CN112190563A - Specific targeting nano vesicle based on chitosan, preparation method and application thereof - Google Patents
Specific targeting nano vesicle based on chitosan, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of nanoparticle anti-tumor, and particularly provides a chitosan-based specific targeting nano vesicle and a preparation method and application thereof. The chitosan-based specific targeting nanovesicle comprises an exosome having tumor cell specific targeting and a composite nanoparticle embedded within the exosome; the composite nano particles contain gold nanoclusters and sulfhydryl-polyethylene glycol-chitosan, and the sulfhydryl of the sulfhydryl-polyethylene glycol-chitosan is combined with the gold nanoclusters through coordination. The chitosan-based specific targeting nano vesicle provided by the invention has good specific targeting property, and can be directly or indirectly used as a medicament for treating cancer so as to specifically target and kill diseased cells.
Description
Technical Field
The invention belongs to the technical field of nano particle anti-cancer, and particularly relates to a chitosan-based specific targeting nano vesicle and a preparation method and application thereof.
Background
Since its emergence, cancer has become the leading cause of death in humans and has developed into a globally important public health problem, and how to effectively treat cancer has also become a major concern for humans. The existing cancer treatment methods mainly comprise surgical excision, chemotherapy, radiotherapy and the like, but the treatment methods generally have the defects that cancer cells are killed, meanwhile, the normal organism is greatly damaged, and the side effect is great, while the common drug treatment methods have the drug effect which is difficult to meet the treatment requirement. With the development of nanotechnology, the efficacy of the nanotechnology applied to medical treatment can be effectively improved, so that the application of nanoparticles to cancer treatment is considered, but most of traditional Nanoparticles (NPs) are rapidly phagocytized and eliminated by a reticuloendothelial system after entering a body, the number of nanoparticles capable of reaching tumor cells is reduced, the efficacy of the nanoparticles is reduced, and the research and the application of the nanoparticles in the field of cancer treatment are greatly limited.
Based on the above current situation, how to ensure that the nanoparticles are not phagocytized and cleared by the reticuloendothelial system after entering the body so as to ensure the effect of the nanotechnology in cancer treatment is very urgent and important.
Disclosure of Invention
The first purpose of the embodiments of the present invention is to provide a specific targeting nano vesicle based on chitosan, which aims to solve the problems of poor targeting effect, reduced drug-loading efficacy, etc. of the existing nanoparticles that are phagocytized by a reticuloendothelial system after entering into the body.
The technical scheme adopted by the embodiment of the invention is as follows:
a chitosan-based specifically targeted nanovesicle comprising an exosome having a tumor cell specific target and a composite nanoparticle embedded within the exosome;
the composite nano particles contain gold nanoclusters and sulfhydryl-polyethylene glycol-chitosan, and the sulfhydryl of the sulfhydryl-polyethylene glycol-chitosan is combined with the gold nanoclusters through coordination.
A second object of the embodiments of the present invention is to provide a method for preparing specific targeting nanovesicles based on chitosan, comprising the following steps:
providing gold nanocluster dispersion, sulfhydryl-polyethylene glycol-chitosan and exosome with tumor cell specific targeting;
adding the sulfydryl-polyethylene glycol-chitosan into the gold nanocluster dispersion liquid, and carrying out coordination combination on the sulfydryl and the gold nanoclusters to obtain composite nanoparticles;
embedding the composite nano-particles into the exosomes with the tumor cell specific targets by adopting an electroporation technology, and then incubating the exosomes with the tumor cell specific targets to recover the exosome membranes of the exosomes with the tumor cell specific targets, thereby obtaining the chitosan-based specific targeted nano-vesicles.
The third purpose of the embodiments of the present invention is to provide an application of the specific targeting nanovesicle based on chitosan, specifically, the specific targeting nanovesicle based on chitosan is used as a drug for treating cancer, or the specific targeting nanovesicle based on chitosan is used as a carrier of a drug for treating cancer.
The embodiment of the invention has the following beneficial effects:
compared with the prior art, the specific targeting nano vesicle based on chitosan provided by the embodiment of the invention comprises an exosome with tumor cell specific targeting and a composite nano particle embedded in the exosome with tumor cell specific targeting, when the specific targeting nano vesicle enters into a body, a reticuloendothelial system has no specific phagocytic reaction on the exosome, so that the composite nano particle embedded in the specific targeting nano vesicle can effectively penetrate through the reticuloendothelial system and is not phagocytized by the reticuloendothelial system, thereby ensuring that the amount of the composite nano particle reaching tumor cells can not be reduced, and as the composite nano particle contains chitosan modified by polyethylene glycol, on one hand, the composite nano particle is effectively conveyed to the surface of the tumor cells to activate lymphocytes with immune function in the body, and simultaneously, the activated lymphocytes with immune function can distinguish normal cells from cancer cells, thereby killing cancer cells, on the other hand, the chitosan modified by the polyethylene glycol has enzyme response, and is slowly released under the enzyme response to prolong the action time on the tumor cells, thereby achieving the effect of dual improvement of drug effect, on the other hand, the slowly released chitosan can inhibit the generation of tumor vascular endothelial cells, so that cancer cannot infiltrate or transfer to surrounding tissues, thereby achieving the purpose of treating early cancer, simultaneously effectively inhibiting the generation of cancer toxin, and also reducing adverse reactions generated in the radiotherapy and chemotherapy processes of cancer patients, in addition, the chitosan has the characteristics of biodegradability and biocompatibility, so that the chitosan can not have other side effects on human bodies, and under the mutual synergistic effect of the characteristics of the exosome, the composite nano-particles and the chitosan loaded on the composite nano-particles, the targeting property and the drug effect of the nano-particles can be improved, the nano vesicle has good specific targeting property, and can be directly or indirectly used as a medicament for treating cancer.
The preparation method of the chitosan-based purposeful targeting nano vesicle has the characteristics of simple production process, stable product performance, suitability for mass production and the like, and the obtained nano vesicle has good targeting property, good drug effect and good drug loading function.
The chitosan-based specific targeting nano vesicle provided by the invention is used as a cancer treatment drug or a cancer treatment drug carrier, has specific targeting property, can ensure the drug effect of the cancer treatment drug, and has no toxic or side effect on a human body.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a transmission electron micrograph of composite nanoparticles provided in example 1;
fig. 2 is a transmission electron microscope image of chitosan-based specific targeting nanovesicles provided in example 1;
FIG. 3 is a schematic diagram of the targeting effect of the chitosan-based specific targeting nanovesicles of example 1 before and after injection into tumor-bearing mice;
FIG. 4 is a photograph of nuclear magnetic images of a tumor-bearing mouse before and after injection of chitosan-based specifically targeted nanovesicles of example 1;
fig. 5 is a therapeutic curve of the composite nanoparticle, chitosan-based specific targeting nanovesicles and PBS for tumor-bearing mice in example 1;
fig. 6 is a body weight change curve of the composite nanoparticle, chitosan-based specific targeting nanovesicles, and PBS tumor-bearing mice of example 1 after treatment.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below 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.
The noun explains:
MMNPs @ Au: the superparamagnetic nano flower-shaped ferroferric oxide nano particles are loaded with gold nanoclusters or the gold nanoclusters are loaded with superparamagnetic nano flower-shaped ferroferric oxide nano particles;
MMNPs-DA: dopamine hydrochloride modified superparamagnetic flower-like ferroferric oxide nanoparticles;
Fe3O4@ Au-PEG-CTS: the superparamagnetic nano flower-shaped ferroferric oxide nano particles load gold nanoclusters, and sulfydryl in sulfydryl-polyethylene glycol-chitosan is coordinated and combined with the gold nanoclusters to obtain composite nano particles;
Exo-Fe3O4@ Au-PEG-CTS: chitosan-based specific targeting nanoparticle obtained by embedding composite nanoparticle in exosomeRice vesicles.
The application relates to three aspects of invention schemes, wherein the first invention scheme is to provide a preparation method of specific targeting nano vesicles based on chitosan.
The preparation method of the specific targeting nano vesicle based on chitosan comprises the following steps:
(A) providing gold nanoclusters and thiol-polyethylene glycol-chitosan;
(B) preparing gold nanoclusters into gold nanocluster dispersion liquid, then adding sulfydryl-polyethylene glycol-chitosan into the gold nanocluster dispersion liquid, and enabling sulfydryl on the sulfydryl-polyethylene glycol-chitosan to be coordinated with the gold nanoclusters to form a strong coordination chemical bond, so that the composite nanoparticles containing the gold nanoclusters and sulfydryl-polyethylene glycol-chitosan are obtained;
(C) providing exosomes with tumor cell specific targeting;
(D) and (C) embedding the composite nano particles obtained in the step (B) into an exosome with tumor cell specific targeting by adopting an electroporation technology, and then incubating the exosome to recover an exosome membrane of the exosome, thereby obtaining the chitosan-based specific targeting nanovesicle.
In the step (a), the gold nanoclusters may be gold nanoclusters having a purity of 99.99% or more, or may be gold nanoclusters supported on the surface of the up-conversion nanomaterial.
The up-conversion nano material is selected from any one of superparamagnetic nano flower-shaped ferroferric oxide nano particles and up-conversion luminescent nano materials.
When the up-conversion nano material is superparamagnetic nano flower-shaped ferroferric oxide particles, on one hand, the agglomeration of gold nanoclusters can be effectively inhibited, the dispersion uniformity of the gold nanoclusters is improved, and further the loading capacity of the mercapto-polyethylene glycol-chitosan on the surfaces of the gold nanoclusters is improved; on the other hand, the nano-vesicle has guidance performance and can play a role in guiding, the composite nano-particles containing the nano-vesicle have a very high enrichment effect in a targeting area under the induction of an external magnetic field, and the efficiency of targeting the tumor cells by the nano-vesicle can be effectively improved by combining with exosomes having tumor cell specificity targeting; on the other hand, the chitosan liposome has a multi-angle structure, can accelerate the damage to pathological cells under the action of an alternating magnetic field, and can greatly improve the treatment effect on tumors by combining with the chitosan which can slowly release and activate immune cells.
When the upconversion nanomaterial is an upconversion luminescent nanomaterial, the upconversion nanomaterial can be a fluorescent nanomaterial so as to facilitate marking and observation, and certainly, the upconversion nanomaterial is not limited to a fluorescent nanomaterial and can also be other upconversion luminescent nanomaterials.
When the gold nanoclusters are loaded on superparamagnetic flower-shaped ferroferric oxide nanoparticles, the gold nanoclusters can be prepared by the following method:
(A1) and adding ferric chloride (FeCl)3) Dissolving the mixture in a mixed solution of Ethylene Glycol (EG) and diethylene glycol, uniformly mixing, then adding polyvinylpyrrolidone (PVP) in a non-reaction atmosphere, reacting at 120-130 ℃ for 0.5-1.5 h, after the reaction is finished, adding sodium acetate (NaAc), uniformly mixing until the sodium acetate is completely dissolved, transferring the mixture into a reaction kettle to react for 10-20 h at 180-250 ℃, after the reaction is finished, alternately washing the product three times by adopting ultrapure water and absolute ethyl alcohol, performing magnetic separation and purification, adding ultrapure water, and uniformly mixing to obtain a superparamagnetic nano flower-shaped ferroferric oxide (MMNPs) dispersion liquid;
(A2) and (D) adding dopamine hydrochloride into the MMNPs dispersion liquid obtained in the step (A1), uniformly mixing, sequentially adding a gold seed solution (the gold seed solution is negatively charged) and a proper amount of aging liquid, and mixing to obtain the MMNPs @ Au dispersion liquid.
In the step (A1), the volume ratio of ethylene glycol to diethylene glycol is (1-5): (15-19); FeCl3And the ratio of the feeding amount of the sodium acetate to the feeding amount of the polyvinylpyrrolidone is (2-15) mmol: (50-100) mmol: (2-10) g. In the reaction process, polyvinylpyrrolidone forms a template in a mixed solution of ethylene glycol and diethylene glycol, so that formation of superparamagnetic nano flower-like ferroferric oxide nanoparticles is promoted.
The specific process of the step (a2) is as follows:
placing the MMNPs dispersion liquid obtained in the step (A1) in a centrifuge tube, separating by using an external magnetic field, removing supernatant, redissolving in 5-10 mL of tetrahydrofuran, performing ultrasonic dispersion, adding dopamine hydrochloride (DA) into the mixture, and performing ultrasonic treatment for 1h to obtain a mixture;
placing the mixture in a shaking table (speed: 100rpm) at room temperature, reacting for 12h, performing magnetic separation to obtain MMNPs-DA, and dispersing in water to obtain MMNPs-DA dispersion liquid;
adding the gold seed solution into the MMNPs-DA dispersion liquid, placing the MMNPs-DA dispersion liquid on a shaking table, shaking the mixture at room temperature, reacting for 10-12 h, carrying out magnetic separation and purification, washing off most DA, and then re-dissolving the DA in 5-10 mL of deionized water to obtain MMNPs @ Au seeds; and finally, adding MMNPs @ Au seeds into the aging solution, dropwise adding a formaldehyde solution (the volume ratio of formaldehyde to deionized water is 1:1), and reacting for 30 min.
The gold seeds in the gold seed solution are selected from one of gold nanoparticles with the particle size of 2 nm-5 nm or colloidal gold with the particle size of 4 nm-20 nm.
The aging solution is HAuCl4And potassium carbonate (K)2CO3) The mixed solution of (1), which is obtained by the following method: 1-3 mL HAuCl with a concentration of 20-30 mM4Adding the mixture into 100-150 mL of 2.5% (mg/mL) potassium carbonate solution, and uniformly mixing.
The feeding proportion of the dopamine hydrochloride and superparamagnetic flower-shaped ferroferric oxide nanoparticles is (10-40) mg: (0.67-5) mmol; the feeding proportion of the gold seeds and the superparamagnetic nano flower-shaped ferroferric oxide nanoparticles is (0.4-3.6) mg: (0.67-5) mmol; HAuCl4And the superparamagnetic nano flower-shaped ferroferric oxide nano particles are added according to the material ratio of (0.0025-0.0075): (0.67-5).
The concentration of the gold seed solution is (4-12) mg/mL, and the HAuCl4The concentration of the solution is (20-30) mM. By adopting a seed synthesis method, gold seeds adsorbed on the surfaces of the superparamagnetic flower-shaped ferroferric oxide nanoparticles grow uniformly and firmly on the surfaces of the superparamagnetic flower-shaped ferroferric oxide nanoparticles, and the obtained gold nanoclusters have a flower-shaped structure, so that the damage effect on pathological cells is improved.
In the step (B), the mass ratio of the gold nanoclusters to the sulfhydryl-polyethylene glycol-chitosan is (1-2): (8-9), under the feeding proportion, the mass content of the obtained composite nano particles, namely the sulfydryl-polyethylene glycol-chitosan, can reach more than 80%, so that the drug effect can be favorably exerted. In some preferred embodiments, the thiol-polyethylene glycol-chitosan is present in an amount of greater than 90% by weight.
The sulfydryl of the sulfydryl-polyethylene glycol-chitosan can be coordinated with the gold nanocluster to form a strong coordination chemical bond, so that the chitosan can be effectively carried, the chitosan can be delivered to a diseased cell through the composite nanoparticles, the steric hindrance of the polyethylene glycol is small, more chitosan can be carried, and the amount of the chitosan delivered to the surface of the diseased cell is increased. Preferably, the molecular weight of the sulfhydryl-polyethylene glycol-chitosan is 2000-5000, such as 2500, 3000, 3500, 4000, 4500 and the like; the gold nanocluster has a particle size of 5nm to 30nm, such as 5nm, 8nm, 10nm, 12nm, 15nm, 18nm, 20nm, 25nm, 30nm, etc.
The sulfhydryl-polyethylene glycol-chitosan of the invention can be obtained by the following method:
(B1) dispersing sulfhydryl-polyethylene glycol-carboxyl (SH-PEG-COOH) in 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, and adding EDC/NHS to activate SH-PEG-COOH carboxyl;
(B2) and (B1) adding Chitosan (CTS) into the activated SH-PEG-COOH, wherein the feeding molar ratio of the SH-PEG-COOH to the CTS is 1: 10-20, reacting the activated carboxyl with amino on chitosan to obtain PEG CTS, namely: thiol-polyethylene glycol-chitosan (SH-PEG-CTS).
In the step (C), the exosome having a tumor cell specific target is selected from at least one of cancer cell exosome, immune cell exosome, tumor stem cell exosome, bone marrow cell exosome, umbilical cord mesenchymal stem cell exosome. These exosomes have specific targeting on tumor cells, can specifically target diseased cells after entering a patient, have extremely strong targeting effect, and reduce the accidental injury to the patient caused by targeting of the drug by means of external conditions.
In step (C), exosomes having tumor cell-specific targeting can be obtained by the following method:
(C1) centrifuging the collected cancer cell suspension or immune cell suspension or tumor stem cell suspension or bone marrow cell suspension or umbilical cord mesenchymal stem cell suspension (3500rpm) at the temperature of 4 ℃, removing cell debris or apoptotic bodies, taking supernatant, continuously centrifuging at the temperature of 4 ℃ (110000g, 35min) for repeating the purification process, centrifuging (110000g, 1h), and precipitating into pellets;
(C2) suspending the pellets obtained in the step (C1) in 200 μ L Phosphate Buffered Saline (PBS) at 4 ℃, and filtering the suspension through a microporous filter membrane with a pore size of 0.22 μm to remove bacteria and residual cell membrane fragments to obtain exosomes;
(C3) and (C) adding the exosome obtained in the step (C2) into a sterile phosphate buffer solution for dilution at the temperature of 4 ℃, and then storing at the temperature of 80 ℃ below zero for later use.
In step (D), the electroporation embedding process is as follows:
(D1) mixing 100-150 mu L of exosome provided in the step (C), 15-20 mu L of composite nano-particles obtained in the step (B) and 85-90 mu L of electroporation buffer solution (total reaction volume: 200-260 mu L), and placing the mixture in an electroporation test tube of 0.2 cm;
(D2) an electroporation treatment was carried out using an electroporation system (Bio-Rad Gene plus Xcell), specifically, electroporation conditions were set at 250V and 100. mu.F; in the electroporation process, an exosome membrane of an exosome is perforated, and composite nanoparticles are gradually loaded into the exosome;
(D3) incubating the product obtained in step (D2) at 37 ℃ for 25min to restore the exosome membrane of the exosome;
(D4) by using OptiPrepTMDensity gradient centrifugation, separating the incubated mixture of step (D3), i.e., using 15-60% OptiPrepTMAnd centrifuging the density gradient centrifugate to remove the composite nano particles to obtain the specific targeting nano vesicle.
If tracing is needed, grafting of a tracing substance, such as sulfonated Cy5.5-N-hydroxysuccinimide ester (NHS-Cy5.5) and other fluorescent markers, can be performed on the obtained specific targeting nano-vesicles after the step (D4), so that traceable specific targeting nano-vesicles are obtained.
Based on the first invention scheme, the second invention scheme of the application is to provide a chitosan-based specific targeting nanovesicle.
The specific targeting nano vesicle based on chitosan comprises exosomes and composite nanoparticles embedded in the exosomes, wherein the exosomes are selected from cancer cell exosomes, immune cell exosomes, tumor stem cell exosomes, bone marrow cell exosomes and umbilical cord mesenchymal stem cell exosomes, and the exosomes have tumor cell specific targeting property; the composite nano particles contain gold nanoclusters and sulfhydryl-polyethylene glycol-chitosan, and the sulfhydryl of the sulfhydryl-polyethylene glycol-chitosan is combined with the gold nanoclusters through coordination. The composite nano-particles are prepared by the preparation method.
In the specific targeting nano vesicle based on chitosan, the exosome is an exosome with specific targeting on tumor cells, and can carry the composite nano particles to enter the body of a patient without being phagocytized or eliminated by a reticuloendothelial system as an invader, so that the quantity of the composite nano particles reaching the tumor cells can not be reduced, and the composite nano particles carry chitosan modified by polyethylene glycol, so that the chitosan has a slow release effect under the action of the polyethylene glycol, and the drug effect is favorably prolonged.
Therefore, the third invention scheme of the present application is to provide the application of the chitosan-based specific targeting nanovesicle, wherein the application comprises using the chitosan-based specific targeting nanovesicle as a drug for treating cancer or a carrier of the drug for treating cancer.
In order to better explain the technical solution of the present application, the following is further explained by several embodiments.
Example 1
A preparation method of specific targeting nano vesicles based on chitosan comprises the following steps:
s11, preparing gold nanoclusters:
a1, adding 15mmol of FeCl3Dissolving in a mixed solution of ethylene glycol and diethylene glycol (the volume ratio of the ethylene glycol to the diethylene glycol is 1:15), uniformly mixing, then adding 10g of PVP in a nitrogen atmosphere, reacting for 1.0h at 130 ℃, after the reaction is finished, adding 50mmol of sodium acetate, uniformly mixing until the sodium acetate is completely dissolved, transferring into a polytetrafluoroethylene reaction kettle for reacting for 15h at 200 ℃, after the reaction is finished, alternately washing the product for three times by adopting ultrapure water and absolute ethyl alcohol, carrying out magnetic separation and purification, adding ultrapure water, and uniformly mixing to obtain an MMNPs dispersion liquid;
a2, placing the MMNPs dispersion liquid obtained in the step a1 into a centrifuge tube, separating by an external magnetic field, removing supernatant, redissolving in 10mL tetrahydrofuran, ultrasonically dispersing, adding dopamine hydrochloride into the mixture, and ultrasonically treating for 1h to obtain a mixture;
a3, placing the mixture in a shaking table (speed is set as 100rpm) at room temperature, reacting for 12h, carrying out magnetic separation to obtain MMNPs-DA, and dispersing in water to obtain MMNPs-DA dispersion liquid;
a4, adding 10mL of gold seed solution into the MMNPs-DA dispersion solution, placing the mixture on a shaking table, shaking the mixture at room temperature, reacting for 10 hours, carrying out magnetic separation and purification, washing most of DA off, and then re-dissolving the DA in 10mL of deionized water to obtain the MMNPs @ Au seed solution;
5, adding 10mL of aging solution into the MMNPs @ Au seed solution, dropwise adding a formaldehyde solution (the volume ratio of formaldehyde to deionized water is 1:1), and reacting for 30min to obtain the gold nanocluster dispersion.
S12, preparing composite nano particles:
b1, mixing the gold nanocluster and the sulfhydryl-polyethylene glycol-chitosan according to the mass ratio of 1: 8, adding mercapto-polyethylene glycol-chitosan with the relative molecular weight of 2000 into the gold nanocluster dispersion obtained from a5, and rotating on an XH-1T type multi-tube adjustable rotary mixer for 25min at the rotating speed of 25 rpm to obtain the composite nanoparticle dispersion.
S13, preparation of the specific targeting nano vesicle based on chitosan:
c1, centrifuging the collected bone marrow cell suspension at 4 ℃ (3500rpm), removing cell debris or apoptotic bodies, taking supernatant, continuing to centrifuge at 4 ℃ (110000g, 35min), repeating the purification process, centrifuging (110000g, 1h), and precipitating into small balls;
c2, suspending the pellet obtained in the step c1 in 200 mu L PBS at the temperature of 4 ℃, and filtering the pellet through a microporous filter membrane with the pore diameter of 0.22 mu m to remove bacteria and residual cell membrane fragments to obtain an exosome;
c3, adding the exosome obtained in the step c2 into a sterile phosphate buffer solution at 4 ℃, diluting, and then storing at-80 ℃;
c4, 100. mu.L of exosome (2.4X 10) obtained in step c39Individual particles/mL, protein content 2.4 μ g) with 20 μ L of the composite nanoparticle dispersion obtained in step S12 and 80 μ L of electroporation buffer (total reaction volume: 200 μ L) were mixed in a 0.2cm electroporation cuvette;
c5, electroporation treatment using electroporation system (Bio-Rad Gene plus Xcell) at 250V and 100. mu.F;
c6, incubating the product obtained in the step c5 at 37 ℃ for 25min so as to recover the exosome membrane of the exosome;
c7, OptiPrep with 50%TMCentrifuging the density gradient centrifugate to remove redundant composite nano particles in the step c6 to obtain specific targeting nano vesicles based on chitosan;
and c8, performing NHS-Cy5.5 grafting on the obtained specific targeting nano vesicle based on the chitosan, thereby obtaining the tracing specific targeting nano vesicle.
And (3) performance testing:
1. topography observation
The composite nanoparticles obtained in step S12 of example 1 and the chitosan-based specific targeting nanovesicles obtained in step S13 were observed by transmission electron microscopy, and the results are shown in fig. 1 and 2, respectively.
As can be seen from fig. 1, the obtained composite nanoparticle has a core-shell structure, wherein the core material is gold nanocluster, and the shell material is thiol-polyethylene glycol-chitosan, which is basically formed by coating thiol-polyethylene glycol-chitosan on the surface of gold nanocluster to form a monodisperse material.
As can be seen from fig. 2, the structure of the vesicle is spherical, wherein the composite nanoparticle is embedded in the bone marrow exosome, and the particle size of the vesicle is about 800 nm.
2. Targeted detection
The obtained specific targeting nano vesicles based on chitosan are injected into a tumor-bearing mouse body, and the obtained specific targeting nano vesicles based on chitosan and the tumor-bearing mouse which is not injected with the specific targeting nano vesicles based on chitosan are used as blank control groups, and the targeting condition of the specific targeting nano vesicles based on chitosan is observed by adopting nuclear magnetic imaging, and the specific results are shown in fig. 3 and fig. 4.
As can be seen from FIGS. 3 and 4, the chitosan-based specific targeting nanovesicles have very strong specific targeting property after being injected into tumor-bearing mice, and can be rapidly aggregated on the surface of diseased cells.
3. Tumor treatment efficacy test
Three tumor-bearing mice with consistent physiological conditions are taken and numbered, and chitosan-based specific targeting nano vesicles (Exo-Fe)3O4@ Au-PEG-CTS), PBS, and composite nanoparticles (Fe)3O4@ Au-PEG-CTS) were injected into tumor-bearing mice, and the changes in tumors were observed in the tumor-bearing mice, as shown in FIGS. 5 and 6.
As can be seen from fig. 5, the specific targeting nanovesicle based on chitosan can significantly inhibit the tumor volume in the tumor-bearing mouse, the tumor volume hardly grows up within 16 days, while the composite nanoparticles have a certain inhibitory effect on the tumor volume in the tumor-bearing mouse, but are not significant, and the tumor volume gradually increases within 16 days.
From fig. 6, after the chitosan-based specific targeting nano-vesicles and the composite nanoparticles are injected into tumor-bearing mice, the weights of the tumor-bearing mice are not obviously changed within 16 days, which indicates that the nano-vesicles have excellent biological safety.
Example 2
A preparation method of specific targeting nano vesicles based on chitosan comprises the following steps:
s21, providing 8mg of gold nanoclusters (the purity is more than or equal to 99.99%) with the average particle size of 5nm and 32mg of sulfhydryl-polyethylene glycol-chitosan with the average relative molecular mass of 3000;
placing the gold nanoclusters, the sulfydryl-polyethylene glycol-chitosan and the deionized water on an XH-1T type multi-tube adjustable rotary mixer to rotate for 25min at the rotating speed of 25 rpm to obtain the composite nanoparticle dispersion liquid.
S22, preparation of the specific targeting nano vesicle based on chitosan:
c1, centrifuging the collected bone marrow cell suspension at 4 ℃ (3500rpm), removing cell debris or apoptotic bodies, taking supernatant, continuing to centrifuge at 4 ℃ (110000g, 35min), repeating the purification process, centrifuging (110000g, 1h), and precipitating into small balls;
c2, suspending the pellet obtained in the step c1 in 200 mu L PBS at the temperature of 4 ℃, and filtering the pellet through a microporous filter membrane with the pore diameter of 0.22 mu m to remove bacteria and residual cell membrane fragments to obtain an exosome;
c3, adding the exosome obtained in the step c2 into a sterile phosphate buffer solution at 4 ℃, diluting, and then storing at-80 ℃;
c4, 100. mu.L of exosome (2.4X 10) obtained in step c39particles/mL, protein content 2.4 μ g) with 18.9 μ L of the composite nanoparticle dispersion obtained in step S21 and 81.1 μ L of electroporation buffer (total reaction volume: 200 μ L) were mixed in a 0.2cm electroporation cuvette;
c5, electroporation treatment using electroporation system (Bio-Rad Gene plus Xcell) at 250V and 100. mu. Fd;
c6, incubating the product obtained in the step c5 at 37 ℃ for 25min so as to recover the exosome membrane of the exosome;
c7, using 60% OptiPrepTMCentrifuging the density gradient centrifugate to remove redundant composite nano particles in the step c6 to obtain specific targeting nano vesicles based on chitosan;
c8, performing NHS-Cy5.5 grafting on the chitosan-based specific targeting nano-vesicle obtained in the step c7, thereby obtaining the chitosan-based specific targeting nano-vesicle with a tracing effect.
Example 3
A preparation method of specific targeting nano vesicles based on chitosan comprises the following steps:
s31, providing 5mg of gold nanoclusters with the average particle size of 10nm (the purity is more than or equal to 99.99%) and 45mg of sulfhydryl-polyethylene glycol-chitosan with the average relative molecular mass of 4000;
and preparing the gold nanoclusters into dispersion, adding sulfydryl-polyethylene glycol-chitosan into the dispersion to obtain mixed liquor, and putting the mixed liquor on an XH-1T type multi-tube adjustable rotary mixer to rotate for 20min at the rotating speed of 30 revolutions per minute to obtain the composite nanoparticle dispersion.
S32, preparation of the specific targeting nano vesicle based on chitosan:
c1, centrifuging the collected bone marrow cell suspension at 4 ℃ (3500rpm), removing cell debris or apoptotic bodies, taking supernatant, continuing to centrifuge at 4 ℃ (110000g, 35min), repeating the purification process, centrifuging (110000g, 1h), and precipitating into small balls;
c2, suspending the pellet obtained in the step c1 in 200 mu L PBS at the temperature of 4 ℃, and filtering the pellet through a microporous filter membrane with the pore diameter of 0.22 mu m to remove bacteria and residual cell membrane fragments to obtain an exosome;
c3, adding the exosome obtained in the step c2 into a sterile phosphate buffer solution at 4 ℃, diluting, and then storing at-80 ℃;
c4, 100. mu.L of exosome (2.4X 10) obtained in step c39particles/mL, protein content 2.4 μ g) with 18.9 μ L of the composite nanoparticle dispersion obtained in step S31 and 81.1 μ L of electroporation buffer (total reaction volume: 200 μ L) were mixed in a 0.2cm electroporation cuvette;
c5, electroporation treatment at 250V and 100. mu.F using electroporation System (Bio-Rad Gene plus Xcell);
c6, incubating the product obtained in the step c5 at 37 ℃ for 25min so as to recover the exosome membrane of the exosome;
c7, OptiPrep with 65%TMDensity ladderCentrifuging the centrifugate to remove redundant composite nano particles in the c6 product to obtain specific targeting nano vesicles based on chitosan;
c8, performing NHS-Cy5.5 grafting on the chitosan-based specific targeting nano-vesicle obtained in the step c7, thereby obtaining the chitosan-based specific targeting nano-vesicle with a tracing effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A specific targeting nanovesicle based on chitosan, comprising exosomes having tumor cell specific targeting and composite nanoparticles embedded within the exosomes;
the composite nano particles contain gold nanoclusters and sulfhydryl-polyethylene glycol-chitosan, and the sulfhydryl of the sulfhydryl-polyethylene glycol-chitosan is combined with the gold nanoclusters through coordination.
2. The chitosan-based specific targeting nanovesicle according to claim 1, wherein the exosomes are selected from at least one of cancer cell exosomes, immune cell exosomes, tumor stem cell exosomes, bone marrow cell exosomes, umbilical cord mesenchymal stem cell exosomes.
3. The specific targeting nanovesicle based on chitosan of claim 2, wherein the mass content of thiol-polyethyleneglycol-chitosan in the composite nanoparticle is not less than 80%.
4. The specific targeting nanovesicle based on chitosan of claim 2, wherein the mass content of thiol-polyethyleneglycol-chitosan in the composite nanoparticle is not less than 90%.
5. The specific targeting nanovesicle based on chitosan according to any of claims 1 to 4, wherein the molecular weight of said thiol-polyethyleneglycol-chitosan is comprised between 2000 and 5000; the grain diameter of the gold nanocluster is 5-30 nm.
6. The specific targeting nanovesicle based on chitosan as claimed in any one of claims 1 to 4, wherein the composite nanoparticle further comprises an up-conversion nanomaterial, and the gold nanoclusters are supported on the surface of the up-conversion nanomaterial.
7. The specific targeting nanovesicle based on chitosan of claim 6, wherein the up-conversion nanomaterial is selected from any one of superparamagnetic, nano flower-like ferroferric oxide nanoparticles, up-conversion luminescent nanomaterials.
8. The preparation method of the chitosan-based specific targeting nanovesicle as claimed in any one of claims 1 to 7, comprising the following steps:
providing gold nanocluster dispersion, sulfhydryl-polyethylene glycol-chitosan and exosome with tumor cell specific targeting;
adding the sulfydryl-polyethylene glycol-chitosan into the gold nanocluster dispersion liquid, and carrying out coordination combination on the sulfydryl and the gold nanoclusters to obtain composite nanoparticles;
embedding the composite nano-particles into the exosomes with the tumor cell specific targets by adopting an electroporation technology, and then incubating the exosomes with the tumor cell specific targets to recover the exosome membranes of the exosomes with the tumor cell specific targets, thereby obtaining the chitosan-based specific targeted nano-vesicles.
9. The method for preparing specific targeting nanovesicles based on chitosan according to claim 8, wherein the ratio of the loading amount of the gold nanoclusters to the loading amount of the thiol-peg-chitosan is (1-2): (8-9).
10. Use of the specific targeted chitosan-based nanovesicle according to any one of claims 1 to 7 or the specific targeted chitosan-based nanovesicle prepared by the method according to any one of claims 8 to 9, as a drug for treating cancer, or as a carrier of a drug for treating cancer.
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