; obtaining the hybrid membrane of the Golgi body and the genetic engineering exosome with the mass ratio of 4:1, 3:1, 2:1, 1:1 or 0:1 by ultrasonic.
Coating a hybrid membrane of Golgi body and genetic engineering exosome on the surface of a nano-carrier of tretinoin; and adding the prepared nano particles of the retinoic acid into the prepared Golgi body and genetic engineering exosome hybrid membrane, and performing ultrasonic treatment to prepare the retinoic acid bionic nano particles coated by the Golgi body and the genetic engineering exosome membrane.
The preparation method comprises the following steps:
the retinoic acid, the nano-carrier material and the hybrid membrane are prepared from the following components in percentage by mass: 1 to 3 percent of tretinoin, 47 to 49 percent of nano carrier material and the balance of a Golgi body and genetic engineering exosome hybrid membrane, preferably 1.5 percent of tretinoin, 48.5 percent of nano carrier material and 50 percent of the Golgi body and genetic engineering exosome hybrid membrane.
The tretinoin can also be a derivative of tretinoin.
The nano carrier material can be any carrier capable of encapsulating tretinoin or derivatives thereof, and comprises one or more of polylactic-co-glycolic acid (PLGA), mesoporous silicon, quantum dots and gold nanocages; preferably a polylactic acid-glycolic acid copolymer.
The particle size of the prepared retinoic acid bionic nanoparticles is 112-142 nm.
The Golgi body and genetic engineering exosome hybrid membrane is of murine origin and is extracted from murine melanoma B16-F10 cells. A retinoic acid series bionic nanoparticle is coated by a Golgi body extracted from a mouse melanoma B16-F10 cell and a genetic engineering exosome hybrid membrane according to the mass ratio of 1:1, for example, a 1mg Golgi body and a 1mg retinoic acid nanoparticle core are coated by a genetic engineering exosome hybrid membrane.
The retinoic acid bionic nano-particles are applied to preparation of antitumor drugs.
The retinoic acid bionic nano-particles are applied to preparation of anti-tumor metastasis medicaments.
A preparation method of a retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi body and genetic engineering exosomes for inhibiting secretion of exosomes PD-L1 and activating immune memory by re-vibrating lymph node immune cells so as to activate systemic immune response comprises the following steps:
preparing the retinoic acid bionic nanoparticles by adopting the preparation method of the retinoic acid bionic nanoparticles coated by the Golgi body and genetic engineering exosome hybrid membrane;
and respectively loading the obtained retinoic acid bionic nanoparticles and PD-L1 monoclonal antibody into polyvinyl alcohol acetoacetate and chitosan to prepare the retinoic acid in-situ spray hydrogel vaccine coated by the Golgi apparatus and genetic engineering exosome hybrid membrane.
The retinoic acid, the nano-carrier material and the hybrid membrane are prepared from the following components in percentage by mass: 1 to 3 percent of tretinoin, 47 to 49 percent of nano carrier material and the balance of a Golgi body and genetic engineering exosome hybrid membrane, preferably 1.5 percent of tretinoin, 48.5 percent of nano carrier material and 50 percent of the Golgi body and genetic engineering exosome hybrid membrane.
The tretinoin can also be a derivative of tretinoin.
The nano carrier material can be any carrier capable of encapsulating tretinoin or derivatives thereof, and comprises one or more of polylactic-co-glycolic acid (PLGA), mesoporous silicon, quantum dots and gold nanocages; preferably a polylactic acid-glycolic acid copolymer.
The particle size of the tretinoin bionic nano-particles is 112-142 nm.
The Golgi body and genetic engineering exosome hybrid membrane is of murine origin and is extracted from murine melanoma B16-F10 cells. A vitamin A acid bionic nanoparticle is coated on a Golgi body and genetic engineering exosome hybrid membrane extracted from a mouse melanoma B16-F10 cell in a mass ratio of 1:1, for example, a vitamin A acid nanoparticle core is coated on 1mg of the Golgi body and genetic engineering exosome hybrid membrane by 1mg of the Golgi body and the genetic engineering exosome hybrid membrane.
The invention provides an application of a retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes in inhibiting and/or treating postoperative recurrence of melanoma.
The invention has the beneficial effects that:
the present invention proposes a technical solution to reduce exosome PD-L1 to attenuate immunosuppression by destroying golgi bodies in cancer cells. To this end, drug-loaded nanoparticles (GENPs) with a golgi-exosome hybrid membrane coating were developed, which can destroy the golgi by targeted delivery of the drug. Disruption of the golgi apparatus disrupts the synthesis of PD-L1 and the release of exosomes, resulting in reduced secretion of exosomes PD-L1. In addition, GENPs can strongly elicit a systemic immune response by mimicking the behavior of exosomes entering draining lymph nodes, activating T cells through vaccine-like actions. By combining the PD-L1 monoclonal antibody with the gel vehicle, a low recurrence rate was successfully achieved in a melanoma incompletely resected mouse model, greatly extending its survival. The method is expected to be transformed in the treatment of the postoperative melanoma and is applied to patients insensitive to the treatment of the immune checkpoint inhibitor. Provides a new idea for the fields of tumor immunotherapy, biomedical engineering, nanotechnology and bionic materials.
Drawings
FIG. 1: a preparation schematic diagram of GENPs;
wherein, the retinoic acid is encapsulated in PLGA, and a hybrid membrane of Golgi body and genetic engineering exosome is encapsulated on the surface of the nano-carrier.
FIG. 2 is a schematic diagram: characterization of GENPs;
and a is B16-F10 cell PD-L1 knockout verification. Scale bar: 20 μm;
b is a western-blot analysis B16-F10 cell PD-L1 knockout;
and c is the electron microscope characterization of the nanoparticles. Scale bar: 500nm (left), 50nm (right);
d is SDS-PAGE protein electrophoretic analysis of the nano-particle, the Golgi apparatus and the genetic engineering exosome membrane material;
e is the grain diameter of the nano-particles and the Zeta potential;
f is the appearance picture of the nanoparticles;
g is the western-blot analysis of key proteins of Golgi apparatus, genetic engineering exosomes and nanoparticles;
h is electron microscope characterization of the nanoparticles. Scale bar: 50 nm;
FIG. 3: the in vitro cellular effects of GENPs;
a is the adhesion of the nanoparticles to B16-F10 cells at 4 degrees in vitro. Scale bar: 10 mu m;
b is quantification of nanoparticle adhesion to B16-F10 cells at 4 degrees in vitro.
c is the uptake of GENPs by B16-F10 cells;
d is the intake of the B16-F10 cells to the GENPs;
e is the in vitro cytotoxicity of B16-F10 cells for 48h after various treatments by tretinoin with different concentrations;
f is western-blot to verify the key protein expression of the nanoparticle GENPs.
h is the co-localization of GENPs in B16-F10 cells in confocal analysis. Scale bar: 10 mu m;
g, quantitatively analyzing the co-localization condition of the GENPs in B16-F10 cells;
FIG. 4: evaluating the inhibition effect of GENPs on exosome PD-L1;
a is a confocal image of different nanoparticles and golgi incubations labeled with BODIPY TR. Scale bar: 5 μm;
b is a confocal image of CD63 immunofluorescence staining in B16-F10 cells treated with GENPs. The scale bar is 10 mu m;
c detecting characteristic expression of PD-L1 and CD63 in the sample by western blotting;
d is the inhibition rate of the nanoparticles with different concentrations on exosome secretion;
e transmission electron microscope images of B16-F10 cells treated with different samples;
FIG. 5: immune gel anti-tumor immune response in C57BL/6 mouse after in situ tumor surgery resection model;
a is a schematic administration diagram of a C57BL/6 mouse orthotopic tumor postoperative excision model;
b flow cytometry image analysis of different drug delivery types versus CD8+A change in the number of T cells;
c is CD8+Quantification of T cells;
d flow cytometry image analysis of different drug delivery types versus CD4+Foxp3+A change in the number of T cells;
e is CD4+Foxp3+Quantification of T cells;
f flow cytometry image analysis of nanoparticles on M1-like macrophages (CD 80)hi) Variation of the quantity；
g is M1-like macrophage (CD 80)hi) Quantifying;
h flow cytometry image analysis of nanoparticles on M2-like macrophages (CD 206)hi) A change in quantity;
i is M2-like macrophage (CD 206)hi) Quantifying;
j flow cytometry image analysis of memory T cells in spleen (CD 3)+CD8+CD62L-CD44+，TEM) (iii) a change in the number of;
k is memory T cell in spleen (CD 3)+CD8+CD62L-CD44+，TEM) Quantifying;
l flow cytometry image analysis of CD8 in draining lymph nodes+A change in the number of T cells;
m is CD8 in lymph node+Quantification of T cells;
n is flow cytometry image analysis of CD4 in draining lymph nodes+Foxp3+The number of T varies;
o is CD4 in draining lymph node+Foxp3+Quantifying T;
FIG. 6: therapeutic potential of GENPs in a post-orthotopic tumor resection model in C57BL/6 mice;
a is in vivo bioluminescence imaging of fluorescently labeled melanoma from different treatment groups after primary tumor removal;
b is a tumor growth curve for different groups;
c is the survival time of different groups of mice;
d is the body weight change of different groups of mice;
e is the tumor growth curve for the different groups;
f is the tumor and lung images of the unused groups.
g is spleen pictures of different groups;
h assessing lymph node accumulation capacity of different groups for in vivo imaging;
i is the quantification of lymph node accumulation capacity in different groups;
j is a photograph of H & E staining of different groups of major organ main organ sections. Red arrows indicate visible metastatic sites;
FIG. 7: immune response and therapeutic potential of immunogels in C57BL/6 mice in situ and distal tumor post-surgical resection models;
a schematic representation of dosing on a post-orthotopic and distal tumor resection model in C57BL/6 mice;
b is in vivo bioluminescence imaging of fluorescently labeled melanoma from different treatment groups after primary tumor removal;
c is tumor and lung images of the unused groups;
d is spleen pictures of different groups;
e is the tumor growth curve of the different groups;
f spleen weight quantification for different groups;
g is in vivo imaging to assess lymph node accumulation capacity of different groups;
h is the quantification of lymph node accumulation capacity in different groups;
i for CD8 for different drug types for flow cytometry image analysis+A change in the number of T cells;
j is CD8+Quantification of T cells;
k flow cytometry image analysis of different drug delivery types versus CD4+Foxp3+A change in the number of T cells;
l is CD4+Foxp3+Quantification of T cells;
m is flow cytometry image analysis of nanoparticles on M1-like macrophages (CD 80)hi) A change in quantity;
n is M1-like macrophage (CD 80)hi) Quantifying;
o flow cytometry image analysis of nanoparticles on M2-like macrophages (CD 206)hi) A change in quantity;
p is M2-like macrophage (CD 206)hi) Quantifying;
q flow cytometry image analysis of CD8 in draining lymph nodes+A change in the number of T cells;
r is CD8 in lymph node+Quantification of T cells;
s flow cytometry image analysis of CD4 in draining lymph nodes+Foxp3+The number of T varies;
t is CD4 in draining lymph node+Foxp3+Quantifying T;
u flow cytometry image analysis of memory T cells in spleen (CD 3)+CD8+CD62L-CD44+，TEM) (iii) a change in the number of;
v is memory T cell in spleen (CD 3)+CD8+CD62L-CD44+，TEM) And (4) quantifying.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto.
Example 1
The preparation method of the vitamin A acid bionic nano-particles (GENPs) coated by the hybrid membrane of the Golgi apparatus and the genetic engineering exosomes comprises the following steps:
details of the preparation of GENPs are shown in FIG. 1.
Step 1: preparing nanoparticles of tretinoin;
the PLGA nano-particles loaded with tretinoin are prepared by an oil-in-water (O/W) emulsion solvent volatilization method. 60mg of PLGA and 2mg of tretinoin were dissolved in 4mL of dichloromethane. The organic phase was transferred to 8mL of 1.5% PVA solution. The solution was then sonicated at 400W in an ice bath for 5 minutes. The emulsion was then shaken overnight at room temperature to eliminate the organic solvent and to form uniformly dispersed nanoparticles. Subsequently, the particle suspension was centrifuged at 16000g for 20 min. The prepared nanoparticles were then washed three times with equal volumes of deionized water.
Step 2: preparing a Golgi body and genetic engineering exosome hybrid membrane (GEM);
extracting the genetic engineering exosome membrane by a differential centrifugation method. Mixing B16-F10Pd-l1-/-Cells were cultured with medium containing 10% exosome-free fetal calf serum at 37 ℃ for 2 days and then centrifuged at 800g for 5 minutes. Thereafter, the mixture was centrifuged at 2000g for 10 minutes and then centrifuged again at 0.2 μmThe filter (Millipore, USA). The filtrate was ultracentrifuged at 100000g for 2 hours to pellet the exosomes. The exosomes were then resuspended in hypotonic buffer rich in protease inhibitor cocktail and the cocktail was stored at 4 ℃ for 12 hours. After cold storage, the mixture was centrifuged at 100000g for 5 hours to obtain a genetically engineered Exosome Membrane (EM).
B16-F10 Golgi apparatus membrane (GM) was extracted. After the B16-F10 cells were collected, they were washed 3 times with PBS (phosphate buffered saline, pH 7.4) and buffer (10mM Tris, protease inhibitor cocktail, 250mM sucrose). The pellet was then diluted in buffer (v: v ═ 1:5) and the cells were homogenized 20 times using a Dounce homogenizer. The homogenate was then transferred to the bottom of the tube using 35% sucrose and 29% sucrose. The cells were then centrifuged at 100000g for 3 h. Finally, the golgi membrane bands (bands between 35% and 29% sucrose gradient) were removed from the cells with a syringe and the resulting golgi membranes were stored at-80 ℃.
The EM and GM proteins were quantified using the BCA protein kit, and the membrane weight was twice the weight of the membrane protein. GM was stained with DiD and DiI. EM was then introduced into DiD/DiI stained GM at different mass ratios. The fluorescence spectrum range of the single sample is between 550-720nm, and the excitation wavelength is 525 nm. EM was mixed into GM at a ratio of 4:1, 3:1, 2:1, 1:1 and 0:1, respectively, by protein mass ratio of EM to GM. The mixture was sonicated at 37 ℃ for 8 minutes to complete membrane fusion. By using
Resonance Energy Transfer (FRET) explores the fusion mechanism. The hybrid membrane obtained by mixing the components in the mass ratio of 1:1 was found to have the best fusion effect.
And step 3: coating a Golgi body and genetic engineering exosome hybrid membrane on the surface of a PLGA copolymer of the retinoic acid;
and (2) mixing the PLGA nano particles (PLGA-RA) carrying the tretinoin obtained in the step 1 and the hybrid membrane (GEM) obtained in the step 2 in a mass ratio of 1: 1. The mixture was sonicated for 8 minutes to cover the membrane. Then, the solution was stirred at 10000rpm for 8 minutes to remove other films, and then stirred for 10 hours. The mixture was extruded 10 times using a polycarbonate membrane with a pore size of 200nm on an extruder (Antos Nano Technology co., Ltd.). The resulting GENPs were then resuspended in water for further evaluation.
Comparative example 1
Nanoparticles ENPs, GNPs and RNPs with genetically engineered exosome membrane EM, golgi membrane GM and endoplasmic reticulum membrane RM coated retinoic acid were prepared, respectively, using the preparation method of example 1. The difference from example 1 is that the membrane coated with tretinoin nanoparticles is a single membrane.
The method for preparing the B16-F10 endoplasmic reticulum membrane specifically comprises the following steps: B16-F10 cells were washed with PBS and then suspended in hypotonic extraction buffer at 4 ℃ for 20 minutes. The cells were then centrifuged at 600g and transferred to a Dounce homogenizer using isotonic extraction buffer. The mixture was then homogenized at 4 ℃ for 10 minutes in a 50-stroke 1000g centrifuge, followed by 15 minutes in 13000g centrifuge. The supernatant was then mixed with a calcium chloride solution. Subsequently, the mixture was stirred at 4 ℃ (8000g for 10 minutes, then 100000g for 60 minutes). Endoplasmic reticulum membranes were finally obtained and stored at-80 ℃.
The nanoparticles prepared in example 1 and comparative example 1 were characterized and examined. Specifically, the polydispersity index (PDI), Zeta potential and nanoparticle size were measured using a Malvern Zetasizer. Tretinoin encapsulated in Nanoparticles (NPs) was quantified using High Performance Liquid Chromatography (HPLC).
To verify the knock-out of the PD-L1 gene, B16-F10Pd-L1 was determined by qRT-PCR-/-Levels of PD-L1 mRNA in the cells. As shown in fig. 2a, PD-L1 is in the same position as the exosome marker CD 63. We note that this is at B16-F10Pd-l1-/-Little fluorescence was shown in the PD-L1 channel of the cells. Furthermore, the western-blot also showed a complete knock-out of PD-L1, as shown in FIG. 2 b. Then, B16-F10Pd-l1 was isolated by several centrifugation and ultracentrifugation steps-/-An exosome. As shown in fig. 2c, it shows a unique "saucer" structure in Transmission Electron Microscopy (TEM). B16-F10Pd-l1 was also tested by CD 63-labeled immunogold using immunoelectron microscopy-/-An exosome. To evaluateGM and B16-F10Pd-l1-/-Fusion of Exosome Membranes (EM) with
The resonance energy transfer (FRET) dyes DiI and DiD stain GM. EM was introduced to initiate membrane fusion between the two dye-doped GMs. As the mass ratio of GM to EM increased, fluorescence recovered at 565nm and decreased at 670 nm. These findings indicate that complete fusion of the two membranes suppresses cross-talk between FRET dye pairs in the original GM. Furthermore, as shown in FIG. 2d, SDS-PAGE protein analysis of membrane protein biomarkers showed that the characteristic proteins inherited from GM and EM in the GEM protein profile were well preserved. The average particle size of the GENPs is 136.7nm, which is slightly larger than the particle size of the core PLGA-RA nanoparticle core. As shown in FIG. 2e, the average Zeta potential of GENPs is-27.7 mV, lower than the average Zeta potential of PLGA-RA nanoparticle inner core. The increase in particle size and the decrease in Zeta potential indicate that the GEM successfully coats PLGA-RA nanoparticles. High Performance Liquid Chromatography (HPLC) showed that the tretinoin encapsulation efficiency of GENPs was 71.4%. As shown in FIG. 2f, the appearance and low polydispersity index (PDI) values of the representative NPs prepared indicate a uniform distribution of sizes. As shown in fig. 2g, western blot analysis was also performed to evaluate the unique protein markers of GENPs. GM130 and γ -adaptin are reported to be marker proteins for the cis-golgi and trans-golgi networks, respectively. These markers coexist on GNPs and GENPs. TSG101, CD63, CD9 are exosome-specific markers, coexisting on EM and GENPs. As shown in FIG. 2h, immunogold staining and transmission electron microscopy imaging showed that PLGA-RA nanoparticles were spherical and GENPs showed both GM and EM biomarkers. These findings suggest successful preparation of hybrid membranes and successful preparation of GENPs nanoparticles. From comparison, it can be seen that GENPs have a combined protein expression compared to ENPs, GNPs.
Example 2
Preparing a dosage form which is a retinoic acid bionic nanoparticle coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes, and loading the retinoic acid bionic nanoparticle with PVAA/CS Gel (aPD-L1@ GENPs @ Gel);
preparing retinoic acid bionic nanoparticles GENPs coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes by using the method in example 1; loading the obtained GENPs and PD-L1 monoclonal antibodies into PVAA and CS respectively to prepare the in-situ spray hydrogel vaccine, namely the retinoic acid system bionic hydrogel vaccine coated by the hybrid membrane of the Golgi body and the genetic engineering exosome for inhibiting secretion of the exosome PD-L1 and triggering immune memory by the heavy-vibrating lymph node immunocytes so as to activate the systemic immune response. The method specifically comprises the following steps:
dried PVA powder (5.0g) was added to a three-necked flask containing 45ml of LDMSO. The mixture was heated to 90 ℃ with magnetic stirring to completely dissolve the PVA. The mixture solution was further heated to 110 ℃ and t-BAA (3.0g,18.98mmol) was added dropwise under nitrogen, and the mixture was stirred for 4h after completion of the addition. The product precipitated and was washed with a mixed solvent of methanol and acetone (v/v ═ 1: 3). Then rinsed with pure ethanol and dried in a vacuum oven at 60 deg.C for 24 hours to obtain PVAA. The prepared PVAA and CS were dissolved in PBS (phosphate buffered saline, pH 7.4), respectively, and the PVAA and CS were uniformly mixed using a two-cylinder nebulizer preparation method to prepare a PVAA/CS hydrogel. The prepared hydrogel comprises 5 mass percent of PVAA and 1.5 mass percent of CS, and the balance is PBS.
The PVAA/CS hydrogel was formed by mixing PVAA and CS solutions under physiological conditions with moderate vortexing. The gel was prepared using a two-cylinder nebulizer. The CS solution comprises GENPs and the PVAA solution comprises aPD-L1. aPD-L1 and nanoparticles were mixed with the precursor solution using a spray gel, encapsulated, and then sprayed easily onto the target site in vitro. In contrast to most other gel formulations that rely on complex conditions (e.g., low temperature, multiple reactions and heat), the gels of the present invention can be made by simply mixing PVAA with CS by spraying at room temperature.
Example 3
Research on Golgi body destruction capability and exosome PD-L1 inhibition capability of Golgi body and genetic engineering exosome hybrid membrane coated retinoic acid biomimetic nanoparticles (GENPs)
To evaluate the cell adhesion of GENPs, B16-F10 cells were incubated with fluorescently labeled nanoparticles for 1 hour at 4 ℃. As shown in FIGS. 3a and 3B, while both GENPs and ENPs showed high fluorescence signals on B16-F10 cells, GNPs showed poor adhesion to B16-F10 cells, indicating that EM coating promoted the affinity of the nanoparticles to tumor cells.
Confocal microscopy was used to study the cellular internalization of GENPs. B16-F10 cells were incubated with FITC-RA, PLGA-RA, GNPs and GENPs, respectively, for 6 hours. As shown in fig. 3c, fluorescence was observed in the cytoplasm at various intensities. In B16-F10 cells, both GNPs and GENPs showed high intracellular fluorescence of FITC-RA (FIG. 3d), suggesting that the increased cellular uptake may be due to GM coating.
The MTT method was used to test the cytotoxicity of GENPs on B16-F10 cells in vitro. As shown in FIG. 3e, at RA concentrations below 5. mu.gmL-1When used, NPs are all less cytotoxic. Thus, 5. mu.gmL was used in subsequent experiments-1RA to shield the cytotoxic effects of NP.
To explore the targeted delivery capacity against the golgi, nanoparticles containing FITC-labeled RA were incubated with B16-F10 cells. The intracellular fate of NP depends on various trafficking cascades following endocytosis, including endosomosomes, and the endosomal-golgi-endoplasmic reticulum. To further determine the intracellular trafficking pathways of PLGA-RA, RNPs, ENPs, GNPs and GENPs, the co-localization ratio of fluorescence determined by Pearson correlation coefficients over different incubation times (fig. 3h) is summarized in fig. 3 g. In the first 2 hours after incubation with B16-F10 cells, the fluorescence co-localization ratio in golgi was found to be higher than 65% for all 5 nanoparticles. Then, in the Golgi, the co-localization of RNPs steadily decreased to 52% (4h) and 31% (6h), while the co-localization of RNPs with ER increased to 38% (4h) and 63% (6h), respectively. Similar trends were also found for ENPs (Golgi: 56% (4h) and 33% (6 h); endoplasmic reticulum: 10% (4h) and 27% (6 h)). In contrast, fluorescence co-localization ratios of GNPs and GENPs coated with golgi membranes were 72% and 67% (4h), 56% and 60% (6h), respectively, which are much higher than RNPs and ENPs. The lysosomal colocalization ratios of GNPs and GENPs were 8% (4h) and 4% (6h), respectively, significantly lower than other NPs. These results indicate that GENPs deliver RA via the endosome-golgi-ER pathway with better golgi retention.
Both VAMP4 and Syn6 were found by study to be expressed in the golgi apparatus and on GM-coated NPs (fig. 3 f). Thus, the phenomena of internalization of GENPs by improvement and long-term retention of GENPs on the Golgi apparatus are likely to be caused by the SNARE proteins present on GM.
The destructive power of GENPs on Golgi bodies and the inhibition rate of an exosome PD-L1 are determined. As shown in fig. 4a, the nanoparticle treated cells exhibited various golgi morphologies compared to the control cells. The cis-golgi bodies of the tested cells were labeled with GM130 antibody, and it was found that GM 130-labeled structures were not found throughout the perinuclear regions in GNPs and GENPs treated cells, and that golgi bodies were disrupted in the cellular plasma. Thus, the polarity of the golgi is decomposed, indicating a possible splitting into small stacks. TEM further confirmed that golgi was broken into small clumps (fig. 4 e).
To explore the exosome inhibitory capacity of GENPs, RA, PLGA-RA, GNPs and GENPs were incubated with B16-F10 cells for 24 hours at 37 ℃. Thereafter, cell-derived exosomes were collected and quantified by measuring their protein concentration. As shown in FIG. 4d, the concentration of GENPs observed by CLSM was 5. mu.gmL-1The release of 40% B16-F10 cell-derived exosomes was almost inhibited. As shown in fig. 4b, the GENPs treated cells exhibited lower fluorescence compared to the control cells. For the exosome PD-L1 inhibition assay, primary antibodies against PD-L1, CD63 and GAPDH were added to the samples. FIG. 4c shows that the CD63 band of the B16-F10 exosome group treated with GENPs was much weaker than the control group. Furthermore, a very weak PD-L1 band was observed for the B16-F10 exosome group treated with GENPs, since disruption of golgi body affects secretion of exosomes and blocks PD-L1 glycosylation modification. These results indicate that GENP can efficiently deliver RA to golgi, completely destroy the structure of golgi and its various physiological processes, and effectively inhibit exosome PD-L1, thereby reversing the tumor immunosuppressive microenvironment in vivo.
aPD-L1@ GENPs @ Gel prepared by the method is used for a mouse model, and the dosage of tretinoin and 2mg of aPD-L1 are used per kilogram of body weight of a mouse based on the body weight of the mouse. Specifically, the examples are shown in test examples 1 to 3.
Test example 1
And (3) inspecting the anti-tumor immune response condition of the vitamin A acid bionic nanoparticles (GENPs) coated by the hybrid membrane of the Golgi apparatus and the genetic engineering exosomes on an excision model of the C57BL/6 mouse orthotopic tumor.
To evaluate the therapeutic effect of aPD-L1@ GENPs @ Gel, the present invention used an incomplete tumor resection model to determine if aPD-L1@ GENPs @ Gel showed a positive effect on tumor immune response (FIG. 5 a). The immunization with spray aPD-L1@ GENPs @ Gel in the tumor resection cavity was examined. An in situ formed Gel comprising aPD-L1@ Gel, GNPs @ Gel, aPD-L1@ GNPs @ Gel, or aPD-L1@ GENPs @ Gel is sprayed into the tumor resection cavity. 5 days post-surgery, residual tumors, spleens and draining lymph nodes were collected and evaluated by flow cytometry. A reduction in regulatory T cells (Tregs) and an increase in the level of tumor infiltrating cytotoxic T lymphocytes was observed in samples treated with GNPs @ Gel or aPD-L1@ Gel. These findings indicate that either masking the B16-F10 surface PD-L1 (external strategy) or delivering the golgi destructive agent RA (internal strategy) can effectively trigger T cell immune responses (fig. 5B and 5c, fig. 5d and 5 e).
Notably, aPD-L1@ GENPs @ Gel (IEB strategy) triggered a strong T cell immune response and T cells were maximally rejuvenated following EM modification. CD8 compared to aPD-L1@ GNPs @ Gel+The increased percentage of T cells and the decreased regulatory T cells may be due to enhanced lymph node activation. To confirm this hypothesis, GNPs and GENPs were first examined for their targeting of DLNs and their effect on T cell responses. C57BL/6 mice with tumor resection cavity were sprayed with GNPs @ Gel 24 hours after treatment and showed low FITC signal in DLNs. In contrast, the GENPs @ Gel group showed a significant increase in FITC signal in DLN (fig. 6h and 6i), indicating that EM-modified s facilitate nanoparticle delivery to Draining Lymph Nodes (DLNs). It was also found that the reduction of regulatory T cells (tregs) and the enhanced infiltration of cytotoxic T lymphocytes in DLNs suggests that GENPs can efficiently enter DLNs and also activate T cells by vaccine-like action (fig. 5l and 5m, fig. 5n and 5 o).
By modulating the phenotypic transformation of tumor-associated M2 macrophages (TAMs), infiltrating M2 macrophages in tumor tissue can be transformed into M1 type, blocking the formation of tumor lymphatic and blood vessels mediated by macrophages (TAMs), and inhibiting tumor development. FIGS. 5f and 5g show M1-like TAM (CD 80)hiCD11b+F4/80+) Increased and M2-like TAM (CD 206)hiCD11b+F4/80+) Decrease (fig. 5h and 5 i). In addition, splenocytes were obtained and stained with fluorescent antibody. T-Effect memory cells (T) were observed between the aPD-L1@ GENPs @ Gel and aPD-L1@ GNPs @ Gel treatment groupsEM) There were significant differences in statistics indicating that the vaccine could develop memory (fig. 5j and 5 k).
These findings suggest that the IEB strategy can reverse the tumor immunosuppressive microenvironment by shielding PD-L1 on the surface and inhibiting secretion of the exosome PD-L1. The sprayed hydrogel vaccine aPD-L1@ GENPs @ Gel can imitate exosomes entering DLNs, activate effective immune cells to activate T cells through vaccine-like action, and improve immune memory, so that the systemic immune response is strongly promoted.
Test example 2
And (3) investigating the treatment potential of the Golgi body and genetic engineering exosome hybrid membrane coated retinoic acid biomimetic nanoparticles (GENPs) on a C57BL/6 mouse orthotopic tumor postoperative excision model.
Tumor progression was followed by bioluminescent signaling from B16-F10 luciferase-labeled cells (FIG. 6 a). Mice treated with aPD-L1@ GENPs @ Gel showed better tumor suppression because three-sixths of mice had no visible tumor (FIGS. 6a, 6b and 6 e). Furthermore, after treatment with aPD-L1@ GENPs @ Gel, 50% of the mice survived for more than 52 days (FIG. 6c), and the body weight of the mice was not affected during the treatment period (FIG. 6 d). In addition, tumors, spleen and lung were collected after 22 days of different treatments, aPD-L1@ GENPs @ Gel eliminated tumor metastasis and showed minimal tumor, consistent with in vivo bioluminescence imaging results (FIG. 6 f). In addition, spleen morphology was normal in mice dosed with aPD-L1@ GENPs @ Gel, while spleen was enlarged in other treatment groups (FIG. 6 g). Furthermore, histological studies of major organ tissue samples showed that the local distribution of tretinoin did not cause severe side effects in mice and no significant tumor metastasis was found after aPD-L1@ GENPs @ Gel treatment (FIG. 6 j). Therefore, aPD-L1@ GENPs @ Gel is a potentially effective hydrogel vaccine for the treatment of postoperative melanoma.
Test example 3
And (3) examining the immune response and the treatment potential of the Golgi body and genetically engineered exosome hybrid membrane coated retinoic acid biomimetic nanoparticles (GENPs) on a C57BL/6 mouse orthotopic tumor and distal tumor postoperative excision model.
To determine whether aPD-L1@ GENPs @ Gel triggered a systemic immune response and inhibited distant tumors, B16-F10 cells were seeded on the other side of the primary tumor to mimic distant metastasis. The primary tumor was then partially resected and aPD-L1@ GENPs @ Gel was then sprayed into the tumor resection cavity (FIG. 7 a). FIG. 7b shows that aPD-L1@ GENPs @ Gel treatment inhibited local tumor recurrence, and tumor progression on the other side was also inhibited (FIGS. 7c and 7 e). After aPD-L1@ GENPs @ Gel treatment, no splenomegaly was found (FIGS. 7d and 7f), and a strong accumulation of DLNs by GENPs was also observed (FIGS. 7g and 7 h).
For flow cytometry analysis, distant tumors were collected, right DLNs and spleens were pooled to form single cell suspensions for testing. The M1-class TAM (CD 80) in distant tumors after aPD-L1@ GENPs @ Gel treatmenthiCD11b+F4/80+) Increased (FIGS. 7M and 7n), TAMs of type M2 (CD 206)hiCD11b+F4/80+) Decreased (FIGS. 7o and 7p), increased tumor-infiltrating cytotoxic T lymphocytes, and decreased regulatory T cells (FIGS. 7i and 7j, FIGS. 7k and 7 l). Furthermore, tregs in the right DLNs decreased and infiltration of cytotoxic T lymphocytes increased (fig. 7q and 7r, fig. 7s and 7T). In addition, splenocytes were obtained and stained with fluorescent antibody. Statistically significant differences were observed between the aPD-L1@ GENPs @ Gel and aPD-L1@ GNPs @ Gel treated groups, indicating that the vaccine can induce immunological memory (TEM) (FIGS. 7u and 7 v).
These results indicate that immune cells stimulated in DLNs in situ are able to migrate and attack the opposite side of the tumor cells. In particular, flow cytometric examination of distant tumors shows a significant increase in the number of aggressive T lymphocytes. These findings elucidate the association between tumors and suggest that sprayed hydrogel vaccines can rejuvenate immune cells in the spleen and DLNs, develop immune memory, and strongly promote systemic T cell activation. Therefore, aPD-L1@ GENPs @ Gel (IEB strategy) is a potential treatment option for melanoma after surgery.
Claims (5)
1. A vitamin A acid bionic nano particle coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes is characterized by comprising: the tretinoin hybrid membrane comprises tretinoin, a hybrid membrane and a nano carrier, wherein the tretinoin hybrid membrane comprises the following components in percentage by mass: 1-3% of tretinoin, 47-49% of nano carrier and the balance of hybrid membrane;
the hybrid membrane is a hybrid membrane of Golgi apparatus and genetic engineering exosomes, is murine and is extracted from murine melanoma B16-F10 cells; coating retinoic acid bionic nanoparticles by using a Golgi body and genetic engineering exosome hybrid membrane according to the mass ratio of 1: 1;
the nano carrier is polylactic acid-glycolic acid copolymer;
the preparation process of the vitamin A acid bionic nano-particles coated by the Golgi apparatus and the genetic engineering exosome membrane comprises the following steps:
preparing a retinoic acid nano-carrier; preparing tretinoin-loaded nanoparticles by volatilizing an oil-in-water emulsifying solvent; the method specifically comprises the following steps: dissolving a nano carrier and tretinoin in dichloromethane, transferring an organic phase into a PVA solution, and performing ultrasonic oscillation in an ice bath to prepare nano particles; the mass volume concentration of the PVA solution is as follows: 0.5% -1.5%;
preparing a hybrid membrane of Golgi apparatus and genetic engineering exosomes; collecting B16-F10 cells, washing with buffer solution, centrifuging, collecting Golgi apparatus membrane, and storing at-80 deg.C; culturing B16-F10Pd-l 1-/-cells and a culture medium with 10% of exosome-free fetal calf serum at 37 ℃, and extracting a genetic engineering exosome membrane by a differential centrifugation method; obtaining a hybrid membrane of the Golgi apparatus and the genetic engineering exosome with the mass ratio of 1:1 by ultrasonic;
adding the prepared retinoic acid nanoparticles into the prepared Golgi body and genetic engineering exosome hybrid membrane, and performing ultrasonic treatment to prepare retinoic acid bionic nanoparticles coated by the Golgi body and the genetic engineering exosome membrane.
2. The retinoic acid bionic nanoparticle coated with a golgi body and genetic engineering exosome hybrid membrane is characterized in that the retinoic acid, the hybrid membrane and the nanocarrier are as follows by mass percent: 1.5% of tretinoin, 48.5% of nano-carrier and 50% of hybrid membrane.
3. A vitamin A acid bionic nano particle in-situ spray hydrogel vaccine formulation coated by a Golgi apparatus and genetic engineering exosome hybrid membrane is characterized in that the vitamin A acid bionic nano particle and PD-L1 monoclonal antibody of claim 1 or 2 are respectively loaded into polyvinyl alcohol acetoacetate and chitosan to prepare in-situ spray hydrogel vaccine;
the particle size of the tretinoin bionic nano-particles is 112-142 nm.
4. The preparation method of the dosage form of the retinoic acid in-situ spray hydrogel vaccine coated by a hybrid membrane of Golgi apparatus and genetic engineering exosomes as claimed in claim 3, which is characterized by comprising the following steps:
preparing a retinoic acid nano-carrier; preparing tretinoin-loaded nanoparticles by volatilizing an oil-in-water emulsifying solvent; the method specifically comprises the following steps: dissolving a nano carrier and tretinoin in dichloromethane, transferring an organic phase into a PVA solution, and performing ultrasonic oscillation in an ice bath to prepare nano particles; the mass volume concentration of the PVA solution is as follows: 0.5% -1.5%;
preparing a hybrid membrane of Golgi apparatus and genetic engineering exosomes; collecting B16-F10 cells, washing with buffer solution, centrifuging, collecting Golgi apparatus membrane, and storing at-80 deg.C; mixing B16-F10Pd-l1-/-Culturing the cells and a culture medium with 10% of fetal calf serum without exosomes at 37 ℃, and then extracting a genetic engineering exosome membrane by a differential centrifugation method; obtaining a hybrid membrane of the Golgi apparatus and the genetic engineering exosome with the mass ratio of 1:1 by ultrasonic;
adding the prepared retinoic acid nanoparticles into the prepared Golgi body and genetic engineering exosome hybrid membrane, and performing ultrasonic treatment to prepare retinoic acid bionic nanoparticles coated by the Golgi body and the genetic engineering exosome membrane;
and respectively loading the obtained retinoic acid bionic nanoparticles and the PD-L1 monoclonal antibody into polyvinyl alcohol acetoacetate and chitosan to prepare the retinoic acid in-situ spray hydrogel vaccine coated by the Golgi body and genetic engineering exosome hybrid membrane.
5. The preparation method of the dosage form of the retinoic acid in-situ spray hydrogel vaccine coated by the hybrid membrane of the Golgi body and the genetic engineering exosome according to claim 4, wherein the prepared retinoic acid bionic nanoparticles coated by the hybrid membrane of the Golgi body and the genetic engineering exosome are applied to preparation of antitumor drugs or anti-tumor metastasis drugs.
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