CN115282286B - Nanometer complex for treating ocular neovascular diseases and application thereof - Google Patents
Nanometer complex for treating ocular neovascular diseases and application thereof Download PDFInfo
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Abstract
The invention discloses a nano-composite for treating ocular neovascular diseases, which is prepared by loading pigment epithelium derived factor PEDF onto small extracellular vesicles sEV derived from umbilical cord mesenchymal stem cells by using an ultrasonic method. The new PEDF@sEV nano-composite material prepared by the ultrasonic method combines the advantages of PEDF and sEV: the PEDF ensures that the nano-composite has better long-term safety in the aspect of inhibiting the proliferation of new blood vessels, and the sEV carrier endows the composite with excellent cross-barrier performance, so that the PEDF@sEV nano-composite has better treatment effect at the same dosage compared with the pure PEDF, and has stronger potential application value compared with the VEGF antibody medicament which is most widely applied clinically at present.
Description
Technical Field
The invention belongs to the technical field of medical nano-composite new materials, and in particular relates to a nano-composite for treating ocular neovascular diseases and application thereof.
Background
Ocular neovascularization is a common pathological feature of a variety of blinding eye diseases, including Age-related macular degeneration (Age-related Macular Degeneration, AMD), proliferative diabetic retinopathy (Proliferative Diabetic Retinopathy, PDR), retinal arteriovenous obstruction, and retinopathy of prematurity (Retinopathy of Prematurity, ROP), as well as a key blinding factor for such diseases.
At present, the clinical treatment of ocular neovascularization mainly comprises vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) antibody medicines, laser photocoagulation and photodynamic therapy, and especially, the vitreous cavity injection of VEGF antibody medicines (abnormal rise of VEGF level is an important factor of ocular neovascularization) has become a gold standard for the first-line clinical treatment of ocular neovascularization diseases. However, due to the nature and mechanism of action of VEGF antibody drugs, patients typically need to inject once every 3 months for 1 to 2 years. Although the short-term efficacy of VEGF antibody drugs is remarkable, since VEGF also has normal neurotrophic effects in the retina, the long-term use of a large amount of VEGF antibody drugs causes the problem that VEGF in the eye is inhibited by "one-knife", thereby causing retinal formation map atrophy.
Pigment epithelium derived factor (Pigment Epithelium Derived Factor, PEDF) is the most potent anti-neovascular factor found in the eye naturally occurring in the eye, and PEDF also has a neurotrophic effect. Under normal physiological conditions, the content of PEDF in the vitreous humor and retina is very high, and PEDF levels decrease when neovascular proliferation occurs. There have been a number of experiments showing that exogenous administration of PEDF, restoring PEDF levels significantly inhibits proliferation of new blood vessels. Methods of exogenously administering PEDF to restore intraocular PEDF levels while inhibiting neovasculature are more biosafety than other methods.
Small extracellular vesicles (Small extracellular vesicles, sEV), due to their natural biological properties, are not only biocompatible but also have a more excellent transport capacity across the barrier than traditional synthetic vectors. In recent years, sEV derived from umbilical cord mesenchymal stem cells has become a star carrier in pharmaceutical carriers because of its anti-inflammatory and neurotrophic effects. PEDF has a molecular weight of 50kDa and belongs to a protein of larger molecular weight. The sEV is used for loading the PEDF to construct the pedf@sEV nano-complex, so that the bioavailability of the PEDF can be effectively improved, namely the loaded PEDF is protected to be degraded and more acts with cells, and the anti-angiogenesis therapeutic effect of the PEDF is enhanced. The PEDF@sEV nano-composite prepared by the invention has better therapeutic effect on resisting new blood vessels by virtue of the superior carrier performance of sEV. However, there is currently no application of pedf@sev nanocomplexes in ocular neovascular diseases.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a nano-composite for treating ocular neovascular diseases and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
a nano-complex for treating ocular neovascular diseases, which is prepared by loading pigment epithelium derived factor PEDF onto umbilical cord mesenchymal stem cell-derived small extracellular vesicles sEV by an ultrasonic method.
Further, the nanocomposite comprises:
feature A: sEV is taken as a carrier, so that the complex is endowed with good barrier-crossing property;
feature B: PEDF is used as a therapeutic drug, so that the complex has better long-term safety in inhibiting new blood vessels;
feature C: PEDF was loaded onto sEV carrier by ultrasonic method to construct the nanocomposite.
Further, sEV derived from umbilical cord mesenchymal stem cells was used as a carrier.
Further, the small extracellular vesicles sEV are sEV obtained by means of gradient centrifugation, i.e. extracellular vesicles secreted by the cells and having a size between 50 and 200 nm.
Further, the final concentration of sEV is 0.05-100mg/ml when the nanocomposite is prepared by ultrasound, and the final concentration of PEDF is 1-100ug/ml when the nanocomposite is prepared by ultrasound.
Further, the mass ratio of sEV to PEDF in ultrasonic is 1-1000:1.
further, the particle size distribution of the PEDF@sEV nano-composite body is between 100 and 200 nm.
Further, the ultrasonic power during ultrasonic treatment is 20% -50%, and the time is as follows: 1-30 minutes, pulse is: the temperature of the mixed solution is controlled between 0 and 4 ℃ during ultrasonic treatment, wherein the time of ultrasonic treatment is 1 to 60 seconds on/1 to 60 seconds off.
Further, the specific preparation steps are as follows:
(1) Preparation and isolation of sEV
Collecting supernatant of a cell condition culture medium by primary culturing umbilical cord mesenchymal stem cells, sequentially carrying out gradient centrifugation on 300g, 10 min, 2000g, 10-15 min, 10000g, 30 min, 110000g and 70 min to obtain a precipitate of sEV, re-suspending by using enough PBS, centrifuging for 110000g and 70 min again, and collecting relatively pure sEV;
(2) Preparation of PEDF@sEV nanocomplex
Configuration of pedf solution and sEV solution
The solution is an equilibrium solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of the PEDF solution is 1-100 mug/mL, the concentration of the sEV solution is 0.05-100mg/mL, the balanced salt solution is PBS balanced salt solution, and the mass ratio of sEV to PEDF is 1-1000:1, a step of;
b. ultrasonic method for preparing PEDF@sEV complex
Performing ultrasonic treatment on the mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic treatment time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, the time is: 1-30 minutes, pulse is: 1-60 seconds on/1-60 seconds off;
repair of PEDF@sEV complexes
After the ultrasonic treatment is finished, the mixed solution is placed in an environment of 25-37 ℃ for incubation, and the incubation time is 30-60 minutes, so that the structure of sEV is recovered;
purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex is finished, performing centrifugation, cleaning and purification for 2-3 times by using an ultrafiltration tube with the molecular weight cutoff of 100-150kDa, taking PBS as a cleaning solution, and completing the recovery of the nano complex to obtain the nano complex for treating ocular neovascular diseases;
wherein, during purification, the centrifugal speed is 2000-20000g, the centrifugal time is 5-45 minutes, and the volume ratio of PBS to complex is 2-10:1, a step of; during recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
The application of the nano-composite for treating ocular neovascular diseases in the aspect of being used as and/or preparing medicines for treating ocular neovascular diseases.
The invention has the advantages and positive effects that:
1. the new PEDF@sEV nano-composite material prepared by the ultrasonic method combines the advantages of PEDF and sEV: the PEDF ensures that the nano-composite has better long-term safety in the aspect of inhibiting the proliferation of new blood vessels, and the sEV carrier endows the composite with excellent cross-barrier performance, so that the PEDF@sEV nano-composite has better treatment effect at the same dosage compared with the pure PEDF, and has stronger potential application value compared with the VEGF antibody medicament which is most widely applied clinically at present.
2. The whole preparation process of the complex is simple and convenient, and can be used for low-cost production. The particle size of the prepared PEDF@sEV nano-composite is mainly distributed between 100 nm and 200nm, the size comparison is concentrated, and the typical lipid bilayer structure is shown in the observation of a transmission electron microscope. ELISA results showed that PEDF was successfully loaded onto sEV and that PEDF@sEV nanocomplexes were successfully constructed. Fluorescence labelling experiments demonstrated that the loading process did not significantly alter the biological behaviour of sEV taken up by the cells.
3. The PEDF@sEV nanometer complex can be prepared by an ultrasonic method during preparation, and the preparation process is simple and convenient. In the preparation process, only the balanced salt solution of PEDF and sEV is mixed according to a certain proportion, and then the ultrasonic treatment is carried out for 1-30 minutes under the condition of 20-50% of power, and the pulse is as follows: 1-60 seconds on/1-60 seconds off. After the ultrasonic treatment is finished, incubating for 30-60 minutes at 25-37 ℃, and then cleaning and purifying by using an ultrafiltration tube to recover the PEDF@sEV nano-complex.
4. The current Vascular Endothelial Growth Factor (VEGF) antibody drug is the gold standard for the clinical first-line treatment of ocular neovascular diseases. VEGF is a key factor in neovascular proliferation, and VEGF antibody drugs mainly neutralize VEGF in ocular endocrine to achieve therapeutic effects, so patients need to perform intraocular injections every 3 months. However, normal physiological levels of VEGF in the eye have normal neurotrophic effects on the retina. With the long-term and large-scale use of VEGF antibody medicines, normal neurotrophic effects of VEGF can be inhibited simultaneously with inhibition of neovascular diseases caused by VEGF.
Pigment Epithelium Derived Factor (PEDF) is the factor with the strongest anti-neovascular ability naturally occurring in the eye, and PEDF levels decrease when suffering from intraocular neovascular diseases. Methods of treating neovasculature by exogenously administering PEDF to restore intraocular PEDF levels are safer than VEGF antibody drugs.
5. Small extracellular vesicles (sEV) are natural drug carriers due to their excellent biocompatibility and transport capacity across barriers. In particular sEV, which is derived from umbilical cord mesenchymal stem cells, has become a star carrier in pharmaceutical carriers because of its anti-inflammatory and neurotrophic effects. According to the invention, the PEDF@sEV nano-complex is constructed by a simple and convenient ultrasonic method, so that the PEDF can be transported to a focus part more effectively, and the therapeutic effect of the PEDF is improved.
In conclusion, compared with the clinical first-line gold standard VEGF antibody medicament, the nano-composite for treating ocular neovascular diseases is prepared by the invention, and has higher biological safety. By virtue of the excellent carrier property of sEV derived from umbilical mesenchymal stem cells, the PEDF@sEV improves the treatment effect of PEDF, and is hopeful to be applied to ocular surface drug administration treatment of retinal neovascular diseases in the future, thereby being hopeful to further complete innovation of the treatment method.
6. The invention aims to design and prepare a nano-composite for treating ocular neovascular diseases. The nanocomposite was a pedf@sev nanocomposite constructed by loading pigment epithelium derived factor (Pigment epithelium derived factor, PEDF) with umbilical cord mesenchymal stem cell-derived small extracellular vesicles (Small extracellular vesicles, sEV). We loaded PEDF onto sEV by the method of ultrasound sEV and PEDF mixture to form a nanocomposite. In the detection of a transmission electron microscope, the nano-composite presents a typical lipid bilayer structure of a biological vesicle, and the loading of PEDF does not cause obvious morphological structure change to sEV. The detection results of the ELISA kit showed that we successfully loaded PEDF onto sEV. Nanosight (particle sizer) measurements showed that PEDF loading did not change the overall particle size distribution of sEV. Fluorescence labelling experiments demonstrated that PEDF loading did not significantly affect sEV bioactivity. In vivo and in vitro experiments prove that the PEDF@sEV nano-complex has obviously improved therapeutic effect compared with the pure PEDF at the cellular level and the animal level respectively.
Drawings
FIG. 1 is a transmission electron micrograph of PEDF@sEV nanocomposite prepared according to example 1 in the present invention; the PEDF@sEV nano-composite presents a complete double-layer film structure diagram under a transmission electron microscope;
FIG. 2 is a graph showing the particle size distribution of PEDF@sEV nanocomposites prepared according to example 1 according to the present invention; the particle size of the PEDF@sEV nano-composite body is mainly concentrated and distributed between 100 nm and 200nm through a particle size analyzer test;
FIG. 3 is a graph showing the effective uptake of PEDF@sEV nanocomposites prepared according to example 1 by cells in the present invention; red dye labeled pedf@sev was incubated with retinal vascular endothelial cells for 24 hours (panel a), 48 hours (panel B), and the results showed efficient uptake of the complex by the cells;
FIG. 4 is a graph showing that PEDF@sEV prepared according to example 1 has a remarkable inhibitory effect on Vascular Endothelial Growth Factor (VEGF) proliferation promoting effect in the present invention; the pedf@sev nanocomposite proliferated more significantly on endothelial cells than PEDF alone and sEV;
FIG. 5 is a graph showing the significant inhibition of retinal neovascularization in OIR model mice by PEDF@sEV prepared according to example 1 of the present invention; compared with pure PEDF and sEV, the pedf@sEV nano-complex has more obvious inhibition on the proliferation of the new blood vessels in the OIR animal model;
FIG. 6 is a graph showing the significant inhibition of the retinal perfusion-free region of OIR model mice by PEDF@sEV prepared according to example 1 of the present invention; compared with pure PEDF and sEV, the pedf@sEV nano-composite has more obvious inhibition on the area of the non-perfusion region in the OIR animal model;
FIG. 7 is a diagram showing a complete double-layer membrane structure of PEDF@sEV nanocomposite (25% power) in the transmission electron microscope;
FIG. 8 is a diagram showing the complete double-layer membrane structure of PEDF@sEV nanocomposite (30% power) in the transmission electron microscope according to the present invention.
Detailed Description
The following describes the embodiments of the present invention in detail, but the present embodiments are illustrative and not limitative, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A nano-complex for treating ocular neovascular diseases, which is prepared by loading pigment epithelium derived factor PEDF onto umbilical cord mesenchymal stem cell-derived small extracellular vesicles sEV by an ultrasonic method.
Preferably, the nanocomposite comprises:
feature A: sEV is taken as a carrier, so that the complex is endowed with good barrier-crossing property;
feature B: PEDF is used as a therapeutic drug, so that the complex has better long-term safety in inhibiting new blood vessels;
feature C: PEDF was loaded onto sEV carrier by ultrasonic method to construct the nanocomposite.
Preferably, sEV derived from umbilical cord mesenchymal stem cells is used as a carrier.
Preferably, the small extracellular vesicles sEV are sEV obtained by means of gradient centrifugation, i.e. extracellular vesicles secreted by the cells and having a size between 50 and 200 nm.
Preferably, the final concentration of sEV is 0.05-100mg/ml when the nanocomposite is prepared by ultrasound, and the final concentration of PEDF is 1-100ug/ml when the nanocomposite is prepared by ultrasound.
Preferably, the mass ratio of sEV to PEDF at ultrasonic treatment is 1-1000:1.
preferably, the particle size distribution of the pedf@sev nanocomposite is between 100 and 200 nm.
Preferably, the ultrasonic power during ultrasonic treatment is 20% -50%, and the time is as follows: 1-30 minutes, pulse is: the temperature of the mixed solution is controlled between 0 and 4 ℃ during ultrasonic treatment, wherein the time of ultrasonic treatment is 1 to 60 seconds on/1 to 60 seconds off.
Preferably, the specific preparation steps are as follows:
(1) Preparation and isolation of sEV
Collecting supernatant of a cell condition culture medium by primary culturing umbilical cord mesenchymal stem cells, sequentially carrying out gradient centrifugation on 300g, 10 min, 2000g, 10-15 min, 10000g, 30 min, 110000g and 70 min to obtain a precipitate of sEV, re-suspending by using enough PBS, centrifuging for 110000g and 70 min again, and collecting relatively pure sEV;
(2) Preparation of PEDF@sEV nanocomplex
Configuration of pedf solution and sEV solution
The solution is an equilibrium solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of the PEDF solution is 1-100 mug/mL, the concentration of the sEV solution is 0.05-100mg/mL, the balanced salt solution is PBS balanced salt solution, and the mass ratio of sEV to PEDF is 1-1000:1, a step of;
b. ultrasonic method for preparing PEDF@sEV complex
Performing ultrasonic treatment on the mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic treatment time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, the time is: 1-30 minutes, pulse is: 1-60 seconds on/1-60 seconds off;
repair of PEDF@sEV complexes
After the ultrasonic treatment is finished, the mixed solution is placed in an environment of 25-37 ℃ for incubation, and the incubation time is 30-60 minutes, so that the structure of sEV is recovered;
purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex is finished, performing centrifugation, cleaning and purification for 2-3 times by using an ultrafiltration tube with the molecular weight cutoff of 100-150kDa, taking PBS as a cleaning solution, and completing the recovery of the nano complex to obtain the nano complex for treating ocular neovascular diseases;
wherein, during purification, the centrifugal speed is 2000-20000g, the centrifugal time is 5-45 minutes, and the volume ratio of PBS to complex is 2-10:1, a step of; during recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
The application of the nano-composite for treating ocular neovascular diseases in the aspect of being used as and/or preparing medicines for treating ocular neovascular diseases.
Specifically, the relevant preparation and detection examples are as follows:
example 1:
a nano-composite for treating ocular neovascular diseases, which is prepared by the following steps:
1. isolation and purification of sEV
Sufficient cell conditioned medium supernatant was collected by primary culturing umbilical cord mesenchymal stem cells. After 300g (10 min), 2000g (10-15 min), 10000g (30 min), a gradient centrifugation of 110000g (70 min) gave a pellet of sEV, which was then resuspended with sufficient PBS and centrifuged again at 110000g (70 min), after which relatively pure sEV was collected.
2. Preparation of PEDF@sEV
Solutions of PEDF and sEV were prepared using PBS (balanced salt solution) respectively, the concentration of PEDF was 5ug/ml, the concentration of sEV was 0.1mg/ml, and the mass ratio of sEV to PEDF was 20:1, and the total volume of the solution was 0.5ml.
The power of the ultrasound was set at 20%, the ultrasound time at 3 minutes, and the pulse parameters at 4 seconds on/2 seconds off. The mixture was placed on ice and sonicated.
3. Repair of PEDF@sEV complexes
After the completion of the sonication, the mixture was incubated in an environment of 37℃for 60 minutes to restore the structure of sEV.
4. Purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex was completed, centrifugation was performed using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa, 4000g for 10 minutes, and then PBS was added for 2 times. Upon recovery, the concentrated pedf@sev nanocomposite was recovered by centrifugation at 1000g for 2 minutes.
Example 2:
a nano-composite for treating ocular neovascular diseases, which is prepared by the following steps:
1. isolation and purification of sEV
Sufficient cell conditioned medium supernatant was collected by primary culturing umbilical cord mesenchymal stem cells. After 300g (10 min), 2000g (10-15 min), 10000g (30 min), a gradient centrifugation of 110000g (70 min) gave a pellet of sEV, which was then resuspended with sufficient PBS and centrifuged again at 110000g (70 min), after which relatively pure sEV was collected.
2. Preparation of PEDF@sEV
Solutions of PEDF and sEV were prepared using PBS (balanced salt solution) respectively, the concentration of PEDF was 10ug/ml, the concentration of sEV was 0.1mg/ml, and the mass ratio of sEV to PEDF was 10:1, and the total volume of the solution was 0.5ml.
The power of the ultrasound was set at 25%, the ultrasound time at 2 minutes, and the pulse parameters at 2s/2s. The mixture was placed on ice and sonicated.
3. Repair of PEDF@sEV complexes
After the completion of the sonication, the mixture was incubated in an environment of 37℃for 30 minutes to restore the structure of sEV.
4. Purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex was completed, centrifugation was performed using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa, 4000g for 10 minutes, and then PBS was added for 2 times. Upon recovery, the concentrated pedf@sev nanocomposite was recovered by centrifugation at 1000g for 2 minutes.
Example 3:
a nano-composite for treating ocular neovascular diseases, which is prepared by the following steps:
1. isolation and purification of sEV
Sufficient cell conditioned medium supernatant was collected by primary culturing umbilical cord mesenchymal stem cells. After 300g (10 min), 2000g (10-15 min), 10000g (30 min), a gradient centrifugation of 110000g (70 min) gave a pellet of sEV, which was then resuspended with sufficient PBS and centrifuged again at 110000g (70 min), after which relatively pure sEV was collected.
2. Preparation of PEDF@sEV
Solutions of PEDF and sEV were prepared using PBS (balanced salt solution) respectively, the concentration of PEDF was 5ug/ml, the concentration of sEV was 0.1mg/ml, and the mass ratio of sEV to PEDF was 20:1, and the total volume of the solution is 1ml.
The power of the ultrasound was set at 30%, the ultrasound time at 2 minutes, and the pulse parameters at 2s/2s. The mixture was placed on ice and sonicated.
3. Repair of PEDF@sEV complexes
After the completion of the sonication, the mixture was incubated in an environment of 37℃for 60 minutes to restore the structure of sEV.
4. Purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex was completed, centrifugation was performed using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa, 4000g for 10 minutes, and then PBS was added for 2 times. Upon recovery, the concentrated PEDF@sEV nanocomplexes were recovered by centrifugation at 1000g for 2 min.
The correlation detection of the invention is as follows:
1. the detection method used was a transmission electron microscope (Transmission Electron Microscope, TEM for short). The sample preparation method comprises the following steps: and (3) dripping 30ul of PEDF@sEV nano-complex solution with proper concentration on a carbon support film, drying at room temperature for 2 minutes, dripping phosphotungstic acid staining solution, standing for 2 minutes, drying under a mercury lamp for 5 minutes, and detecting by a transmission electron microscope. The results are shown in figures 1, 7 and 8, and it can be seen that the ultrasound loading did not significantly alter the relatively intact vesicle structure of sEV.
2. The particle size and distribution of the pedf@sev nanocomposite were detected using a nanoparticle analyzer (Naonosight, NTA for short). As shown in the detection result in FIG. 2, the particle size distribution of the PEDF@sEV nano-composite body is mainly concentrated between 100 nm and 200nm, and the particle size distribution of sEV is not obviously changed by ultrasonic drug delivery.
3. The red fluorescent lipophilic dye pKH26 is used for labeling sEV, and then the nano complex is constructed. The red fluorescent labeled pedf@sev nanocomplexes were co-cultured with cells. Cell fixation and staining were performed at 24 hours and 48 hours, respectively, and the uptake of the nanocomposite by cells was observed by fluorescence microscopy. Blue is nucleus and red is nanocomposite. The results are shown in FIG. 3, and it can be seen that the PEDF@sEV nanocomposite can be efficiently taken up by cells and taken into cells.
4. Upregulation of vascular endothelial growth factor (VEGF for short) levels is an important factor in the pathogenesis of neovascular eye diseases. Therefore, the cell experiment adopts VEGF to promote the proliferation of human retina vascular endothelial cells (HREC for short) as an in vitro experiment model. As shown in fig. 4, pedf@sev nanocomplexes showed more remarkable proliferation of endothelial cells than PEDF alone and sEV at the same concentration, and the pedf@sev nanocomplexes had a stronger ability to inhibit cell proliferation. This suggests that sEV enhances the therapeutic effect of PEDF on proliferation inhibition after loading with PEDF, and it can also be seen that PEDF and sEV in the pedf@sev nanocomposite have a synergistic effect.
5. The Oxygen-induced retinopathy model (Oxygen-Induced Retinopathy, OIR for short) is a very widely used model of ocular neovascular animals. The OIR model is to put the young mice which are born for 7 days into an oxygen cabin (high oxygen environment) for 5 days, and then return to normal environment for 5 days for continuous breeding. The retinal blood vessels of mice begin to develop after birth, and the OIR model has two pathological features: 1. after entering the oxygen cabin for feeding on the 7 th day, the microvasculature at the central part of the retina of the mouse is degenerated under the stimulation of the high oxygen environment to form a perfusion-free area (a vascular-free area); 2. when the medium is fed back to normal environment, the central non-perfused area is severely anoxic, so that the retina is stimulated to form a great number of new blood vessels with incomplete structures and morphology around the non-perfused area, and peaks at the 17 th day after birth. Therefore, aiming at the above 2 pathological characteristics, the ideal therapeutic drug should have 2-point effect, 1: the area of the non-perfused area is effectively reduced, namely, the remodeling of the non-perfused blood vessel is recovered; 2: effectively inhibit the proliferation of pathological new blood vessels.
As shown in fig. 5 and 6, the pedf@sev nanocomposite has more obvious inhibition of neovascular proliferation in an OIR animal model than PEDF and sEV alone, the pedf@sev nanocomposite has more obvious inhibition of the area of the non-perfused area in the OIR animal model than PEDF and sEV alone, and pedf@sev nanocomposite has a synergistic effect compared with PEDF alone, and PEDF and sEV in the pedf@sev nanocomposite have stronger effects of inhibiting pathological neovascular proliferation and reducing non-perfusion. This advantageously suggests that sEV strongly enhances the therapeutic effect of PEDF after loading with PEDF.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (2)
1. A nano-complex for treating ocular neovascular diseases, which adopts sEV from umbilical cord mesenchymal stem cells as a carrier;
the small extracellular vesicles sEV are sEV obtained by using a gradient centrifugation method, namely extracellular vesicles secreted by cells and having a size of between 50 and 200 nm;
the preparation method comprises the following specific steps:
(1) Preparation and isolation of sEV
Collecting supernatant of a cell condition culture medium by primary culturing umbilical cord mesenchymal stem cells, sequentially carrying out gradient centrifugation on 300g, 10 min, 2000g, 10-15 min, 10000g, 30 min, 110000g and 70 min to obtain a precipitate of sEV, re-suspending by using enough PBS, centrifuging for 110000g and 70 min again, and collecting relatively pure sEV;
(2) Preparation of PEDF@sEV nanocomplex
a. Preparation of PEDF solution and sEV solution
The solution is an equilibrium solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of the PEDF solution is 1-100 mug/mL, the concentration of the sEV solution is 0.05-100mg/mL, the balanced salt solution is PBS balanced salt solution, and the mass ratio of sEV to PEDF is 1-1000:1, a step of;
b. ultrasonic method for preparing PEDF@sEV complex
Performing ultrasonic treatment on the mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic treatment time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, the time is: 1-30 minutes, pulse is: 1-60 seconds on/1-60 seconds off;
c. repair of PEDF@sEV complexes
After the ultrasonic treatment is finished, the mixed solution is placed in an environment of 25-37 ℃ for incubation, and the incubation time is 30-60 minutes, so that the structure of sEV is recovered;
purification of PEDF@sEV complexes
After the repair of the PEDF@sEV complex is finished, performing centrifugation, cleaning and purification for 2-3 times by using an ultrafiltration tube with the molecular weight cutoff of 100-150kDa, taking PBS as a cleaning solution, and completing the recovery of the nano complex to obtain the nano complex for treating ocular neovascular diseases;
wherein, during purification, the centrifugal speed is 2000-20000g, the centrifugal time is 5-45 minutes, and the volume ratio of PBS to complex is 2-10:1, a step of; during recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
2. The use of the nanocomposite for the treatment of ocular neovascular diseases according to claim 1 for the preparation of a medicament for the treatment of ocular neovascular diseases.
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| CN111511377A (en) * | 2017-07-31 | 2020-08-07 | 谱系细胞疗法公司 | Compositions and methods for restoring or preventing vision loss from disease or traumatic injury |
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| CN110123839B (en) * | 2018-02-02 | 2021-02-19 | 上海睿泰生物科技股份有限公司 | Human pluripotent stem cell exosome loaded with photosensitive drug, preparation and application thereof |
| EP3705132A1 (en) * | 2019-03-04 | 2020-09-09 | Curebiotec GmbH | Method for the treatment of a disease using pigment epithelium-derived factor (pedf) |
| KR102198942B1 (en) * | 2019-07-02 | 2021-01-05 | 의료법인 성광의료재단 | Genetically engineered mesenchymal stem cell for expressing pedf or overexpressing pedf compared to mother cell |
| CN110295142B (en) * | 2019-07-11 | 2021-04-27 | 中国医学科学院北京协和医院 | Bone marrow mesenchymal stem cell exosome for promoting angiogenesis and preparation method and application thereof |
| CN111494341A (en) * | 2020-05-26 | 2020-08-07 | 南开大学 | Nano cell membrane drug-loaded vesicle and preparation method and application thereof |
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| CN105267240A (en) * | 2014-12-16 | 2016-01-27 | 天津医科大学眼科医院 | Applications of exosome from mesenchymal stem cells |
| CN109890964A (en) * | 2016-09-09 | 2019-06-14 | 康奈尔大学 | The delivering of nucleic acid, albumen and small molecule in hyaloid capsule foam |
| CN111511377A (en) * | 2017-07-31 | 2020-08-07 | 谱系细胞疗法公司 | Compositions and methods for restoring or preventing vision loss from disease or traumatic injury |
| US10758571B1 (en) * | 2019-04-09 | 2020-09-01 | Combangio, Inc. | Processes for making and using a mesenchymal stem cell derived secretome |
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