CN116966317A - Targeting drug and preparation method and application thereof - Google Patents

Targeting drug and preparation method and application thereof Download PDF

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Publication number
CN116966317A
CN116966317A CN202310766852.0A CN202310766852A CN116966317A CN 116966317 A CN116966317 A CN 116966317A CN 202310766852 A CN202310766852 A CN 202310766852A CN 116966317 A CN116966317 A CN 116966317A
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evs
microneedle
extracellular vesicles
targeted drug
drug
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李秋柏
曹玉林
陈智超
吴迪
胡萱
余娅丽
王珊
屈姣
杨汴蕾
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Tongji Medical College of Huazhong University of Science and Technology
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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    • A—HUMAN NECESSITIES
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    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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Abstract

The invention discloses a targeting drug, a preparation method and application thereof, wherein the targeting drug comprises extracellular vesicles and active ingredients, and the active ingredients are encapsulated in the extracellular vesicles; the extracellular vesicles are surface-modified with antibodies that specifically bind to antigens on the surface of the target cells. The targeted drug is not easy to gather at the lung and liver parts, and the proportion of the drug reaching the target part is high, so that the dosage can be greatly reduced under the same treatment effect. The targeted drug is embedded into the dissolvable substrate to prepare the microneedle patch, which can replace tail vein injection and avoid the risk of pulmonary embolism death caused by tail vein large-dose extracellular vesicle injection.

Description

Targeting drug and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a targeted drug and a preparation method and application thereof.
Background
Multiple Myeloma (MM) is the second largest tumor of the blood system that is currently incurable, and is mainly manifested by clonal proliferation of malignant plasma cells in Bone Marrow (BM) and associated organ damage. Also susceptible to complications of extramedullary lesions (Extramedullary disease, EMD) include extramedullary infiltration (extramedullary infiltration) and extramedullary plasmacytomas (extramedullary plasmacytoma, EMP). Current MM leading edge treatment strategies mainly include: CD38 monoclonal antibodies, proteasome inhibitors, histone deacetylase inhibitors, immunomodulators, anthracycline antitumor agents (liposomal doxorubicin), cyclic nonspecific agents (cyclophosphamide), hematopoietic Stem Cell Transplantation (HSCT), chimeric antigen receptor T cell immunotherapy (CAR-T), and the like. With the continuous advent of new drugs and the improvement of detection means, the diagnosis and treatment of MM are continuously improved and perfected, and nevertheless, most patients eventually progress to a recurrent refractory stage, and the antitumor efficiency thereof remains a great challenge.
In the current drug therapy setting, from small molecules to nucleic acids to proteins, drug design and efficacy have progressed over the last few decades, but a key problem remains to be solved: how can they be delivered most efficiently to target cells? The drug delivery system (drug delivery system, DDS) is utilized to enhance the accumulation of drugs in tumor tissues (target tumor cell strategy), so that the anti-cancer effect is improved, the side effect is reduced, and the method is a popular field of current anti-tumor treatment. Extracellular Vesicles (EVs) are natural nanoscale vectors containing proteins, lipids, nucleic acids secreted by cells. At present, extracellular vesicles have become a favorable carrier for anti-tumor treatment due to the advantages of low immunogenicity, good tissue compatibility (the barrier effect of EVs biological membranes avoids the influence of enzyme in body fluid on the degradation or activity of drugs), capability of overcoming natural barriers (such as blood brain barriers), long circulation time, natural targeting, improvement of water solubility, toxicity and the like of drugs such as chemistry, genes and the like.
The prior report proves that EVs are extremely easy to gather at the lung and liver parts after being injected into a tumor animal model by tail vein, the effective concentration of the EVs reaching the target part is greatly limited, and the model animal is easy to be killed by pulmonary embolism caused by the tail vein high-dose application of the EVs, so how to safely enter the blood circulation and effectively reach the tumor target tissue part becomes a difficult problem for the person skilled in the art.
Disclosure of Invention
The invention provides a targeting drug and a preparation method and application thereof, in order to solve the problem of low concentration of drug-carrying extracellular vesicles reaching a target site.
The technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a targeted drug comprising an extracellular vesicle and an active ingredient, the active ingredient being encapsulated within the extracellular vesicle; the extracellular vesicles are surface-modified with antibodies that specifically bind to antigens on the surface of the target cells.
The invention modifies the antibody capable of specifically binding the target cell surface antigen on the surface of the extracellular vesicles encapsulating the active ingredients, so that the cell quantity of the extracellular vesicles entering the multiple myeloma cells is obviously higher than that of the extracellular vesicles which are not modified, and the concentration of the drug-carrying extracellular vesicles at the target part is greatly improved.
Based on the technical scheme, the target cells are human multiple myeloma cells, and the active ingredients are medicines for treating the human multiple myeloma cells.
Based on the technical scheme, the antigen is CD38 antigen, and the antibody is CD38 polypeptide. The invention modifies CD38 polypeptide on the surface of the extracellular vesicle which encapsulates the active ingredient, which can effectively improve the active ingredient in the multiple myeloma cell.
Based on the above technical scheme, the amino acid sequence of CD38 polypeptide is ARGDYYGSNSLDYW, which is linked to the outer surface of extracellular vesicles by polyethylene glycol.
Based on the technical scheme, the molecular weight of the polyethylene glycol is 1500-3000, preferably 2000.
In a second aspect, the present invention provides a microneedle patch, which is prepared by using a dissolvable base material to prepare dissolvable microneedles, loading the targeted drugs into the microneedles, dissolving the microneedles in interstitial fluid and releasing the targeted drugs after the microneedles are inserted into the skin of a model animal.
The microneedle patch provided by the invention comprises: a microneedle array and the targeted drug described above; wherein: the microneedle array adopts a dissolvable substrate; the targeted drug is embedded within the dissolvable substrate of the microneedle array. In vivo animal experiments prove that the microneedle patch can more safely and effectively deliver the targeted drug to the tumor part than a tail vein, and meanwhile, compared with the delivery of unmodified extracellular vesicles, the microneedle patch can more effectively realize targeted accumulation of the drug at the tumor part, thereby enhancing the anticancer curative effect.
On the basis of the technical scheme, the dissolvable base material is gelatin.
In a third aspect, the present invention provides a method for preparing the targeted drug, which is characterized by comprising:
incubating the active ingredient with the extracellular vesicles to obtain extracellular vesicles encapsulated with the active ingredient;
and incubating the extracellular vesicles with the encapsulated active ingredients with phospholipid-polyethylene glycol-antibody to obtain the targeted drug.
Based on the technical proposal, the weight ratio of the extracellular vesicles encapsulating the active ingredients to the phospholipid-polyethylene glycol-antibody is 10:0.5-1.5.
In a fourth aspect, the present invention provides a method for preparing the microneedle patch described above, comprising:
dispersing a targeted drug in a solution of a dissolvable substrate to obtain a precursor solution;
filling the precursor solution into a microneedle mould, and drying in vacuum to form a microneedle array;
filling the microneedle mould with a solution capable of dissolving the substrate, and taking down the microneedle mould after drying to obtain the microneedle patch.
The micro-needle prepared by the method has complete appearance, no empty needle or broken needle, good skin penetration performance and good solubility, and can efficiently deliver targeted drugs.
Based on the technical scheme, the weight ratio of the soluble substrate to the targeted drug is 1000:1.0-5.0, preferably 1000:2.5.
compared with the prior art, the invention has the following beneficial effects: the targeted drug provided by the invention is not easy to gather at the lung and liver parts, and the proportion of the drug reaching the target part is high, so that the dosage can be greatly reduced under the same treatment effect. The targeted drug is embedded into the dissolvable substrate to make the microneedle patch, which can deliver the targeted drug to the tumor site more safely and effectively than the tail vein.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings that are required to be used in the embodiments of the present invention will be briefly described below. It is evident that the drawings described below are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a schematic representation of the construction of CD38 polypeptide modified drug-loaded extracellular vesicles.
FIG. 2 shows extracellular vesicle surface CD38 polypeptide expression as detected by flow cytometry; wherein, FIG. 2a shows the expression of CD38 polypeptide on the surface of unmodified, non-drug-loaded Extracellular Vesicles (EVs); FIG. 2b shows the expression of CD38 polypeptide on the surface of CD38 polypeptide modified non-drug loaded extracellular vesicles (hereinafter referred to as CD 38-EVs).
FIG. 3 shows the CD38-EVs surface CD38 expression observed by fluorescence microscopy; wherein, FIG. 3a indicates red fluorescent dye PKH-26 labeled EVs, FIG. 3b indicates green fluorescent dye FITC labeled anti-CD 38 polypeptide antibodies, and FIG. 3c is a merge fluorescent staining pattern of FIGS. 3a and 3 b.
FIG. 4 shows extracellular vesicles detected by flow cytometry entering multiple myeloma cells; wherein, fig. 4a and 4c indicate the amount of EVs and CD38-EVs entering the myeloma cell line RPMI8226 with high expression of CD38 antigen at 4h, 8h, 12h and the statistical graph thereof;
FIGS. 4b and 4d are graphs indicating the amount of EVs and CD38-EVs entering the myeloma cell line U266 at 4h, 8h, 12h, which is highly expressed by the CD38 antigen, and statistics thereof.
FIG. 5 shows the entry of extracellular vesicles into multiple myeloma cells as detected by laser confocal microscopy; wherein fig. 5a illustrates the case where EVs enter RPMI 8226; FIG. 5b shows the CD38-EVs entering the RPMI 8226; FIG. 5c shows the EVs entering U266; figure 5d shows the CD38-EVs entering U266.
FIG. 6 shows the effect of various extracellular vesicles detected by the cell proliferation CCK-8 assay on the proliferative activity of RPMI8226 and U266; wherein, figure 6a shows the proliferative activity effect of various extracellular vesicles on RPMI8226 cells; FIG. 6b shows the effect of various extracellular vesicles on the proliferative activity of U266 cells.
FIG. 7 shows the effect of various extracellular vesicles detected by EdU experiments on proliferation of RPMI8226 and U266; wherein fig. 7a and 7b are graphs showing the effect of the EdU experiment on RPMI8226 proliferation activity of different extracellular vesicles and their statistics; FIGS. 7c and 7d are graphs showing the effect of EdU experiments on the proliferation activity of U266 and their statistics for different extracellular vesicles.
FIG. 8 demonstrates the construction and therapeutic effects of microneedles carrying drug-loaded extracellular vesicles modified with a CD38 polypeptide; wherein, FIG. 8a shows a schematic of a drug-loaded extracellular vesicle modified with a CD38 polypeptide; FIG. 8b shows a schematic flow chart of a microneedle constructed with a drug-loaded extracellular vesicle modified with a CD38 polypeptide; FIG. 8c is a photograph of a model animal's subcutaneous tumor treated with a microneedle patch; FIG. 8d is a microneedle electron micrograph; FIG. 8e is a microneedle scanning confocal microscopy image; FIG. 8f is a Fluorescence Molecular Tomography (FMT) detection of tail vein injection, delivery of fluorescently labeled EVs by microneedle patches, and arrival of CD38-EVs at tumor target sites; FIG. 8g shows the effect of tail vein and microneedle delivery on tumor volume in mice for different modes of treatment, EV-Dox and CD 38-EV-Dox.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the essential aspects of the present invention will be further described in detail with reference to the following specific examples and the accompanying drawings. The examples are only for explaining the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available.
Example 1: preparation of targeted drugs
1. Acquisition of umbilical cord mesenchymal stem cell-derived extracellular vesicles
(1) Acquisition and culture of human umbilical cord mesenchymal stem cells
The umbilical cord donor was granted and the healthy maternal term (38-40 weeks gestation) fetal umbilical cord was collected. Under the aseptic condition, the umbilical cord is fully washed for 2-3 times by PBS (phosphate buffered saline) solution containing 100U/mL penicillin and 100U/mL streptomycin, and residual blood stains are removed; dividing the umbilical cord into 4-5cm umbilical cord segments, longitudinally separating each part, and carefully removing the arteriovenous; further separating the mixture, and shearing the mixture into tissue blocks with the size of 1-2 mm; the tissue pieces were planted in 10cm dishes and a small amount of DMEM/F-12 medium (DMEM/F-12 containing 10% FBS and 1% diabody) was added and placed in forward direction at 37℃with 5% CO 2 Adding 5mL of culture medium after 24h in a saturated humidity cell incubator, changing the liquid once every 3 days for about 10 days, removing umbilical cord tissue, changing the liquid completely, observing the growth condition of cells every other day, and using 0.2 when the cells are converged to 70% -80%Digesting with 5% trypsin/EDTA, centrifuging at 250g for 5min, and discarding supernatant to obtain human umbilical cord mesenchymal stem cells; resuspension was performed with complete medium and subculture was performed at a ratio of 1:3.
(2) Extraction of umbilical cord mesenchymal stem cell-derived extracellular vesicles
Culturing umbilical cord mesenchymal stem cells by adopting a DMEM/F12 culture medium for removing EVs in serum, taking 40 mL/tube of culture supernatant, adding 8 tubes, and obtaining EVs by adopting a differential centrifugation method: 750g×15min, collecting supernatant; 2000 g.times.20 min, the supernatant was taken into Beckman tubes, extracellular vesicle pellet was obtained at 4℃for 16000 g.times.1 h, extracellular vesicle pellet was resuspended with PBS buffer, and the aspirated fraction was used for protein quantification and the remainder was used for subsequent experiments.
(3) Quantification of extracellular vesicles
The BCA kit detects the content of EVs and protein standards are added to 96-well plates separately according to kit instructions, PBS being used to make up the total volume to 20 μl. Extracting EVs by the method, re-suspending the sucked part with PBS, centrifuging for 1h to obtain EVs precipitate, lysing the EVs precipitate by RIPA lysate for about 30min, centrifuging for 10min at 16000g at 4deg.C to obtain protein supernatant, and adding appropriate amount of protein supernatant into 96-well plate. 200 μLBCA working solution (prepared in advance, B: A=1:50) was added to each well of the 96-well plate, and absorbance was measured at 562nm after incubation at 37℃for 30 min.
2. Construction of doxorubicin-carrying extracellular vesicles (hereinafter referred to as EV-Dox)
1mg of doxorubicin hydrochloride was dissolved in 1.7242 mM LDMSO to prepare a stock solution with a concentration of 1mM, 30. Mu.g of doxorubicin hydrochloride and 250. Mu.g of EVs were co-incubated at 37℃for 1 hour, and then centrifuged at 16000g at 4℃for 1 hour, the doxorubicin hydrochloride which did not enter EVs in the supernatant was removed, and the obtained precipitate was EV-Dox, and dissolved with PBS for use.
Construction of drug-loaded extracellular vesicles modified with CD38 Polypeptides
(1) Synthesis of phospholipid-polyethylene glycol-CD 38
Weighing 100mg of phospholipid-polyethylene glycol-N-hydroxysuccinimide (DSPE-PEG-NHS, PEG molecular weight 2000) and dissolving in 3 mM LDMF, adding CD38 peptide (1.1 eq.) with an amino acid sequence of ARGDYYGSNSLDYW and triethylamine (3.0 eq.) to dissolve completely, and reacting for 12h at room temperature; the reaction solution is transferred to a dialysis bag (with a molecular weight cut-off of 2000 Da) and dialyzed in pure water for 24 hours, the dialysate is collected, and the product obtained by freeze drying is phospholipid-polyethylene glycol-CD 38 (DSPE-PEG-CD 38).
(2) Construction of CD38 polypeptide modified drug-free extracellular vesicles (hereinafter referred to as CD 38-EVs)
Mu.g of EVs was taken, 25. Mu.L of DSPE-PEG-CD38 solution at a concentration of 1. Mu.g/. Mu.L was added, the volume was set to 1mL with PBS, gently swirled with a gun head, and incubated at room temperature for 1 hour. And transferring the dispersion liquid into a special centrifuge tube, centrifuging for 60 minutes at 16000g and 4 ℃, sucking the upper layer solution, removing unbound DSPE-PEG-CD38, obtaining precipitate which is CD38-EVs, adding a precooled PBS solution into the precipitate for blowing and re-dissolving for later use, and detecting the expression of CD38 on the surface of the CD38-EVs by using a follow-up flow cytometry and a fluorescence microscope.
(3) Construction of CD38 polypeptide modified drug-loaded extracellular vesicles
Mu. gEV-Dox was taken, 25. Mu.L of DSPE-PEG-CD38 solution at a concentration of 1. Mu.g/. Mu.L was added, the volume was set to 1mL with PBS, gently swirled with a gun head, and incubated at room temperature for 1 hour. And then transferring the dispersion liquid into a special centrifuge tube, centrifuging for 60 minutes at the temperature of 16000g and 4 ℃, sucking the upper solution, removing unbound DSPE-PEG-CD38, obtaining a precipitate which is CD38 polypeptide modified doxorubicin-carrying extracellular vesicles (hereinafter referred to as CD 38-EV-Dox), adding precooled PBS solution into the precipitate for blowing and re-dissolving for later use, and detecting the killing effect of the CD38 modified drug-carrying vesicles on multiple myeloma cells and for preparing microneedles in the subsequent CCK-8 and EdU cell proliferation experiments.
Example 2: characterization and identification of CD38-EVs
1. Flow cytometry to detect CD38-EVs surface CD38 expression
After incubating CD38-EVs and EVs with FITC-labeled anti-CD 38 polypeptide antibodies for 20min at 4 ℃ in dark, 16000g is centrifuged for 1h, unbound antibodies are discarded, PBS is resuspended, and the ratio of FITC-positive expression extracellular vesicles is detected by a nanofluidic instrument.
2. Fluorescent microscope for observing CD38 expression on CD38-EVs surface
After incubating PKH-26 fluorescence-labeled CD38-EVs with FITC-labeled anti-CD 38 polypeptide antibody for 20min at 4 ℃ in dark condition, 16000g is centrifuged for 1h, unbound antibody is discarded, a small amount of PBS is used for resuspension and smear, and the fluorescent expression condition of extracellular vesicles is observed by a fluorescence microscope.
As shown in FIG. 2, the nano-flow meter detects that CD38-EVs expresses CD38 higher than EVs, and the positive rate is 31.6%.
As shown in FIG. 3, FIG. 3a shows the red fluorescent dye PKH-26 labeled EVs, FIG. 3b shows the green fluorescent dye FITC labeled anti-CD 38 polypeptide antibodies, FIG. 3c shows that after FIG. 3a and FIG. 3bMerge, the red fluorescence of PKH-26 overlaps with the green fluorescence of FITC, confirming the expression of CD38 polypeptide on the extracellular vesicle surface membrane, i.e., demonstrating the success of the construction of CD 38-EVs.
Example 3: in vitro targeting verification of CD38 polypeptide modified extracellular vesicles 1. Acquisition and culture of human myeloma cell line RPMI8226 and U266
Human multiple myeloma cell lines RPMI8226 and U266 were purchased from ATCC in the united states. NESTT 75cm in 15mL complete 1640 medium (containing 10% fetal bovine serum and 1% diabody) 2 In a culture flask, at 37deg.C, 5% CO 2 Culturing in a cell culture box. The cell morphology was observed daily with a phase contrast microscope and cultured until the cell abundance reached 75%, at which time the cell concentration was approximately 6X 10 5 Cell suspension was collected, supernatant was removed by low-speed centrifugation, cell pellet was collected, and medium was supplemented after re-suspension with 1mL of complete medium, pmi1640, according to 1:2, subculture was performed at a ratio of 2.
2. Flow cytometry for detecting CD38-EVs and cases of EVs entering multiple myeloma cells
The red fluorescence marked CD38-EVs and EVs are co-cultured with myeloma cell lines RPMI8226 and U266 expressing CD38 antigen for 4 hours, 8 hours and 12 hours, each group of cells are collected, the cells are centrifuged at 1200rpm for 5 minutes, the supernatant is discarded, the cells are washed by PBS for 1 time, finally, the cell sediment is resuspended by 200 mu LPBS, and the flow detection is carried out to observe the difference of the expression quantity of the cell fluorescence of each group.
3. Laser confocal observation of CD38-EVs and cases of EVs entering multiple myeloma cells
The green fluorescence labeled CD38-EVs and EVs were co-cultured with RPMI8226 for 8h, the cells of each group were collected, centrifuged at 1200rpm for 5min, the supernatant was discarded, the cells were washed 1 time with PBS, and finally the cell pellet was resuspended with 10. Mu.LPBS, smeared and DAPI-sealed, and the difference in the amount of extracellular vesicles entering the RPMI8226 cells was observed with confocal microscopy.
Red fluorescent-labeled CD38-EVs and EVs were co-cultured with U266 for 8h, each group of cells was collected, centrifuged at 1200rpm for 5min, the supernatant was discarded, the cells were washed 1 more times with PBS, and finally the cell pellet was resuspended with 10 μlpbs, smeared and DAPI-sealed, and confocal microscopy was performed to observe the difference in the amount of extracellular vesicles entering the U266 cells.
As shown in fig. 4, fig. 4a and 4c show that the amount of CD38-EVs entering RPMI8226 at 8h, 12h is significantly higher than EVs, the difference being statistically significant; FIGS. 4b and 4d show that CD38-EVs entered U266 cells at 4h, 8h, 12h in significantly higher amounts than EVs, and that the differences were statistically significant.
As shown in fig. 5, laser confocal microscopy measurements showed that CD38-EVs entered RPMI8226, U266 cells in higher amounts than EVs.
EXAMPLE 4 killing of multiple myeloma cells by CD38 polypeptide modified drug-loaded extracellular vesicles in vitro
CCK-8 cytotoxicity test to detect killing effect of various extracellular vesicles on RPMI8226 and U266
RPMI8226 and U266 were inoculated into 96-well plates at 2000 wells, different types of extracellular vesicles (EVs, CD38-EVs, EV-Dox, CD 38-EV-Dox) and the same volume of PBS were added as Controls (CTRL), 6 duplicate wells were set up per group, 10. Mu. LCCK-8 solution was added per well at 24h, 48h and 72h, and the plates were gently shaken to mix the liquids and incubated in a 37℃incubator. After 2h, the sample was taken out and the Optical Density (OD) at a wavelength of 450nm was read by an ELISA reader.
EdU experiments to examine the effects of various extracellular vesicles on proliferation of RPMI8226 and U266
RPMI8226 and U266 were combined to 2X 10 5 The amount of each well was inoculated into a 12-well plate, and different kinds of extracellular vesicles (EVs, CD38-EVs, EV-Dox, CD 38-E) were addedV-Dox) and the same volume of PBS as Control (CTRL), 3 duplicate wells were set per group, smeared after 24h labeling of each group of cells EdU, and then fixed with 4% paraformaldehyde for 30min; adding 50 mu LApolo staining reaction liquid into the fixed cells, and incubating for 30min by a decolorizing shaker at room temperature in the dark; after staining, 100. Mu.L of penetrant (PBS of 0.5% Triton X-100) was added to the mixture and incubated for 10 minutes in a destaining shaker, and the mixture was washed 1 time with PBS for 5 minutes; 100 mu L of 1XHOechst33342 reaction solution is added to each group of smears, PBS is clear after light shielding, room temperature and decoloration and shaking table incubation for 30 minutes, and the smears are observed on a fluorescence microscope.
As shown in FIG. 6, the effect of the different treatment groups, namely blank (CTRL), EVs (EV), CD38-EVs (CD 38-EV), EVs doxorubicin-loaded (EV-Dox), CD38-EVs doxorubicin-loaded (CD 38-EV-Dox), on the proliferation activity of RPMI8226, U266 at 24h, 48h and 72h was shown in the cell proliferation CCK-8 experiment. Compared with CTRL group, EV group and CD38-EVs group, the EV-Dox group and CD38-EV-Dox group have obvious inhibition effect on RPMI8226 and U266 proliferation, wherein the CD38-EV-Dox group has stronger inhibition effect on RPMI8226 cells in 24H, 48H and 72H than the EV-Dox group, and the difference has statistical significance; the CD38-EV-Dox group has stronger inhibition effect on U266 cells at 24h than the EV-Dox group, and the difference has statistical significance.
As shown in fig. 7, fig. 7a and 7b are graphs showing the effect of the EdU experiments on RPMI8226 cell proliferation activity and statistics thereof in different treatment groups, i.e., blank group (CTRL), EVs group (EV), CD38-EVs group (CD 38-EV), EVs doxorubicin-loaded group (EV-Dox), CD38-EVs doxorubicin-loaded group (CD 38-EV-Dox); fig. 7c and 7d are graphs showing the effect of the EdU experiment on the proliferation activity of U266 in different treatment groups and their statistics. Compared with CTRL group, EV group and CD38-EVs group, the EV-Dox group and CD38-EV-Dox group have obvious inhibition effect on RPMI8226 proliferation, wherein the CD38-EV-Dox group has stronger inhibition effect on RPMI8226 cells than the EV-Dox group, and the difference has statistical significance; the CD38-EV-Dox group has stronger inhibition effect on U266 than the EV-Dox group, and the difference has statistical significance. Example 5: preparation and characterization of microneedles carrying drug-loaded extracellular vesicles modified by CD38 polypeptide (hereinafter referred to as CD38-EV-Dox microneedles)
1. In this example, the soluble polymer was gelatin and the CD38-EV-Dox microneedle was prepared as follows:
dispersing CD38-EV-Dox in deionized water to prepare vesicle aqueous solution with the concentration of 1 g/L; weighing 1g of gelatin, adding 9mL of deionized water, and stirring at 60 ℃ to fully mix and re-dissolve the gelatin; at 40 ℃, the reconstituted gelatin solution and the vesicle aqueous solution are mixed according to the following 4:1, uniformly mixing the components according to the volume ratio to prepare uniform precursor solution; the microneedle mould is selected as follows: needle length 600m, bottom diameter 280 μm, needle tip distance 600 μm, base groove depth 2.5mm,15×15 array, size 11×11mm 2 The method comprises the steps of carrying out a first treatment on the surface of the Dripping 200 mu L of precursor solution into a microneedle mould, placing in a vacuum drying dish, vacuumizing to-0.06 MPa, and keeping for 30min; heating and concentrating for 1h in a baking oven at 40 ℃; dripping 500 mu L of re-dissolved gelatin solution into a concentrated mould to prepare a microneedle patch base; and (3) drying at 40 ℃ for 12 hours, and then gently separating the microneedle patch from the microneedle mould.
2. The microneedle patch prepared by the method is observed by a scanning electron microscope and a scanning confocal microscope.
On the basis of successful construction of the aCD38-EV-Dox in FIG. 8, the microneedle patch is prepared by a die method, and the operation flow is as shown in FIG. 8b, and vacuum pumping and drying separation are required. First adding a gelatin solution loaded with CD38-EV-Dox to the microneedle mould surface; then vacuumizing to remove bubbles, centrifuging the die, and pushing the gelatin solution carrying CD38-EV-Dox into the microneedle channels of the die; then adding a blank gelatin solution for centrifugation to form a base; finally, the gelatin microneedle patch was dried at 40 ℃ and separated from the mold.
Fig. 8d and 8e are overall views of the microneedle patch taken through a scanning electron microscope and a scanning confocal microscope, respectively. The microneedle patch was a 15 x 15 array with a total of 225 microneedles. The needle is conical and has uniform size. Each needle was about 600 μm in height, 280 μm in bottom diameter, and 600 μm in needle-to-needle spacing. FIG. 8d is a micro-needle Scanning Electron Microscope (SEM) image showing a uniform and defect-free micro-needle patch; fig. 8e is a fluorescent image of a microneedle made of DIR dye-labeled gelatin.
Example 6: in vivo targeting and therapeutic effects of CD38-EV-Dox micro-target model mouse subcutaneous tumor
1. Establishment of NOD/SCID immunodeficiency mouse subcutaneous tumor model
The experimental animals were SPF-grade NOD/SCID mice purchased from Fukang Biotechnology Co., ltd. Of Beijing, and raised in SPF-grade animal houses at the laboratory animal center of the university of science and technology, shangji medical college of China. All animal experiment processes accord with the regulations of the relevant regulations of the national Ministry of the people and the republic of China.
The animals of the 4 week old new mice were kept at the test center for 2 weeks. The mice were weighed and anesthetized with 1.25% avermectin 0.02mL/g of anesthetic for intraperitoneal anesthesia, and after anesthesia, the mice were prepared for skin preparation at the site to be molded, and dehairing was performed with an electric dehairing instrument and dehairing paste. Then, the skin of the right upper back of the mouse is rubbed by 75% alcohol disinfectant, and 100 mu L of a 1mL syringe filled with matrigel/RPMI 8226 cell mixture is injected into the skin of the right upper back, wherein the cell content of the syringe is 5 multiplied by 10 6 Is to prevent the pinhole from overflowing after the needle is drawn. After completing the subcutaneous injection, the mice were placed on a heat-insulating pad until they were revived. Mice were observed daily for mental and active status, and changes in body weight, tumor volume, etc. were recorded every 3-4 days.
NOD/SCID model mice grouping and in vivo targeting validation
At about 1 month after subcutaneous inoculation and molding, the tumor-bearing mice were randomly divided into 4 groups: the tail vein injection EVs group, the tail vein injection CD38-EVs group, the microneedle patch delivery EVs group and the microneedle patch delivery CD38-EVs group, the CD38-EVs and the EVs are all subjected to fluorescent labeling in advance, and the number of mice in each group is 3.
When each treatment group is treated for 1h and 3h, fluorescence detection is carried out on subcutaneous tumor parts of mice in each group by adopting a Fluorescence Molecular Tomography (FMT), imaging data obtained by an FMT/CT dual-mode imaging system are integrated, and a distribution image of EVs fluorophores on the subcutaneous tumor parts of the mice is constructed.
NOD/SCID model mice grouping and therapeutic treatment
At about 1 month after subcutaneous inoculation and molding, the tumor-bearing mice were randomly divided into 5 groups: blank, tail vein EV-Dox, tail vein CD38-EV-Dox, microneedle patch delivered CD38-EV-Dox, 4 mice per group. Each group of mice was treated 1 time a week for 4 times a total treatment, and after the treatment was completed, the mental and active states of the mice were dynamically observed, and the changes in body weight, tumor volume, and the like were recorded.
Fig. 8c shows a schematic diagram of a microneedle therapy, in which the microneedle can be firmly inserted into the skin of the back, the position of the microneedle can be firmly fixed by using skin adhesive, the movement of the mouse is unlimited after the therapy, and the microneedle is not easy to fall off.
FIG. 8f shows that FMT detects tail vein injection, microneedle patch delivers fluorescently labeled EVs and CD38-EVs to tumor target site, microneedle group delivers EVs to tumor site more effectively than tail vein injection group, wherein microneedle-carried CD38-EVs have stronger targeting delivery capability than microneedle-carried EVs.
FIG. 8g shows the effect of different treatment modes such as tail vein and microneedle delivery EV-Dox and CD38-EV-Dox on the tumor volume of mice, and the results show that the effect of microneedle patch delivery CD38-EV-Dox group, microneedle patch delivery EV-Dox group and tail vein injection CD38-EV-Dox group can effectively inhibit the tumor growth of mice, wherein the effect of microneedle patch delivery CD38-EV-Dox group has stronger tumor growth inhibition effect than microneedle patch delivery EV-Dox group.
The above examples are only for illustrating the detailed method of the present invention, and the present invention is not limited to the above detailed method, i.e., it does not mean that the present invention must be implemented depending on the above detailed method. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (10)

1. A targeted drug, characterized by: comprises an extracellular vesicle and an active ingredient, wherein the active ingredient is encapsulated in the extracellular vesicle; the extracellular vesicles are surface modified with antibodies that specifically bind to antigens on the surface of the target cells.
2. The targeted drug of claim 1, wherein: the target cells are human multiple myeloma cells.
3. The targeted drug of claim 1, wherein: the antigen is a CD38 antigen and the antibody is a CD38 polypeptide.
4. A targeted drug according to claim 3, characterized in that: the amino acid sequence of the CD38 polypeptide is ARGDYYGSNSLDYW, which is linked to the outer surface of the extracellular vesicle by polyethylene glycol.
5. The targeted drug of claim 1, wherein: the molecular weight of the polyethylene glycol is 1500-3000.
6. A microneedle patch, characterized by: comprising the following steps: a microneedle array and the targeted drug of any one of claims 1 to 5; wherein:
the microneedle array adopts a dissolvable substrate; the targeted drug is embedded within the dissolvable substrate of the microneedle array.
7. A method of preparing a targeted drug of any one of claims 1 to 5, comprising:
incubating the active ingredient with the extracellular vesicles to obtain extracellular vesicles encapsulated with the active ingredient;
and incubating the extracellular vesicles with the encapsulated active ingredients with phospholipid-polyethylene glycol-antibody to obtain the targeted drug.
8. The method of preparing a targeted drug of claim 7, wherein: the weight ratio of the extracellular vesicles packed with the active ingredients to the phospholipid-polyethylene glycol-antibody is 10:0.5-1.5.
9. A method of making the microneedle patch of claim 6, wherein: comprising the following steps:
dispersing a targeted drug in a solution of a dissolvable substrate to obtain a precursor solution;
filling the precursor solution into a microneedle mould, and drying in vacuum to form a microneedle array;
filling the microneedle mould with a solution capable of dissolving the substrate, and taking down the microneedle mould after drying to obtain the microneedle patch.
10. The method of making a microneedle patch of claim 9, wherein: the weight ratio of the soluble substrate to the targeting drug is 1000:1.0-5.0.
CN202310766852.0A 2023-06-27 2023-06-27 Targeting drug and preparation method and application thereof Pending CN116966317A (en)

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