CN109568269B - Exosome with diagnosis and treatment functions and preparation method thereof - Google Patents
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Abstract
The invention discloses an exosome with a diagnosis and treatment function and a preparation method thereof, and belongs to the technical field of nano-medicine. The method comprises the following steps: 1) exosome extraction and purification: extracting exosome secreted by the cell from the culture solution of the mesenchymal stem cell of the C57 mouse, and suspending the obtained exosome in 10mM PBS solution for storage at the temperature of minus 80 ℃ for later use; 2) preparing a sample: mixing the prepared curcumin or indocyanine green solution and the exosomes obtained in the step 1) in 10mM PBS to obtain a sample; 3) electric conversion: performing electroporation on the sample obtained in the step 2) to obtain an electroporated sample; 4) and (3) incubation: placing the electroporated sample in the step 3) in a cell culture box and incubating for 30min in a dark place to obtain an incubated liquid; 5) and (3) purification: centrifuging the liquid incubated in the step 4) for 60-100min, and taking out the precipitate to obtain the exosome with the diagnosis and treatment function. The invention has simple operation and high repetition rate.
Description
Technical Field
The invention belongs to the technical field of nano medicine, and particularly relates to an exosome with a diagnosis and treatment function and a preparation method thereof.
Background
Chemotherapy remains one of the major modes of tumor treatment. However, most chemotherapy drugs have the defects of poor water solubility, fast metabolism, poor biocompatibility, unsatisfactory distribution in vivo, low cell permeability and the like, and the effect and the utilization rate of the chemotherapy drugs are seriously limited. Therefore, constructing a novel drug carrier to improve drug solubility and stability, improve tissue penetration, improve drug concentration at tumor sites, and avoid being captured by immune cells is a current research hotspot.
The unique advantages of exosomes in drug transport make them one of the research hotspots. Exosomes are nanovesicles derived from mammalian cells, with diameters around 40-200 nm. Exosomes are recognized by the host as "self," and thus act as an active carrier, delivering certain cellular RNAs, drug molecules, etc. into mammalian cells. The vesicular bodies have unique advantages as carriers, such as low immunogenicity, high stability in blood, high efficiency of drug delivery to cells, stronger effect of enhancing osmotic retention, and the like. At present, the existing researchers use exosome to load drugs, and the result shows that the exosome loaded drugs have better anti-tumor activity compared with free drugs. However, the efficiency of loading the exosome with the medicament is low, and patent CN108114290A discloses a preparation method of the exosome simultaneously loading the chemical medicament and the nano material, curcumin and nano ferroferric oxide are loaded into the exosome by an electrotransformation method, but the encapsulation rate of the exosome is not expressed in the whole text. Exosomes can be used for loading of many different types of drugs, including nucleic acid molecules (mRNA, siRNA, etc.), chemical drugs (curcumin, indocyanine green, etc.), and nanomaterials (nanosilver, nanoiron, etc.).
Curcumin is a fat-soluble phenolic substance extracted from rhizomes of curcuma longa of curcuma genus of zingiberaceae family, and contains phenolic hydroxyl groups, and the phenolic hydroxyl groups can generate oxidation reaction and effectively terminate free radical reaction when lipid peroxidation reaction occurs on cell membranes, so that the curcumin can show various physiological activities, such as multiple effects of oxidation resistance, tumor resistance, aging prevention and the like. In vitro experiments show that curcumin can remarkably inhibit proliferation, migration and invasion of tumor cells and promote apoptosis of the tumor cells. Therefore, curcumin has wide application prospects in various cancers. Indocyanine green (ICG) is currently the only Near Infrared (NIR) dye approved by the FDA in the united states. The study shows that ICG has photodynamic property and can generate active oxygen after being excited by NIR light with strong penetrating power. Further, the medicine can act on tumor parts through combination of photothermal therapy and oxidative free radicals, so that the effect of killing tumor cells is achieved. ICG is therefore currently considered the most promising drug for photothermal therapy.
The traditional tumor treatment can only inhibit the growth of the tumor through medicines, but the diagnosis and the disease evaluation of the interior of the tumor are not timely and accurate. By discussing different electrotransformation parameters and medicine mass ratios, the method for efficiently preparing the exosome with the diagnosis and treatment integrated function is provided, the medicine is quickly and efficiently encapsulated in the exosome, and meanwhile, the method can be used for combined encapsulation of a plurality of medicines, so that the cooperativity and consistency of tumor treatment and diagnosis are realized.
Disclosure of Invention
The invention aims to overcome the technical defects at present and provides a preparation method of an exosome with a diagnosis and treatment function. The method has simple operation and high repetition rate. The prepared exosome has higher encapsulation efficiency and diagnosis and treatment integrated function, and can be suitable for encapsulation of multiple single drugs and co-loading of multiple combined drugs. The exosome prepared by the method has the functions of chemotherapy, photothermal therapy, photodynamic therapy, imaging and the like, and provides a new carrier and a preparation method thereof for diagnosis and treatment of various diseases such as tumors and the like.
The invention also aims to provide the exosome with the diagnosis and treatment function obtained by the preparation method.
The purpose of the invention is realized by the following technical scheme:
a preparation method of exosome with diagnosis and treatment functions comprises the following steps:
1) exosome extraction and purification: extracting exosome secreted by the cell from a C57 mouse mesenchymal stem cell culture solution by adopting a differential ultracentrifugation method, and suspending the obtained exosome in a 10mM PBS solution to store at-80 ℃ for later use;
2) preparing a sample: mixing the prepared curcumin or indocyanine green solution and the exosomes obtained in the step 1) in 10mM PBS, and uniformly mixing to obtain a sample;
3) electric conversion: placing the sample obtained in the step 2) in an electroporator for electroporation to obtain an electroporated sample;
4) and (3) incubation: placing the electroporated sample in the step 3) in a cell culture box and incubating for 30min in a dark place to obtain an incubated liquid;
5) and (3) purification: centrifuging the liquid incubated in the step 4) for 60-100min by a centrifugal force of 140,000g and 100,000g, and taking out the precipitate to obtain the exosome with the diagnosis and treatment function.
In one embodiment, the C57 mouse is perfused with mesenchymal stem cell culture fluid with or without exosome-free serum.
In one embodiment, in step 2), 0-100 μ g exosome, 0-300 μ g curcumin or 0-300 μ g indocyanine green is added to 200 μ L of the 10mM PBS solution, respectively.
In one embodiment, in step 3), the conditions for electroporation are: voltage 0-350V, pulse width 100 mus, interval 1000 mus, number of discharges: 0-9 times.
An exosome with diagnosis and treatment functions is obtained by the preparation method. The prepared exosome with the diagnosis and treatment function has the drug slow-release performance.
Compared with the prior art, the invention has the following advantages and effects:
(1) the preparation method provided by the invention is simple to operate, short in preparation time and high in repetition rate. The prepared exosome with diagnosis and treatment functions has the drug slow-release performance, and the encapsulation rate is up to 90%; it can improve solubility, stability and permeability of hydrophobic drugs, and prevent phagocytosis and clearance by immune system in vivo.
(2) The prepared exosome with the diagnosis and treatment function can keep the complete structure and the fluidity of the nano vesicles, simultaneously retains the physicochemical properties of the loaded drug, such as chemotherapeutic activity, photothermal effect, photodynamic effect, fluorescence imaging effect and the like, and realizes the diagnosis and treatment integration.
Drawings
Fig. 1 is a fluorescence diagram for qualitatively and quantitatively detecting the generation of exosomes with diagnosis and treatment functions excited by near infrared light, wherein: a is exosome, B is curcumin, C is indocyanine green, and D is curcumin and indocyanine green;
fig. 2 is a graph showing the encapsulation efficiency results of exosomes having diagnostic function, wherein: a is exosome carrying curcumin, B is exosome carrying indolizine green, C is exosome carrying curcumin and indolizine green, and D is exosome carrying curcumin and indolizine green;
fig. 3 is a graph showing the results of measuring the average particle size of exosomes having diagnostic function, in which: a is an exosome, B is an electrotransfer exosome, C is an exosome carrying curcumin, D is an exosome carrying indolizine green, and E is an exosome carrying curcumin and indolizine green;
fig. 4 is a graph showing the results of measuring the average potential of exosomes having diagnostic function, in which: a is an exosome, B is an electrotransfer exosome, C is an exosome carrying curcumin, D is an exosome carrying indolizine green, and E is an exosome carrying curcumin and indolizine green;
fig. 5 is a diagram of the detection result of the photothermal effect of the exosome with diagnosis function in vitro, wherein: a is phosphate, B is curcumin, C is indocyanine green, D is curcumin and indocyanine green, E is exosome, F is exosome carrying curcumin, G is exosome carrying indocyanine green, and H is exosome carrying curcumin and indocyanine green;
fig. 6 is a graph showing the results of detecting the release of the exosomal accumulated drugs with diagnostic function, wherein: a is curcumin, B is indocyanine green, C is exosome-carrying curcumin, D is exosome-carrying indocyanine green, E is exosome-carrying curcumin and indocyanine green (curcumin), and F is exosome-carrying curcumin and indocyanine green (indocyanine green).
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
In the present application, the exosomes are first isolated, purified:
1) exosome extraction and purification: 200mL of supernatant of C57 mouse mesenchymal stem cells was collected for differential centrifugation: taking the supernatant after 10min at 300 g; taking supernatant after 10min at 2,000 g; taking supernatant after 30min at 10,000 g; resuspend pellet at 100,000g for 70 min; 100,000g, 70min resuspended in 200. mu.L 10mM PBS, 30. mu.L of sample was taken for BCA assay for protein content;
2) preparing a curcumin solution: weighing 4mg curcumin powder, adding 200 mu L DMSO solution, mixing well, supplementing to 1mL with 10mM PBS, and preparing into 2mg/mL curcumin solution;
3) preparing an indocyanine green solution: weighing 4mg of indocyanine green powder, adding 200 mu L of DMSO solution, fully and uniformly mixing, supplementing 1mL with 10mM PBS, and preparing into 2mg/mL indocyanine green solution;
then drugs such as curcumin, indocyanine green and the like are loaded into exosomes through electroporation:
4) electric conversion: adding exosome, curcumin solution and indocyanine green solution into 10mM PBS solution according to a certain proportion; after mixing uniformly, adding the mixture into a 96-hole cell culture plate, putting the cell culture plate into an electroporator, and performing electroporation according to certain reaction conditions;
5) and (3) incubation: after perforation, putting the 96-well plate into an incubator at 37 ℃ in a dark place, and incubating for 30 min;
6) and (3) purification: centrifuging the perforated exosome twice at 100,000-140,000g ultracentrifuge for 70-90min each time, removing supernatant, and obtaining precipitate which is the exosome (drug-loaded exosome) with diagnosis and treatment functions;
7) fluorescence detection: diluting 10 mu L of exosome with diagnosis and treatment functions in the step 6) to 100 mu L with 10mM PBS, and then dripping the exosome onto a fluorescence imager to respectively detect the fluorescence absorption values at 700nm and 800 nm;
8) uv-vis detection: detecting curcumin at 421nm and indocyanine green at 784nm respectively by using the ultraviolet-visible spectrophotometer with the diagnosis and treatment function of the exosome in the step 6), and comparing the values with a standard curve to calculate the encapsulation rate;
9) particle size and potential analysis: analyzing the particle size and the potential of the exosome with the diagnosis and treatment function in the step 6), and detecting the particle size distribution and the potential;
10) photothermal effect: adding the exosome with the diagnosis and treatment function in the step 6) into a 3mL quartz vessel, radiating for 5min by using an infrared laser with the wavelength of 808nm, and recording the temperature change every 30 s;
11) drug slow release: and (4) carrying out in-vitro accumulation release on the exosome with the diagnosis and treatment function in the step 6), and calculating the drug slow release efficiency.
Example 1
Adding 80 mu g of exosome into 10mM PBS solution, mixing uniformly, adding into a 96-well plate, putting into an electroporator, and operating according to the following parameters: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, and the discharge times are 6 times. After electroporation, the suspension can be put into a cell culture box to be incubated for 30min, then ultracentrifugation is carried out twice at 100,000g for 70 min/time, supernatant is removed, and 200 mu L PBS is used for resuspension and precipitation to obtain the exosome after electroporation. The particle size was-150 nm, as shown in FIG. 3. The Zeta potential is-8 mV, as shown in FIG. 4. Storing at 4 ℃ in a dark place for subsequent detection and experiments.
Example 2
Adding 80 μ g of exosome and 80 μ L of curcumin solution (concentration is 2mg/mL) into 10mM PBS solution, mixing uniformly, adding into a 96-well plate, placing into an electroporator, and operating according to the following parameters: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, and the discharge times are 6 times. After electroporation, the suspension can be put into a cell culture box for incubation for 30min, then ultracentrifugation is carried out twice at 100,000g for 70 min/time, supernatant is removed, and 200 mu L PBS is used for resuspension and precipitation, namely the exosome loaded with curcumin. The encapsulation efficiency was 86.64%, as shown in FIG. 2; the particle size is 170nm, as shown in FIG. 3; the Zeta potential is-17 mV, as shown in FIG. 4; the prepared curcumin-loaded exosome has drug slow-release performance, as shown in fig. 6. Storing at 4 ℃ in a dark place for subsequent detection and experiments.
Example 3
Adding 80 mu g of exosome and 40 mu L of indocyanine green solution (the concentration is 2mg/mL) into 10mM PBS solution, mixing uniformly, adding into a 96-well plate, placing into an electroporator, and operating according to the following parameters: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, and the discharge times are 6 times. After electroporation, the suspension can be put into a cell culture box for incubation for 30min, then ultracentrifugation is carried out twice at 100,000g for 70 min/time, supernatant is removed, 200 mu L PBS is used for resuspension and precipitation, namely the indocyanine green-loaded exosome, and the encapsulation rate is 97.79 percent, as shown in figure 2; the particle size is 180nm, as shown in FIG. 3; the Zeta potential is-6 mV, as shown in FIG. 4. The prepared indocyanine green-loaded exosome has fluorescence imaging, photo-thermal effect and drug slow-release performance, and is shown in figures 1, 5 and 6. Storing at 4 ℃ in a dark place for subsequent detection and experiments.
Example 4
Adding 80 mu g of exosome, 40 mu L of curcumin solution (with the concentration of 2mg/mL) and 40 mu L of indocyanine green solution (with the concentration of 2mg/mL) into 10mM PBS solution, uniformly mixing, adding into a 96-well plate, placing into an electroporator, and operating according to the following parameters: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, and the discharge times are 6 times. After electroporation, putting the suspension into a cell culture box to incubate for 30min, then ultracentrifuging for 70 min/time twice at 100,000g, removing supernatant, and carrying out heavy suspension precipitation by using 200 mu L PBS (phosphate buffer solution) to obtain the exosome loading curcumin/indocyanine green together, wherein the encapsulation rate of curcumin is-87.87%, and the encapsulation rate of indocyanine green is-96.97%, as shown in FIG. 2; the particle size was-200 nm, as shown in FIG. 3; the Zeta potential is-15 mV, as shown in FIG. 4. The prepared exosome loading curcumin/indocyanine green together has fluorescence imaging, photo-thermal effect and drug slow-release performance, and is shown in figures 1, 5 and 6. Storing at 4 ℃ in a dark place for subsequent detection and experiments.
Example 5
Adding 80 mu g of exosome, 80 mu L of curcumin solution (with the concentration of 2mg/mL) and 40 mu L of indocyanine green solution (with the concentration of 2mg/mL) into 10mM PBS solution, uniformly mixing, adding into a 96-well plate, placing into an electroporator, and operating according to the following parameters: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, and the discharge times are 6 times. After electroporation, the suspension can be put into a cell culture box for incubation for 30min, then ultracentrifugation is carried out twice at 100,000g for 70 min/time, supernatant is removed, and 200 mu L PBS is used for heavy suspension precipitation, thus obtaining the exosome co-loading curcumin/indocyanine green. Storing at 4 ℃ in a dark place for subsequent detection and experiments.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. A preparation method of exosome with diagnosis and treatment functions is characterized by comprising the following steps:
1) exosome extraction and purification: extracting exosome secreted by the cell from a C57 mouse mesenchymal stem cell culture solution by adopting a differential ultracentrifugation method, and suspending the obtained exosome in a 10mM PBS solution to store at-80 ℃ for later use;
2) preparing a sample: mixing the prepared curcumin and indocyanine green solution and the exosome obtained in the step 1) in 10mM PBS, and uniformly mixing to obtain a sample;
3) electric conversion: placing the sample obtained in the step 2) in an electroporator for electroporation to obtain an electroporated sample; the parameters are as follows: the voltage is 250V, the pulse width is 100 mus, the interval is 1000 mus, the discharging times is 6 times;
4) and (3) incubation: placing the electroporated sample in the step 3) in a cell culture box and incubating for 30min in a dark place to obtain an incubated liquid;
5) and (3) purification: centrifuging the liquid incubated in the step 4) for 60-100min by a centrifugal force of 140,000g and 100,000g, and taking out the precipitate to obtain the exosome with the diagnosis and treatment function.
2. The method for preparing exosomes with diagnosis and treatment functions according to claim 1, wherein the culture solution of mesenchymal stem cells of the C57 mouse is exosome-free serum or serum-free.
3. The method for preparing exosomes with diagnosis and treatment functions according to claim 1, wherein in the step 2), 100 μ g of exosomes, 300 μ g of curcumin and 300 μ g of indocyanine green are respectively added into 200 μ L of the 10mM PBS solution.
4. An exosome having a diagnostic function, which is obtained by the production method according to any one of claims 1 to 3.
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| CN111407742B (en) * | 2020-03-30 | 2021-10-22 | 西南交通大学 | Anti-tumor nano-particles and preparation method and application thereof |
| CN113430165B (en) * | 2021-06-17 | 2022-02-08 | 山东省齐鲁细胞治疗工程技术有限公司 | THC-loaded stem cell drug delivery system and preparation method thereof |
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