CN112933113A - Immune-enhanced exosome hydrogel compound and preparation method and application thereof - Google Patents
Immune-enhanced exosome hydrogel compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immune enhancement type exosome hydrogel compound and a preparation method and application thereof, wherein the compound is a hydrogel drug delivery system formed by loading a drug-M1 type macrophage exosome by an M1 type macrophage exosome, then forming hydrogel through chemical crosslinking of mannatide and hyaluronic acid, and loading the drug-M1 type macrophage exosome in the hydrogel. The mannatide-hyaluronic acid hydrogel prepared by the invention not only has the advantages of acid-sensitive drug release, injectability, self-healing and the like of Schiff base gel, but also has the effects of enhancing anti-tumor immunity, increasing exosome stability, tumor adhesion, biodegradability, biosafety and the like, and can release the drug-M1 type macrophage exosome in a sustained manner after local injection administration of tumors, permeate the tumor and tumor inflammation areas in a targeted manner, activate tumor immunity and kill tumors in a synergistic manner.
Description
Technical Field
The invention discloses an immune-enhanced exosome hydrogel compound and a preparation method and application thereof, and belongs to the technical field of hydrogel compounds.
Background
Malignant tumor seriously threatens human health, and the existing treatment schemes such as surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy have certain limitations. Although chemotherapy and immune tumor combined treatment is a common means, a systemic administration scheme is adopted, and the defects of low tumor selectivity, weak immune response efficiency, large toxic and side effects and the like exist, so that the clinical application is limited.
In recent years, local tumor administration as an effective improvement scheme of systemic treatment is one of the hot spots in research and development in the anti-tumor field. The exosome is a nano vesicle with a double-layer lipid membrane structure secreted by cells, is used as a drug delivery carrier with great application prospect, and has the advantages of low toxicity, no immunogenicity, good permeability and the like. Research proves that M1 type macrophages have tumor and tumor inflammation tendencies and can be used for targeted delivery of antitumor drugs. It is worth noting that the M1 type macrophage exosome has a membrane structure similar to that of M1 type macrophages, and has more significant tumor targeting compared with exosome secreted by unpolarized macrophages. Nevertheless, the drug-loaded exosomes after topical administration still have the defects of low stability, poor retention property and the like, and the application of the drug-loaded exosomes as a local sustained-release carrier is hindered.
On the other hand, the hydrogel has unique advantages as a local tumor drug delivery carrier: (1) the unique three-dimensional network structure is beneficial to the high-efficiency entrapment and the slow-controlled drug release of various drugs; (2) the appropriate gel material is selected to realize minimally invasive, good biocompatibility and biodegradable characteristics; (3) local injection of tumors is beneficial to the high tumor accumulation property and low systemic toxicity of the drug. However, based on the complex characteristics of tumor and tumor microenvironment, the design of the hydrogel for local tumor drug delivery still faces great challenges, such as non-responsive drug release of the gel in the tumor region, whether the drug can be further specifically targeted and accumulated in the tumor tissue after being released from the gel, non-injectability of the gel material, low tumor adhesion, no immune enhancement activity, and the like.
Hyaluronic acid is an important component of cell matrix, has excellent biocompatibility and degradability, and particularly, the hyaluronic acid is highly expressed by CD (compact disc) of tumor cells44The receptor has higher affinity and certain tumor adhesiveness. The patent application No. 201910145974.1 discloses an exosome-loaded composite hydrogel and a preparation method thereof, wherein aldehyde groups of oxidized hyaluronic acid and amino groups of oxalic dihydrazide are crosslinked to form gel, but the gel does not consider the problems of in-vivo and in-vitro stability of exosomes and adhesion of gel tumors in the gel.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention provides an immune-enhanced exosome hydrogel compound and a preparation method and application thereof, the hydrogel compound is a brand-new chemotherapy and immunopotentiator combination scheme, can overcome the defects of poor stability, low targeting efficiency, weak retention and the like of a drug-loaded exosome and the defects of low adhesion, non-injection, non-response drug release and the like of a common hydrogel tumor, improves the effective accumulation concentration of the drug in tumor and tumor inflammation environment, reduces the toxic and side effects of the whole body, exerts a synergistic killing effect, and has a remarkable treatment effect on various solid tumors.
The technical scheme is as follows: in order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an immune enhancement type exosome hydrogel compound is a hydrogel drug delivery system formed by loading a drug by M1 type macrophage exosomes to form a drug-M1 type macrophage exosomes, then forming a hydrogel through chemical crosslinking of mannatide and hyaluronic acid, and loading the drug-M1 type macrophage exosomes in the hydrogel.
Preferably, the drug is selected from doxorubicin, paclitaxel, curcumin, ganoderic acid a, ginsenoside, quercetin, alkannin or houttuynin.
The chemical crosslinking is to oxidize hyaluronic acid and then react with mannatide in solution to chemically crosslink aldehyde group of oxidized hyaluronic acid and amino group of mannatide, preferably reacting at room temperature for 15s-2min, and more preferably 30 s.
The preparation method of the immune enhancement type exosome hydrogel compound comprises the following steps:
s1: extracting and purifying M1 type macrophage exosome;
s2: preparing a medicament-M1 type macrophage exosome;
s3: preparation of mannan peptide-hyaluronic acid hydrogel of drug-M1 type macrophage exosome:
and (4) mixing the drug-M1 type macrophage exosome in the step (S2) with the mannan peptide solution, adding an oxidized hyaluronic acid solution, uniformly mixing, and reacting at room temperature to form the mannan peptide-hyaluronic acid hydrogel loading the drug-M1 type macrophage exosome.
Preferably, the step S1 includes inducing macrophage M1 polarization, extracting exosome from M1 polarized macrophage culture solution, and finally obtaining M1 polarized macrophage exosome, namely the M1 type macrophage exosome, by ultracentrifugation.
Specifically, the method comprises the following steps: using LPS and IFN-gamma to induce mouse macrophage M1 polarization, extracting exosome from M1 polarized macrophage culture solution, obtaining M1 polarized macrophage exosome by ultracentrifugation, and suspending in 1 × PBS for storage at-80 ℃ for later use. Preferably, the mouse macrophage is RAW264.7 cell and bone marrow-derived macrophage.
Preferably, in step S2, the drug M1-type macrophage exosome is prepared by electroporation, and the mass ratio of the M1-type macrophage exosome (calculated as protein) to the drug is 1:1 to 5, and more preferably 1: 1-2, more preferably 1: 2.
specifically, the method comprises the following steps: and (3) uniformly mixing the drug solution and the M1 type macrophage exosome under the condition of keeping out of the sun, and carrying out drug loading according to set parameters by using an electroporator. The obtained sample is ultracentrifuged for 2 times to remove free drugs, and the obtained drug-loaded exosome precipitate is resuspended by 1 XPBS for standby. Preferably, the electroporator has the following set parameters: voltage 300-1000V, discharge time 1-10 ms, discharge frequency: 1 time. More preferably: voltage 700 ~ 1000V, discharge time 5 ~ 10ms, the number of times of discharging: 1 time, more preferably: voltage 1000V, discharge time 5ms, number of discharges 1 time.
Preferably, in step S3, hyaluronic acid is oxidized with sodium periodate to prepare oxidized hyaluronic acid, and the molecular weight of hyaluronic acid is 50-1000 kDa, more preferably 100-200 kDa.
Preferably, in step S3, the concentration of the oxidized hyaluronic acid solution is 5 to 20% (W/V), more preferably 6 to 8% (W/V); the molecular weight of the mannatide is 40-90 kDa, and the optimized molecular weight is 51-72 kDa; the concentration of the mannan peptide solution is 5-20% (W/V), more preferably 6-10% (W/V); the volume ratio of the mannan peptide solution to the oxidized hyaluronic acid solution is 1: 1-3, reacting at room temperature for 15s-2min, preferably 30 s.
The invention finally provides the application of the immune enhanced exosome hydrogel compound in preparing antitumor drugs.
Preferably, the tumor comprises breast cancer, prostate cancer, bladder cancer, skin cancer, liver cancer, stomach cancer, brain cancer or ovarian cancer.
In the immune enhancement type exosome hydrogel compound, the content of a drug in the compound is 0.1-5 mg/mL. The compound can be administered by tumor in-situ injection or tumor-surrounding local injection, and the administration dose is 1-20 mL/kg.
Mannatide, alpha-mannatide, is a glycoprotein separated from 33# streptococcus metabolite, and has the functions of increasing peripheral leukocyte level, enhancing reticuloendothelial system phagocytosis function, activating macrophage and lymphocyte, inducing thymic lymphocyte to generate active substance, improving and enhancing organism immunity and stress capability, etc. In the compound, mannatide is used as an immunopotentiator for the adjuvant treatment of tumors. In addition, the mannan peptide is composed of mannan chain and basic polypeptide, amino rich in the basic polypeptide can be crosslinked with aldehyde group in the oxidized hyaluronic acid to form Schiff base type gel, and the mannan has affinity with macrophage surface mannose receptor highly infiltrated in tumor microenvironment, and has adhesion and retention of the tumor microenvironment.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) mannan peptides enhance the tumor adhesion of the gel: mannan in mannan peptide has very high affinity with mannose receptor over-expressed by tumor cells and peripheral macrophages, hyaluronic acid and tumor cell CD44The receptor has extremely high affinity, realizes excellent tumor adhesion through the design of a gel material, and can prolong the time to further improve the accumulation of the drug in the tumor.
(2) Mannatide enhances dispersion stability of macrophage exosome: the affinity of the mannan peptide and a mannose receptor on the surface of macrophage exosome is also benefited, and the dispersion stability of exosome in gel is obviously improved. When the gel mass concentration is extremely low, the invention can realize uniform dispersion and long-term non-coagulation of macrophage exosomes in the system.
(3) The gelation of the mannatide is beneficial to the local controlled release of the drug in the tumor: the Schiff base type hydrogel is prepared by utilizing the amino design of the basic polypeptide chain in the structure of the mannatide, has the advantages of acid-sensitive degradation, injectability, self-healing and the like, can be self-degraded in a tumor slightly acidic environment, and slowly releases the mannatide and the adriamycin-exosome, so that the defects of uneven drug distribution, quick metabolism and the like during local injection of the mannatide and the adriamycin-exosome are overcome.
(4) Macrophage exosome M1 polarization further enhanced tumor-targeted permeability: compared with unpolarized macrophage exosomes, the macrophage exosome M1 has obvious tumor and tumor inflammation tendencies, and can assist the drug adriamycin carried by the macrophage exosomes in further targeting and permeating to the tumor central area.
(5) The exosome gel compound obviously enhances the anti-tumor curative effect: based on the characteristics, after the exosome gel compound is administrated in situ on the tumor, the exosome gel compound can be acid-sensitive degraded, slowly release mannatide and a medicament-M1 type macrophage exosome, penetrate to the tumor and a tumor inflammation area in a targeted mode, activate tumor immunity and kill the tumor in a synergistic mode.
Drawings
FIG. 1 is a schematic diagram of the structure of an exosome-gel complex of the present invention;
FIG. 2 is a schematic diagram of the preparation of an exosome-gel complex of the present invention;
FIG. 3 is a diagram showing injectability of exosome-gel complexes in the present invention;
FIG. 4 is a TEM image of M1-type macrophage exosomes in the present invention;
FIG. 5 is a TEM image of an exosome of doxorubicin-M1-type macrophages in the present invention;
FIG. 6 is a schematic particle size diagram of macrophage exosomes of type M1 and macrophage exosomes of type adriamycin-M1 in the present invention;
FIG. 7 is a graph showing the rheometer of the present invention to verify the colloidal properties of mannatide-hyaluronic acid hydrogel;
FIG. 8 is an SEM showing that the mannatide-hyaluronic acid hydrogel has a loose and porous network structure according to the present invention;
FIG. 9 is the dispersion stability of exosome hydrogel complexes of the present invention;
FIG. 10 shows the toxicity of the blank mannatide-hyaluronic acid hydrogel of the present invention on different cell lines;
FIG. 11 is a graph showing the cytotoxicity of doxorubicin formulations of the present invention (DOX, DOX @ exos, DOX @ M1-exos-HA-gel and DOX @ M1-exos-OHA/Man gel) against 4T1 cells;
FIG. 12 is a graph showing the degradation curve of mannatide-hyaluronic acid hydrogel according to the present invention;
FIG. 13 is a graph showing the release of exosomes from mannatide-hyaluronic acid hydrogels of the present invention;
FIG. 14 shows the tumor adhesion test of mannatide-hyaluronic acid hydrogel according to the present invention;
FIG. 15 shows the effect of mannatide on macrophage NO production in mannatide-hyaluronic acid hydrogel according to the present invention;
FIG. 16 shows the effect of mannatide on macrophage phagocytosis rate in mannatide-hyaluronic acid hydrogel according to the present invention;
FIG. 17 is a graph showing the accumulation of doxorubicin in tumor tissues at various time points of local tumor injection for each doxorubicin preparation of the present invention;
FIG. 18 is a graph of tumor volume changes in mice at various time points after treatment with each formulation of the present invention;
FIG. 19 is a graph of body weight of mice at various time points after treatment with each formulation of the present invention;
FIG. 20 is a HE slice of mouse tumor tissue treated with each formulation of the present invention;
FIG. 21 shows Ki67 sections of mouse tumor tissue treated with each formulation of the present invention;
FIG. 22 is a CD31 section of mouse tumor tissue after treatment with each formulation of the present invention;
FIG. 23 is a HE slice of mouse heart tissue treated with each formulation of the present invention;
FIG. 24 is an HE slice of mouse liver tissue treated with each formulation of the present invention;
FIG. 25 is a HE slice of mouse kidney tissue treated with each formulation of the present invention;
FIG. 26 is a graph of the kidney index Blood Urea Nitrogen (BUN) values of mice treated with each of the formulations of the present invention;
FIG. 27 is a graph of the kidney index Creatinine (CRE) values for mice treated with each of the formulations of the present invention;
FIG. 28 is a graph showing liver index of blood-glutamic-oxaloacetic transaminase (AST) values of mice treated with each formulation of the present invention;
FIG. 29 shows the liver index of mice treated with each formulation of the present invention, the value of alanine Aminotransferase (ALT).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the above figures and in the following examples, DOX represents doxorubicin; man represents mannatide; m1-exos represents an M1-type macrophage exosome; HA means hyaluronic acid, OHA means oxidized hyaluronic acid, DOX @ M1-exos means macrophage exosome of adriamycin-M1 type; DOX @ exos denotes doxorubicin-macrophage exosomes; DOX @ M1-exos-HA gel represents a cross-linked hyaluronic acid gel loaded with macrophage exosomes of the doxorubicin-M1 type; DOX @ M1-exos-OHA/Man gel represents a mannatide-hyaluronic acid hydrogel loaded with macrophage exosomes of doxorubicin-M1 type; OHA/Man gel represents blank mannatide-hyaluronic acid hydrogel; HA gel represents a cross-linked hyaluronic acid gel; CS gel represents chitosan-glutaraldehyde hydrogel; OHA/CS gel represents an oxidized hyaluronic acid-chitosan hydrogel.
Example 1: preparation and characterization of mannatide-hyaluronic acid hydrogel loaded with adriamycin-M1 type RAW264.7 macrophage exosome
(1) High-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody under the condition of 5% CO237 ℃ constant temperature incubatorAfter the mouse macrophage is cultured and the cells are completely attached and the growth state is good, 500ng/mL LPS and 2ng/mL IFN-gamma are added, and the RAW264.7 macrophage with M1 polarization induced to form is cultured for 24 hours. The original culture medium is aspirated, washed with PBS for 2 times, and then added with serum-free high-sugar DMEM culture medium for 48 hours. 100mL of the medium was collected and M1-macrophage exosomes were isolated using ultracentrifugation: centrifuging at 4 deg.C for 15min at 300g, and collecting supernatant; centrifuging at 3000g for 30min, and collecting supernatant; centrifuging at 10000g for 90min, and resuspending the precipitate with 1 × PBS; centrifuging at 100000g for 90min, suspending the precipitate with 100. mu.L of 1 XPBS to obtain M1-macrophage exosome (M1-exos), and measuring the content of the exosome protein by BCA method.
(2) Under the dark condition, dissolving adriamycin in 1 XPBS by ultrasonic waves, and mixing the adriamycin and the PBS according to a mass ratio of 1: 2 and M1-macrophage exosome are uniformly mixed, added into an electroporation cuvette, and subjected to drug loading according to set parameters (voltage 1000V, discharge time 5ms and discharge times 1 time) by using an electroporation method. After the obtained sample is placed in a cell culture box to be incubated for 60min, the cell culture box is ultracentrifuged for 90min at 150000g, and the precipitate is resuspended by 1 XPBS; centrifuging at 150000g for 90min, suspending the precipitate with 100 μ L of 1 XPBS to obtain adriamycin-M1 type macrophage exosome (DOX @ M1-exos), and measuring the adriamycin drug loading by a microplate reader.
(3) Preparing 2 percent (W/V) hyaluronic acid (molecular weight 100kDa) solution, slowly dripping 4.4 percent (W/V) sodium periodate solution, stirring uniformly, and reacting for 24 hours at room temperature in a dark place. 2mL of ethylene glycol was added and stirred for 1h to terminate the oxidation reaction. Adding the above mixture into dialysis bag (molecular weight cut-off is 3500Da), dialyzing for 1 week, and lyophilizing to obtain Oxidized Hyaluronic Acid (OHA). After doxorubicin-M1 type macrophage exosome and 6% (W/V) mannatide (molecular weight 72kDa) solution are mixed, 6% (W/V) oxidized hyaluronic acid solution with the same volume as the mannan peptide solution is added and uniformly mixed, and the mixture reacts for 30s at room temperature to form mannatide-hyaluronic acid hydrogel (DOX @ M1-exos-OHA/Man gel) carrying the doxorubicin-M1 type macrophage exosome, wherein the structural schematic diagram is shown in figure 1, the formation schematic diagram is shown in figure 2, and the injectability performance is shown in figure 3.
(4) Transmission Electron Microscopy (TEM) is used for proving that the M1-macrophage exosome (shown in figure 4) obtained in the step 1 and the adriamycin-M1-type macrophage exosome (shown in figure 5) obtained in the step 2 have typical exosome vesicle-type structures, and the DOX drug loading in the exosome is 8.79% measured by an enzyme-linked immunosorbent assay. And (3) measuring the particle sizes of the M1-macrophage exosomes obtained in the step (1) and the adriamycin-M1 type macrophage exosomes obtained in the step (2) by using a particle size analyzer (figure 6), wherein the particle size ranges from 70 nm to 100 nm.
(5) The product obtained in the step (3) is proved to have colloidal properties by using a rheometer (figure 7), and the successful synthesis of the Schiff base hydrogel is proved by the invention; microscopic imaging of the hydrogel using Scanning Electron Microscopy (SEM) (fig. 8) revealed that the hydrogel had a loose porous microstructure.
Example 2: preparation and characterization of mannan peptide-hyaluronic acid hydrogel loaded with adriamycin-M1 type bone marrow-derived macrophage exosome
(1) Taking leg bones of a mouse with the age of 4-6 weeks, flushing bone marrow with PBS buffer solution, and centrifuging; erythrocytes were lysed using ACK and DMEM medium centrifuged. Culturing in high-glucose DMEM medium (10% fetal calf serum, M-CSF 20ng/ml)1.5ml six-well plate for 3 days, washing off floating cells, and replacing with new culture medium to obtain adherent mouse bone marrow-derived macrophage. High-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody under the condition of 5% CO2After the mouse macrophage is cultured in a constant temperature incubator at 37 ℃, the cells are completely attached to the wall and grow well, 500ng/mL LPS and 2ng/mL IFN-gamma are added, and M1 polarized bone marrow-derived macrophage is induced and formed after 24 hours of culture. The original culture medium is aspirated, washed with PBS for 2 times, and then added with serum-free high-sugar DMEM culture medium for 48 hours. 100mL of the medium was collected and M1-macrophage exosomes were isolated using ultracentrifugation: centrifuging at 4 deg.C for 15min at 300g, and collecting supernatant; centrifuging at 3000g for 30min, and collecting supernatant; centrifuging at 10000g for 90min, and resuspending the precipitate with 1 × PBS; centrifuging at 100000g for 90min, suspending the precipitate with 100 μ L of 1 × PBS to obtain M1-macrophage exosome, and measuring the protein content of the exosome by BCA method.
(2) Under the dark condition, dissolving adriamycin in 1 XPBS by ultrasonic waves, and mixing the adriamycin and the PBS according to a mass ratio of 1:1 and M1-macrophage exosome are uniformly mixed, an electroporation cuvette is added, and medicine loading is carried out according to set parameters (voltage 700V, discharge time 10ms and discharge frequency 1 time) by using an electroporation method. After the obtained sample is placed in a cell culture box to be incubated for 60min, the cell culture box is ultracentrifuged for 90min at 150000g, and the precipitate is resuspended by 1 XPBS; centrifuging at 150000g for 90min, suspending the precipitate with 100 μ L1 × PBS to obtain adriamycin-M1 type macrophage exosome, and measuring DOX drug loading with microplate reader.
(3) Preparing 2 percent (W/V) hyaluronic acid (molecular weight 200kDa) solution, slowly dripping 4.4 percent (W/V) sodium periodate solution, stirring uniformly, and reacting for 24 hours at room temperature in a dark place. 2mL of ethylene glycol was added and stirred for 1h to terminate the oxidation reaction. Adding the above mixture into dialysis bag (molecular weight cut-off is 3500Da), dialyzing for 1 week, and lyophilizing to obtain Oxidized Hyaluronic Acid (OHA). Mixing the adriamycin-M1 type macrophage exosome with 10% (W/V) mannatide (molecular weight 51kDa) solution, then adding 8% (W/V) oxidized hyaluronic acid solution with the same volume as the mannatide solution, uniformly mixing, and reacting at room temperature for 30s to form the mannatide-hyaluronic acid hydrogel carrying the adriamycin-M1 type macrophage exosome.
Example 3: dispersion stability, biosafety, degradability and drug release profile of exosome-gel complexes
(1) DOX @ M1-exos-HA gel was prepared by ultrasonically dispersing the doxorubicin-M1 type RAW264.7 macrophage exosomes prepared in example 1 into a commercially available cross-linked hyaluronic acid gel (HA gel, huaxi organism, cat No. TL100, chinese patent No. 2015zl 101091144). In order to examine the dispersion stability of DOX @ M1-exos in a mannatide-hyaluronic acid hydrogel and a common cross-linked hyaluronic acid gel, the prepared DOX @ M1-exos-HA-gel and DOX @ M1-exos-OHA/Man gel are placed in a refrigerator at 4 ℃, the sample condition is observed at 0d and 7d respectively, as shown in FIG. 9, after 7d, the DOX @ M1-exos in the common cross-linked hyaluronic acid gel have aggregation and precipitation phenomena, while the DOX @ M1-exos in the mannatide-hyaluronic acid hydrogel are uniformly dispersed, and the fact that the dispersion stability of the DOX @ M1-exos in the gel is obviously improved due to the fact that macrophage exosome surface mannose receptors have good affinity with mannan in the gel is presumed.
(2) Mixing 6% (W/V) mannan peptide solution and 6% (W/V) oxidized hyaluronic acid solution uniformly, reacting for a certain time at room temperature, and preparing blank mannan peptide-hyaluronic acid hydrogel (OHA/Man gel). In order to examine the cytotoxicity of the blank mannatide-hyaluronic acid hydrogel, 4T1 cells and RAW264.7 cells are respectively inoculated on a 96-well plate, blank mannatide-hyaluronic acid hydrogels with different masses are respectively given after the cells are attached to the wall, the incubation is carried out for 48 hours, and the cell survival rate is measured by an MTT method. Referring to fig. 10, the blank gel is not significantly toxic to cells, and shows good biological safety of the gel, as shown in fig. 10.
(3) To examine the toxicity of free DOX, DOX @ exos, DOX @ M1-exos-HA-gel and DOX @ M1-exos-OHA/Man gel to 4T1 cells, 4T1 cells were seeded on a 96-well plate, the above preparation groups were administered after attachment to the wall, incubated for 48h, and the cell viability was determined by the MTT method. Conclusion of this example referring to FIG. 11, DOX @ M1-exos-OHA/Man gel is shown in FIG. 11 to have significant cytotoxicity, IC, against 4T1 cells50The value was 455 ng/mL.
(4) And (3) investigating the degradation performance of the mannatide-hyaluronic acid hydrogel and the common cross-linked hyaluronic acid gel under different pH conditions, respectively soaking the two hydrogels in PBS solutions with different pH values, weighing and recording at different time points, and periodically replacing the PBS solutions. The mass loss rate (mass loss ratio) is calculated as follows: etam＝(mt-m0)/m0X 100. As shown in fig. 12, at pH 7.4, the mass loss rates of the mannatide-hyaluronic acid hydrogel and the common crosslinked hyaluronic acid gel are not significantly different, and at pH 6.5 and pH5.0, the mass loss of the mannatide-hyaluronic acid hydrogel is significantly increased, because the imine bond in the gel is degraded under acidic conditions, it is proved that the mannatide-hyaluronic acid hydrogel can be acid-sensitively degraded under the slightly acidic environment of tumor, and can rapidly release the mannatide and the exosomes entrapped in the hydrogel.
(5) In order to evaluate the capacity of the mannatide-hyaluronic acid hydrogel to release exosomes under in-vitro physiological conditions, the exosomes were labeled with Dir, and exosome release was investigated by using a transwell cell. The gel exosomes prepared in example 1 were placed in the upper chamber with 100mL of PBS buffer pH 7.4 as release medium. Releasing at 37 ℃ and 100r/min, measuring the fluorescence content of the exosome outside the cell at each time point by an enzyme-labeling instrument, and the conclusion of the example is shown in figure 13, as shown in figure 13, the mannatide-hyaluronic acid hydrogel has obvious acid-sensitive drug release and slow release characteristics, and the exosome is almost completely released within 7d when the pH is 5.0.
(6) To evaluate the in vivo tumor adhesion of the mannatide-hyaluronic acid hydrogel of the present invention, first, 2 schiff base-based self-assembled gels were synthesized in the reference: chitosan-glutaraldehyde hydrogel (CS-gel) and oxidized hyaluronic acid-chitosan hydrogel (OHA CS gel). The Dir modified hydrogel was further prepared with a CS-gel formulation of 3% wt chitosan and 3% wt glutaraldehyde, and an OHA/CS gel formulation of 3% wt chitosan and 6% wt oxidized hyaluronic acid. 4T1 cells (10)6/only) injected into mammary gland of BALB/c mice, tumor models were prepared, each hydrogel group was injected around the tumor after 7d, and distribution of the gel around the mouse tumor was observed using in vivo imaging of the mice at 0, 3, 7 d. OHA/Man gel, OHA/CS gel and CS gel are Schiff base type gels, and have certain acid-sensitive degradation injectability and fluidity. As shown in FIG. 14, after 7d, OHA/CS gel was slightly more concentrated in the tumor area than CS-gel, while OHA/Man gel could clearly adhere to the tumor area and achieve the targeted release of the tumor, presumably because mannatide had better affinity with mannose receptor highly expressed in the tumor area tissue.
Example 4: effect of exosome gel complexes on macrophage Activity
(1) In order to examine the influence of blank mannatide-hyaluronic acid hydrogel on the generation of NO by macrophages, the abdominal cavity of a mouse is flushed by RPMI1640 culture medium, the abdominal cavity macrophages are collected, nonadherent cells are washed out 6h after cell plating, the mouse is cultured for 24h after each preparation group is added (the concentration of mannan peptide is 200 mug/mL), and the generation amount of NO is determined by Griess reaction, please refer to FIG. 15 for the conclusion of the example, as shown in FIG. 15, blank gel may induce the abdominal cavity macrophages of the mouse to generate NO and enhance the activity of the macrophages.
(2) In order to investigate the influence of the blank mannatide-hyaluronic acid hydrogel on the phagocytic function of macrophages, SPF mice were injected subcutaneously with each preparation group for 7 days, injecting 2% chicken erythrocyte suspension 1mL into abdominal cavity of each mouse, killing the mouse by cervical dislocation after 30min, fixing on mouse plate, cutting abdominal wall skin at the center, injecting 2mL physiological saline into abdominal cavity, slightly rotating mouse plate for 1min, then sucking out 1mL of abdominal solution, dripping on 2 glass slides, placing in an enamel box filled with wet gauze, incubating at 37 deg.C for 30min, rinsing with normal saline, air drying, fixing with 1:1 acetone methanol solution, dyeing with Giemsa dye solution for 3min, rinsing with distilled water, air drying, observing with blank mannatide-hyaluronic acid hydrogel high power lens, recording the number of macrophages and phagocytosed chicken red blood cells, and calculating phagocytosis rate. Referring to fig. 16, the blank mannatide-hyaluronic acid hydrogel can modulate the immunity of the body by enhancing the phagocytic function of macrophages, as shown in fig. 16.
Example 5: evaluation of tumor accumulation in vivo by exosome-gel complex
4T1 cells (10)6/one) was injected into mammary gland of BALB/c mouse to prepare tumor model with tumor mass of 500mm3Thereafter, the preparation groups were injected peritumorally, the doxorubicin dose was 5mg/kg, the mice were sacrificed after 1, 2, 3, 5, 7, 14d, tumor tissues were taken, doxorubicin was extracted by homogenization, and the doxorubicin concentration was measured by HPLC, for the conclusion of this example, see FIG. 17. As shown in fig. 17, tumor doxorubicin concentration measurements showed that both DOX-loaded unpolarized exosomes (DOX @ exos) and M1 polarized exosomes (DOX @ M1-exos) increased accumulation of DOX in tumors compared to free DOX, however DOX @ M1-exos had greater tumor retention, confirming that M1 polarized exosomes had greater tumor targeting compared to unpolarized macrophage exosomes. Further, after DOX @ M1-exos is encapsulated in mannatide-hyaluronic acid hydrogel and common cross-linked hyaluronic acid gel, DOX @ M1-exos-HA gel HAs a certain DOX slow release effect but slow and incomplete release, while DOX @ M1-exos-OHA/Man gel HAs the strongest and most stable slow and controlled release effect and the most obvious tumor targeting property and the strongest tumor accumulation capacity in each preparation group.
Example 6: in vivo therapeutic experiments with exosome-gel complexes
4T1 cells (10)6/one) was injected into mammary gland of BALB/c mouse, tumor model was prepared, administration was started after 7d, each preparation group (DOX 5mg/kg) was injected once a week around tumor, and therapeutic evaluation was performedAnd (4) price. Mice were monitored for tumor volume and weight changes. After 14 days, blood was taken from orbital veins, BUN, CRE, AST, ALT indices were measured, mice were sacrificed, and heart, liver, kidney, and tumor tissues were sectioned for analysis. As shown in FIG. 18, the DOX @ M1-exos-OHA/Man gel group showed little progress in tumor growth and had a significant antitumor effect. As shown in FIGS. 20-22, DOX @ M1-exos-OHA/Man gel has stronger anti-tumor cell proliferation, anti-tumor angiogenesis and tumor killing effects than those of the free adriamycin group, and as shown in FIGS. 19 and 23-29, mice of the DOX @ M1-exos-OHA/Man gel group have no obvious weight reduction during treatment, no obvious toxicity to heart, liver and kidney and good safety.
Finally, it should be noted that the above mentioned embodiments are only preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, several modifications and equivalents may be made without departing from the technical solution of the present invention, and these modifications and equivalents should also be considered as the protection scope of the present invention.
Claims (10)
1. An immune enhancement type exosome hydrogel compound is characterized in that a drug-M1 type macrophage exosome is formed by encapsulating a drug by M1 type macrophage exosome, then a hydrogel is formed by chemical crosslinking of mannatide and hyaluronic acid, the drug-M1 type macrophage exosome is loaded in the hydrogel, and a hydrogel drug delivery system is formed.
2. The immune-enhancing exosome hydrogel complex according to claim 1, wherein the drug is selected from doxorubicin, paclitaxel, curcumin, ganoderic acid a, ginsenoside, quercetin, alkannin or houttuynin.
3. The immune-enhanced exosome hydrogel complex according to claim 1, wherein said chemical cross-linking is by oxidation of hyaluronic acid followed by reaction with mannatide in solution.
4. A method of preparing an immune-enhancing exosome hydrogel complex according to any one of claims 1-3, comprising the steps of:
s1: extracting and purifying M1 type macrophage exosome;
s2: preparing a medicament-M1 type macrophage exosome;
s3: preparation of mannan peptide-hyaluronic acid hydrogel of drug-M1 type macrophage exosome:
and (4) mixing the drug-M1 type macrophage exosome in the step (S2) with the mannan peptide solution, adding an oxidized hyaluronic acid solution, uniformly mixing, and reacting at room temperature to form the mannan peptide-hyaluronic acid hydrogel loading the drug-M1 type macrophage exosome.
5. The method for preparing an immune-enhancing exosome hydrogel complex according to claim 4, wherein the step S1 comprises inducing macrophage M1 to be polarized, extracting exosomes from macrophage culture solution polarized by M1, and finally obtaining the macrophage exosomes polarized by M1 by ultracentrifugation, namely the macrophage exosomes of M1 type.
6. The method for preparing an immune-enhancing exosome hydrogel complex according to claim 4, wherein in step S2, the drug-M1 type macrophage exosome is prepared by electroporation, and the mass ratio of the M1 type macrophage exosome (calculated as protein) to the drug is 1: 1-5, preferably 1: 2.
7. the method for preparing an immune-enhanced exosome hydrogel complex according to claim 4, wherein in step S3, hyaluronic acid is oxidized by sodium periodate to prepare oxidized hyaluronic acid, and the molecular weight of hyaluronic acid is 50-1000 kDa.
8. The method for preparing an immune-enhanced exosome hydrogel complex according to claim 4, wherein in the step S3, the concentration of the oxidized hyaluronic acid solution is 5-20% (W/V), the molecular weight of the mannatide is 40-90 kDa, the concentration of the mannatide solution is 5-20% (W/V), and the volume ratio of the mannatide solution to the oxidized hyaluronic acid solution is 1: 1-3, reacting at room temperature for 15s-2 min.
9. Use of the immuno-enhanced exosome hydrogel complex of any one of claims 1-3 for the preparation of an anti-tumor drug.
10. The use of claim 9, wherein the tumor comprises breast cancer, prostate cancer, bladder cancer, skin cancer, liver cancer, stomach cancer, brain cancer or ovarian cancer.
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