CN113398282B - Delivery system of exosome biomimetic modified cerium oxide nanoparticles and application of exosome biomimetic modified cerium oxide nanoparticles in hair cells - Google Patents
Delivery system of exosome biomimetic modified cerium oxide nanoparticles and application of exosome biomimetic modified cerium oxide nanoparticles in hair cells Download PDFInfo
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Abstract
The invention belongs to the technical field of medical biology, and particularly relates to a delivery system of exosome biomimetic modified cerium oxide nanoparticles and application of the delivery system in hair cells. The components of the delivery system include: cerium oxide nanoparticles, neural stem cell exosomes, the neural stem cell exosomes comprising surface proteins for inhibiting apoptosis, the cerium oxide nanoparticles having peroxidase activity; the technical problem that the ROS of the cells cannot be efficiently eliminated in the prior art is solved.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a delivery system of exosome biomimetic modified cerium oxide nanoparticles and application of the delivery system in hair cells.
Background
Protection and repair of the capillaries is critical to the prevention and treatment of hearing loss. Numerous studies have shown that hearing loss is closely related to the increase of Reactive Oxygen Species (ROS), which are involved in the apoptotic process of hair cells. It has been shown that ROS interact with hair cell membrane phospholipids to produce lipid peroxides, some of which initiate hair cell apoptosis as mediators of acoustic nerve and hair cell apoptosis. When the ROS is increased under the conditions of noise exposure, ischemia or using ototoxic drugs and the like, and the ROS is beyond the scavenging capacity of a cochlear antioxidant system, a series of oxidative stress injuries such as hair cell apoptosis, death and the like are triggered. Defourny et al found that excessive noise exposure caused vasoconstriction, resulting in the production of large amounts of ROS, and that excessive amounts of ROS caused a dramatic increase in intracellular oxidation, resulting in damage to auditory hair cells. Taken together, ROS are the major cause of hair cell apoptosis.
With the continuous and deep understanding of ROS (reactive oxygen species) damaging cochlear hair cells, various antioxidant drugs are also sequentially applied to clinic, mainly comprising ROS scavengers, antioxidant enzyme inducers and the like. In recent years, with the rapid development of nanotechnology and the continuous penetration of nanotechnology into the medical field, nanomaterials with catalytic activity similar to natural enzymes, particularly with antioxidant enzyme activity such as superoxide dismutase (SOD) and Catalase (CAT), can simulate endogenous antioxidant systems to play a role in protecting cells, and open up a new way for removing cellular ROS. At present, a plurality of nano catalytic materials are found to be used as mimics of natural antioxidant enzymes, and are applied to cell protection, regulation and control of ROS levels in various cells, and relief of the burden of the antioxidant enzymes under pathological conditions.
Therefore, how to effectively eliminate ROS is urgent to research a substance or drug carrier or method for protecting hair cells and recovering hearing.
Disclosure of Invention
The application provides an exosome biomimetic modification cerium oxide nanoparticle and application thereof in hair cells of outer ears and inner ears, so as to solve the technical problem that cell ROS cannot be efficiently cleared in the prior art.
In a first aspect, the present application provides a delivery system for exosome biomimetic modified ceria nanoparticles, the components of the delivery system comprising: cerium oxide nanoparticles, neural stem cell exosomes, the neural stem cell exosomes comprising surface proteins for inhibiting apoptosis, the cerium oxide nanoparticles having peroxidase activity.
Optionally, the delivery system secretes cytokines for repair of hair cells.
A method for preparing a delivery system of an exosome biomimetic modified ceria nanoparticle, the method comprising the steps of:
obtaining the neural stem cell exosomes;
mixing the neural stem cell exosome and a cerium ion solution, incubating and purifying to obtain a purified solution;
and contacting and reacting the purified solution with a reducing agent to obtain the cerium oxide nanoparticle modified exosome, namely the delivery system.
Optionally, the reducing agent includes any one of ammonia water, urea and pure ammonia.
Optionally, the preparation method of the neural stem cell exosome comprises the following steps:
constructing and transfecting the neural stem cells by plasmids to obtain a cell strain modified by a targeting molecule;
and (3) after the cell strain is cultured, breaking cells, and purifying to obtain the neural stem cell exosome.
Optionally, the targeting molecule comprises a 666.
An exosome biomimetic modified cerium oxide nanoparticle delivery system is applied to external ear and internal ear hair cells.
Alternatively, the delivery system is used to eliminate ROS alone or in combination with receptor proteins on hair cells to inhibit apoptosis.
Optionally, the HSP70 protein expressed by the delivery system is combined with Toll-like receptor 4 (TLR 4) on hair cells to be used for inhibiting apoptosis.
Optionally, the delivery system is used to scavenge excess ROS to protect hair cells and/or the delivery system secretes multiple cytokines for modulating the cochlear microenvironment.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the components of the delivery system provided by the embodiments of the present application include: the nerve stem cell exosome comprises surface protein, can be combined with receptors in hair cells in a targeting mode and is used for inhibiting apoptosis, the cerium oxide nanoparticles have peroxidase activity and extremely strong anti-oxidation characteristics, excessive ROS is eliminated, the effect of protecting the hair cells is achieved, meanwhile, a delivery system can secrete cell secretion factors, a cochlea microenvironment is regulated and controlled, the hair cells are repaired and protected, the delivery system is used for cooperative protection and repair of oxidative damage of the hair cells, and hearing protection, recovery or partial recovery of auditory function can be effectively achieved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments of the invention
Technical solutions in the prior art will be briefly described below for the drawings required to be used in the embodiments or the description of the prior art, and it is obvious for a person skilled in the art that other drawings can be obtained according to these drawings without inventive labor.
FIG. 1 is a flow chart of construction of engineered neural stem cell stable transformants and separation and extraction of engineered exosomes and performance thereof;
FIG. 2 is a graph of the A666-NSC-Exo in situ modified cerium oxide nanoparticles performance;
fig. 3 is a graph of the effect of ANEN on hair cell protection and repair.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In a first aspect, the present application provides a delivery system for exosome biomimetic modified ceria nanoparticles, the components of the delivery system comprising: cerium oxide nanoparticles, neural stem cell exosomes, the neural stem cell exosomes comprising surface proteins for inhibiting apoptosis, the cerium oxide nanoparticles having peroxidase activity.
In the examples of the present application, cerium oxide (CeO)2) The nano particles have two mixed valence states of trivalent cerium and tetravalent cerium and oxygen vacancies, have the properties similar to natural SOD and CAT, show excellent catalytic performance, can effectively catalyze and eliminate ROS, and are widely proven antioxidant nano materials. The oxidation resistance of the cerium oxide nano-particles is utilized to remove excessive ROS, so that the effect of protecting hair cells is realized.
In an embodiment of the application, the neural stem cell exosome comprises a surface protein comprising at least one of BIRC1/NAIP, BIRC2/cIAP1, BIRC3/cIAP2 for inhibiting apoptosis.
As an alternative embodiment, the delivery system secretes cytokines for repair of hair cells.
A method for preparing a delivery system of an exosome biomimetic modified ceria nanoparticle, the method comprising the steps of:
obtaining the neural stem cell exosomes;
mixing the neural stem cell exosome and a cerium ion solution, incubating and purifying to obtain a purified solution;
and (3) contacting and reacting the purified solution with a reducing agent to obtain an exosome modified by the cerium oxide nanoparticles, namely the delivery system, namely the engineered neural stem cell exosome (ANEN) embedded with the antioxidant nanomaterial.
In the embodiment of the application, the neural stem cell exosome and the cerium ion solution are mixed and incubated, and the purification comprises the following steps: exosomes were associated with Ce (NO) in PBS (phosphate) aqueous solution3)3·6H2O (0.1 mM) was incubated at 37 ℃ for 16 h to allow Ce to react3+Adsorbing on the surface of exosome sufficiently, centrifuging the mixed solution at 4 deg.C and 120,000 rpm for 2 h, removing supernatant, and removing free Ce3+The bottom pellet was resuspended in PBS.
In embodiments of the present application, contacting and reacting the purified solution with a reducing agent comprises: rapidly adding 2 mL of ammonia water (28-30%) to react for 5 h at 37 ℃; carrying out ultra-separation at the speed of 120,000 rpm for 2 h at the temperature of 4 ℃, and washing for 3 times by using PBS to obtain the exosome modified in situ by the cerium oxide nanoparticles, (A666-NSC-Exo-CeO2Nanoparticles, ANEN), i.e. the delivery system. And the ANE with the capacity of targeting hair cells has the particle size of about 100 nm.
In the examples of the present application, cerium oxide nanoparticles synthesized on the ANE surface have natural antioxidant enzyme-like activity and a particle size of about 4 nm.
In the embodiment of the application, the ANE is administered by tympanogram injection, firstly, the exosome carries cerium oxide nanoparticles to penetrate through round window membrane to reach the inner ear, and then the hair cells are targeted under the action of a 666; secondly, cerium oxide nanoparticles on the surface of ANEN protect hair cells by scavenging excess ROS; in addition, HSP70 protein expressed by ANEN exosome can combine with Toll-like receptor 4 (TLR 4) on hair cells to react, so that apoptosis is inhibited, and the hair cells are protected; meanwhile, ANEN secretes various cytokines to regulate a cochlear microenvironment and promote the recovery of hair cell functions by activating autophagy to inhibit inflammatory reaction, thereby realizing multi-effect synergy of targeting, protection and repair of hair cells and reducing hearing loss. Namely, the ANEN can actively target hair cells and improve the bioavailability of the nano material; the cerium oxide can effectively remove excessive free radicals and protect hair cells, and meanwhile HSP70 on ANE is combined with TLR4 on hair cells to inhibit apoptosis and cooperatively protect hair cells.
As an alternative embodiment, the reducing agent includes any one of ammonia water, urea, and pure ammonia.
In the embodiment of the application, the reducing agent can be used for mildly synthesizing the cerium oxide nanoparticles on the surface of the exosome of the neural stem cell, and the reducing agent needs to have the following characteristics: the reaction conditions and the process are mild.
As an alternative embodiment, the preparation method of the neural stem cell exosome comprises the following steps:
constructing and transfecting the neural stem cells by plasmids to obtain a cell strain modified by a targeting molecule;
and (3) after the cell strain is cultured, breaking cells, and purifying to obtain the neural stem cell exosome.
In the examples of the present application, the construction and transfection of neural stem cells by plasmids include: construction of plasmid and establishment of stable cell line: the selected vector is a lentiviral vector PLVX, the expression sequences of mCherry and A666 are obtained by PCR amplification, Palm is connected to two ends of mCherry through primer design to construct a fusion protein Palm-mChery-A666, wherein Palm is a membrane localization peptide and can interact with a cytoplasmic membrane through a thioester bond. mCherry is used to indicate the expression and localization of the fusion protein. When the neural stem cells were grown to 50% density, the objective plasmid and helper plasmid (pLP 1, pLP2, VSVG) were transfected into the cells at a ratio of 3:1:1:1 by lipofectamine 3000, the medium was changed after 6 hours, the cell supernatant was collected after 48 hours, and centrifuged at 1,000 g for 10 min at 4 ℃ to remove the residual cell debris. Adding the cell supernatant into cells with the density of about 60%, simultaneously adding 2 mug/mL polybrene, culturing for 24 h, removing the culture medium, adding a fresh culture medium, and adding 4 mug/mL puromycin to screen positive cells to obtain an A666 modified engineering stable transformed cell strain.
In the examples of the present application, the cell line is cultured, and then the cells are disrupted and purified, including: isolation and extraction of targeting peptide modified neural stem cell exosomes (ANE). Carrying out expanded culture on the engineered stem cells, then carrying out continuous culture for 48 h by using serum without exosomes, collecting supernatant, centrifuging for 15 min at the temperature of 4 ℃ at 300 g, then centrifuging for 15 min at the temperature of 3,000 g to remove cell debris, centrifuging for 70 min at the temperature of 20,000 g to remove large vesicles, then centrifuging for 120 min at the temperature of 120,000 g to obtain exosome precipitate, washing by using PBS, and centrifuging for 120 min at the temperature of 120,000 g to obtain relatively pure exosomes.
In the embodiment of the application, the neural stem cell exosome (A666-NSC-Exo, ANE) is modified by a genetic engineering means to construct an engineered stable neural stem cell transformant, and the surface of the transformant is modified with hair cell targeting molecules.
As an alternative embodiment, the targeting molecule comprises a 666.
In the embodiment of the application, the targeting molecule can be specifically combined with the receptor protein on the receptor cell, and the targeting molecule is brought to the exosome modified in situ by the cerium oxide nanoparticle to act on the predetermined hair cell.
Example 1 construction of engineered neural Stem cell Stable transformants and isolation and extraction of engineered exosomes
Firstly, a sequence design of a Palm membrane targeting peptide is utilized to be connected with mCherry on a primer, an A666 targeting peptide (CLEPRWGFGWWLH) is connected with mCherry on an enzyme cutting mode, an expression vector is a PLVX vector for packaging lentivirus, a promoter is CMV (cytomegavirus), and a stable transfer cell line is constructed by utilizing a lentivirus infection mode (a-b in figure 1). After the stable transformation line is constructed, an A666 modified neural stem cell exosome (ANE) with the size of about 100 nm is separated and screened out by an ultracentrifugation method through expanded culture, the morphology, the particle size and the components of the A666 modified neural stem cell exosome (ANE) are analyzed by a TEM, an NTA, a flow cytometer and a WB, TEM results show that the A666 modified neural stem cell exosome (ANE) presents an obvious saucer-shaped structure, the particle size of the NTA modified neural stem cell exosome (ANE) is about 100 nm when the NTA is detected, the exosome is successfully extracted (c-d in figure 1), the Simple Wes result shows that the exosome contains specific Marker of CD9 and HSP70 exosomes, and the A666 expression is increased by combining the results of the flow cytometer, and the construction of the engineered exosome is successfully established (e-f in figure 1). Wherein, a in figure 1 is the construction of a stable cell line plasmid; b in FIG. 1 is a fluorescence imaging chart of the stable transformant; c in FIG. 1 is a TEM image of A666-NSC-Exo; FIG. 1, d is the characterization of the particle size potential of A666-NSC-Exo, indicating that the potential of A666-NSC-Exo in the range of 90-110nm fluctuates in the range of-45-30 mv, indicating that the particle size of A666-NSC-Exo has obvious uniformity, and e is the WB analysis of A666-NSC-Exo, wherein A666-NSC-Exo represents the neural stem cell exosome targeting hair cells, A666-NSC represents the neural stem cell targeting hair cells, TSG101 represents the tumor susceptibility gene 101 protein, GAPDH represents glyceraldehyde-3-phosphate dehydrogenase, and CD9 represents the specific marker genome of exosome; the A666-NSC-Exo group is proved to have better hair cell regeneration control advantage than the A666-NSC group; in FIG. 1, f is the expression of the targeting peptide in flow cytometry analysis, control represents a control group, A666-NSC-Exo represents a neural stem cell exosome group targeting hair cells, the Cherry expression level of the A666-NSC-Ex on the surface is obviously higher than that of the exosome group, and the Cherry is used as the basis for later-stage repairing and protecting the hair cells.
Example 2A 666-NSC-Exo in situ modified cerium oxide nanoparticles
Cerium oxide nanoparticles are synthesized in situ on a neural stem cell exosome, the cerium oxide nanoparticles with uniform particle size and moderate size are preliminarily synthesized by optimizing synthesis conditions, ANEN is constructed, TEM results are shown in a figure 2, and obvious cerium oxide nanoparticles are seen on the exosome. Meanwhile, WB analysis shows that the exosome modified with the cerium oxide nanoparticles contains Marker of neural stem cells (Sox 2, Bmil, Nestin), which indicates that the modified exosome does not influence the physiological activity (b in figure 2) and has little influence on the particle size (c in figure 2), and the exosome has good stability in aqueous solution (d in figure 2), wherein A666-NSC-Exo representsThe surface of the neural stem cell exosome of the hair cell targeting molecule is modified, and AMNE represents A666-NSC-Exo in-situ modified cerium oxide nanoparticles. In addition, by comparing the activities before and after in situ synthesis of cerium oxide nanoparticles (e-g in fig. 2), the performance of the neural stem cell exosome (ANE) group was weaker in all aspects than that of the modified ANEN, indicating that the modified ANEN has catalytic activities of SOD and CAT, and the ability to scavenge free radicals. ANEN was shown to have the ability to protect hair cells by hair cell in vitro experiments (h in fig. 2). A in FIG. 2 is a TEM representation of ANEN; b in fig. 2 is WB analysis of neural stem cell function maintenance in ANEN; c in FIG. 2 is a particle size potential characterization of ANEN; d in FIG. 2 is ANEN stability analysis; in FIG. 2, e is the SOD-like enzyme catalytic activity of different concentrations of ANEN and ANE; in FIG. 2 f is ANEN and ANE at different H2O2The ability to produce oxygen at concentrations that exert CAT-like enzyme activity; g in FIG. 2 is the different concentrations of ANEN and ANE versus O2-The ability to clear; in FIG. 2 h shows that ANEN has similar SOD and CAT enzyme activities and can eliminate O2-. The last graph in H of FIG. 2 illustrates the control group, H2O2Group, ANEN + H2O2Normalized total count rate of inactive oxygen free radicals in the group.
Example 3 protection and repair of hair cells by ANE, NEN and ANEN
NEN represents neural stem cell exosome of hair cells; saline represents normal Saline, PBS is a phosphate buffer control group, and ROS is a radical control group. A in FIG. 3 is the effect of ANEN on ROS-induced morphology of mouse inner ear hair cells; in FIG. 3, b is the effect of ANEN on the survival rate of hair cells outside the inner ear of mice caused by ROS, and c is the effect of AHEN (neural stem cell exosome targeting hair cells) prevention on hearing threshold of ROS-induced deafness mouse model.
As can be seen from a-b in fig. 3, the inner ear outer hair cell loss rate of the bottom loop (bottom) of the ANE + ROS group reaches about 10%, and the second loop (middle) also has about 5% hair cell loss, compared to the treatment group containing cerium oxide nanoparticles (NEN + ROS and ANEN + ROS) which has a significant hair cell damage prevention effect. C in FIG. 3 shows the effect of this prevention on hearing thresholds in ROS-induced deafness model in mice, where ABR thresholds in the ears of ROS group mice were significantly increased at various stimulation frequencies, thus indicating successful modeling. The ABR threshold values of the ANE + ROS, NEN + ROS and ANEN + ROS treatment groups are obviously different from those of the ROS group, so that the treatment system can reduce the influence of the ROS on the hearing threshold value of the mouse, and particularly the advantages of the ANEN + ROS group are more obvious.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A delivery system for an exosome biomimetic modified ceria nanoparticle, the delivery system comprising the components: cerium oxide nanoparticles, neural stem cell exosomes, the neural stem cell exosomes comprising surface proteins for inhibiting apoptosis, the cerium oxide nanoparticles having peroxidase activity;
the HSP70 protein expressed by the delivery system is combined and reacted with Toll-like receptor 4 (TLR 4) on hair cells and is used for inhibiting apoptosis;
a method of making the delivery system comprising the steps of:
obtaining the neural stem cell exosomes;
mixing the neural stem cell exosome and a cerium ion solution, incubating and purifying to obtain a purified solution;
contacting and reacting the purified solution with a reducing agent to obtain a cerium oxide nanoparticle modified exosome, namely the delivery system;
the reducing agent comprises any one of ammonia water, urea and pure ammonia.
2. The delivery system according to claim 1, wherein the delivery system secretes cytokines for repairing hair cells.
3. The delivery system according to claim 1, wherein the method for preparing the neural stem cell exosomes comprises the steps of:
constructing and transfecting the neural stem cells by plasmids to obtain a cell strain modified by a targeting molecule;
and (3) after the cell strain is cultured, breaking cells, and purifying to obtain the neural stem cell exosome.
4. The delivery system of claim 3, wherein the targeting molecule comprises A666.
5. Use of the delivery system of claim 1 in the manufacture of a medicament for scavenging ROS and protecting the external and internal ear hair cells.
6. The use according to claim 5, wherein the delivery system is used to eliminate ROS alone or in combination with receptor proteins on hair cells to inhibit apoptosis.
7. The use according to claim 5, wherein the delivery system is used to scavenge excess ROS protecting hair cells and/or the delivery system secretes multiple cytokines for modulating the cochlear microenvironment.
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