CN115282286A - Nano complex for treating ocular neovascular diseases and application thereof - Google Patents
Nano complex for treating ocular neovascular diseases and application thereof Download PDFInfo
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Abstract
The invention discloses a nano complex for treating ocular neovascular diseases, which is prepared by loading pigment epithelium derived factor PEDF onto a small extracellular vesicle sEV derived from umbilical mesenchymal stem cells by using an ultrasonic method. The novel PEDF @ sEV nano-composite material prepared by the ultrasonic method combines the advantages of PEDF and sEV: PEDF enables the nano complex to have better long-term safety in the aspect of inhibiting the proliferation of new vessels, the sEV carrier endows the complex with excellent cross-barrier performance, and compared with pure PEDF, the PEDF @ sEV nano complex has better treatment effect under the same dosage and has stronger potential application value compared with VEGF antibody medicaments which are clinically applied most widely at present.
Description
Technical Field
The invention belongs to the technical field of new medical nano-complex materials, and particularly relates to a nano-complex for treating eye neovascularization diseases and application thereof.
Background
Ocular neovascularization is a common pathological feature of various blinding eye diseases, including Age-related Macular Degeneration (AMD), proliferative Diabetic Retinopathy (PDR), retinal artery and vein occlusion, retinopathy of Prematurity (ROP), and is also a key blinding factor of such diseases.
At present, the clinical treatment of ocular neovascularization mainly comprises Vascular Endothelial Growth Factor (VEGF) antibody drugs, laser photocoagulation and photodynamic therapy, and especially, intravitreal injection of VEGF antibody drugs (the abnormal increase of VEGF level is an important factor for intraocular neovascularization) becomes the 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 require injections every 3 months for 1 to 2 years. Although the short-term efficacy of VEGF antibody drugs is significant, because VEGF also has normal neurotrophic effects in the retina, the long-term use of large amounts of VEGF antibody drugs causes the problem that intraocular VEGF is inhibited by "one-time cutting", thereby causing retinal geographic atrophy.
Pigment Epithelium Derived Factor (PEDF) is the Factor which is found to be naturally present in the eye with the strongest capacity for resisting new blood vessels, and the PEDF also has the neurotrophic effect. Under normal physiological conditions, the content of PEDF in the vitreous humor and in the retina is very high, and when neovascularization occurs, the level of PEDF decreases. A large number of experiments show that exogenous PEDF administration can obviously inhibit the proliferation of new blood vessels when the content of PEDF is recovered. Methods of exogenously administering PEDF to restore intraocular PEDF levels while inhibiting neovascularization are safer than other methods.
Small extracellular vesicles (sEV) have good biocompatibility and superior cross-barrier transport capacity compared with conventional synthetic vectors due to their natural biological properties. In recent years, the umbilical cord mesenchymal stem cell-derived sEV has become a star carrier in drug carriers due to the anti-inflammatory and neurotrophic effects of the sEV. PEDF has a molecular weight of 50kDa and is a protein of larger molecular weight. PEDF is loaded by sEV to construct PEDF @ sEV nano-complex, so that the bioavailability of PEDF can be effectively improved, namely the loaded PEDF is protected from degradation and further acts with cells, and the treatment effect of PEDF on neovascularization is enhanced. The PEDF @ sEV nano-complex prepared by the invention enables PEDF to exert a better anti-angiogenesis therapeutic effect by virtue of the excellent carrier performance of sEV. However, the application of the PEDF @ sEV nano-complex in ocular neovascular diseases is not provided at present.
Through 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 complex for treating ocular neovascular diseases and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a nanocomposite for treating ocular neovascular diseases is prepared by loading pigment epithelium derived factor PEDF onto umbilical cord mesenchymal stem cell-derived small extracellular vesicle sEV by using an ultrasonic method to prepare a PEDF @ sEV nanocomposite.
Further, the nanocomposite comprises:
the characteristics are as follows: the sEV is used as a carrier, so that a complex is endowed with good cross-barrier property;
and (B) is as follows: PEDF is used as a therapeutic drug, so that the complex has better long-term safety in the aspect of inhibiting the neovascularization;
and (C) feature: PEDF was loaded onto the sEV vector by means of sonication to construct the nanocomplex.
Further, umbilical cord mesenchymal stem cell-derived sEV was used as a vector.
Further, the small extracellular vesicles slev are shev obtained by means of gradient centrifugation, i.e. extracellular vesicles secreted by cells and having a size between 50 and 200 nm.
Further, the final concentration of sEV in the ultrasonic preparation of the nano-composite is 0.05-100mg/ml, and the final concentration of PEDF in the ultrasonic preparation of the nano-composite is 1-100ug/ml.
Further, the mass ratio of sEV to PEDF in ultrasound is 1-1000:1.
further, the particle size distribution of the PEDF @ sEV nano-composite 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, pulses are: 1-60 seconds on/1-60 seconds off, and the temperature of the mixed solution is controlled at 0-4 ℃ during ultrasonic treatment.
Further, the preparation method comprises the following specific steps:
(1) Preparation and isolation of sEV
Carrying out primary culture on umbilical cord mesenchymal stem cells, collecting cell conditioned medium supernatant, carrying out gradient centrifugation for 300g, 10 minutes, 2000g, 10-15 minutes, 10000g, 30 minutes, 110000g and 70 minutes in sequence to obtain sEV precipitate, carrying out heavy suspension by using sufficient PBS again, centrifuging for 110000g and 70 minutes again, and collecting relatively pure sEV;
(2) Preparation of PEDF @ sEV nanocomposite
Preparation of PEDF solution and sEV solution
The solution is a balanced solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of PEDF solution is 1-100 mug/mL, the concentration of sEV solution is 0.05-100mg/mL, the balanced solution of the salts is PBS balanced salt solution, and the mass ratio of sEV to PEDF is 1-1000:1;
b. preparation of PEDF @ sEV complex by ultrasonic method
Carrying out ultrasonic treatment on the mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, for: 1-30 minutes, pulses are: 1-60 seconds on/1-60 seconds off;
repair of PEDF @ sEV complexes
After the ultrasonic treatment is finished, putting the mixed solution into an environment of 25-37 ℃ for incubation for 30-60 minutes to recover the structure of the sEV;
purification of PEDF @ sEV Complex
After the repair of the PEDF @ sEV complex is finished, an ultrafiltration tube with the molecular weight cutoff of 100-150kDa is used for centrifuging, cleaning and purifying for 2-3 times, PBS is used as a cleaning solution, and the recovery of the nano complex is finished, so that the nano complex for treating the ocular neovascular diseases is obtained;
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; when in recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
The application of the nano-complex for treating the ocular neovascular diseases in the aspect of serving as and/or preparing a medicament for treating the ocular neovascular diseases.
The invention has the advantages and positive effects that:
1. the novel PEDF @ sEV nano-composite material prepared by the ultrasonic method combines the advantages of PEDF and sEV: PEDF enables the nano complex to have better long-term safety in the aspect of inhibiting the proliferation of new vessels, the sEV carrier endows the complex with excellent cross-barrier performance, and compared with pure PEDF, the PEDF @ sEV nano complex has better treatment effect under the same dosage and has stronger potential application value compared with VEGF antibody medicaments which are clinically applied most widely at present.
2. The whole preparation process of the complex is simple and convenient, and the complex 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 sizes of the PEDF @ sEV nano-composite are concentrated, and a typical lipid bilayer structure is shown in the observation of a transmission electron microscope. ELISA results show that PEDF is successfully loaded on sEV, and PEDF @ sEV nano-complex is successfully constructed. Fluorescence labeling experiments confirmed that the loading process did not significantly alter the biological behavior of the uptake of sEV by cells.
3. The PEDF @ sEV nano complex can be prepared by an ultrasonic method during preparation, and the preparation process is simple and convenient. During the preparation process, only the balanced salt solutions of PEDF and sEV are mixed according to a certain proportion, and the mixture is subjected to ultrasonic treatment for 1-30 minutes under the condition of 20-50% of power, wherein 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 washing and purifying by using an ultrafiltration tube to recover the PEDF @ sEV nanometer complex.
4. At present, vascular Endothelial Growth Factor (VEGF) antibody drugs are the gold standard for the first-line treatment of ocular neovascular diseases in clinical practice. VEGF is a key factor for the proliferation of new blood vessels, and VEGF antibody drugs mainly neutralize intraocular secreted VEGF to achieve therapeutic effects, so patients need to perform intraocular injection every 3 months. However, VEGF at normal physiological levels in the eye has normal neurotrophic effects on the retina. With the long-term heavy use of VEGF antibody drugs, the normal neurotrophic effects of VEGF can be inhibited while the VEGF-induced neovascularization is inhibited.
Pigment Epithelium Derived Factor (PEDF) is the most potent factor naturally present in the eye against neovasculature, and when suffering from neovasculature in the eye, PEDF levels are reduced. Methods of treating neovasculature by exogenous administration of PEDF to restore intraocular PEDF levels are safer than VEGF antibody drugs.
5. Small extracellular vesicles (sevs) are natural drug carriers due to their excellent biocompatibility and ability to transport across barriers. Especially, the umbilical cord mesenchymal stem cell-derived sEV has become a star carrier in drug carriers due to the 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 more effectively transported to a focus part, and the treatment effect of the PEDF is improved.
In conclusion, compared with the first-line clinical gold standard VEGF antibody drug, the nano complex for treating the ocular neovascularization diseases prepared by the invention has higher biological safety. By virtue of the excellent carrier characteristic of the sEV derived from the umbilical cord mesenchymal stem cells, the PEDF @ sEV improves the treatment effect of the PEDF, is expected to be applied to ocular administration treatment of retinal neovascular diseases in the future, and is expected to further complete the innovation of the treatment method.
6. The invention aims to design and prepare a nano complex for treating ocular neovascular diseases. The nano composite object is a PEDF @ sEV nano composite constructed by loading Pigment Epithelium Derived Factor (PEDF) by using Small extracellular vesicles (sEV) derived from umbilical cord mesenchymal stem cells. We loaded PEDF onto the smev by means of sonication of the mixture of smev and PEDF, forming a nanocomplex. The nano-complex presents a typical lipid bilayer structure of a biological vesicle in the detection of a transmission electron microscope, and PEDF loading does not cause obvious morphological structure change on sEV. The detection results of the ELISA kit indicated that we successfully loaded PEDF onto the sov. Nanosight (particle sizer) measurements showed that the loading of PEDF did not alter the overall particle size distribution of the sov. Fluorescence labeling experiments confirmed that PEDF loading did not have a significant effect on the biological activity of the sEV. In vitro and in vivo experiments prove that the PEDF @ sEV nano complex has obviously improved treatment effect compared with 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 of the particle size distribution of the PEDF @ sEV nanocomposite prepared according to example 1 of the present invention; the PEDF @ sEV nano-composite is tested by a particle sizer, and the particle size of the PEDF @ sEV nano-composite is mainly and intensively distributed between 100 nm and 200 nm;
FIG. 3 is a graph showing the cellular efficient uptake of PEDF @ sEV nanocomposites prepared according to example 1 of the present invention; incubation of red dye-labeled pedf @ sov with retinal vascular endothelial cells for 24 hours (panel a) and 48 hours (panel B) revealed efficient uptake of the complex by the cells;
FIG. 4 is a graph showing that PEDF @ sEV, prepared according to example 1, has a significant inhibitory effect on the endothelial growth factor (VEGF) proliferation-promoting effect; the PEDF @ smev nanocomplexes proliferated endothelial cells more significantly than pure PEDF and smev;
FIG. 5 is a graph showing that PEDF @ sEV prepared according to example 1 of the present invention has a significant inhibitory effect on retinal neovascularization in OIR model mice; compared with pure PEDF and sEV, the PEDF @ sEV nano complex has more obvious inhibition on the proliferation of new vessels in an OIR animal model;
FIG. 6 is a graph showing the significant inhibitory effect of PEDF @ sEV prepared in accordance with example 1 of the present invention on the retinal non-perfusion areas of OIR model mice; compared with pure PEDF and sEV, the PEDF @ sEV nano complex has more obvious inhibition on the area of a non-perfusion area in an OIR animal model;
FIG. 7 is a structural diagram of a complete double-layer film of the PEDF @ sEV nano-composite (25% power) in the invention under a transmission electron microscope;
FIG. 8 is a structural diagram of a complete bilayer membrane of the PEDF @ sEV nanocomposite (30% power) in the present invention under a transmission electron microscope.
Detailed Description
The present invention is described in detail below with reference to the following examples, which are intended to be illustrative and not limiting, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A nano-complex for treating ocular neovascular diseases is prepared by loading pigment epithelium derived factor PEDF onto a small extracellular vesicle sEV (extracellular vesicle) derived from umbilical cord mesenchymal stem cells by using an ultrasonic method to prepare a PEDF @ sEV nano-complex.
Preferably, the nanocomposite comprises:
the characteristics are as follows: the sEV is used as a carrier, so that a complex is endowed with good cross-barrier property;
and (B) is as follows: PEDF is used as a therapeutic drug, so that the complex has better long-term safety in the aspect of inhibiting the neovascularization;
and (C) feature: PEDF was loaded onto the sEV vector by means of sonication to construct the nanocomplexes.
Preferably, umbilical cord mesenchymal stem cell-derived sEV is used as a vector.
Preferably, the small extracellular vesicles shev are shev 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 preparing the nano-composite by ultrasonic wave, and the final concentration of PEDF is 1-100ug/ml when preparing the nano-composite by ultrasonic wave.
Preferably, the mass ratio of sEV to PEDF during ultrasonic treatment is 1-1000:1.
preferably, the particle size distribution of the PEDF @ sEV nano-composite is between 100 and 200 nm.
Preferably, the ultrasonic power in the ultrasonic process is 20% -50%, and the time is as follows: 1-30 minutes, pulses are: 1-60 seconds on/1-60 seconds off, and the temperature of the mixed solution is controlled at 0-4 ℃ during ultrasonic treatment.
Preferably, the preparation method comprises the following steps:
(1) Preparation and isolation of sEV
Carrying out primary culture on umbilical cord mesenchymal stem cells, collecting cell conditioned medium supernatant, carrying out gradient centrifugation for 300g, 10 minutes, 2000g, 10-15 minutes, 10000g, 30 minutes, 110000g and 70 minutes in sequence to obtain sEV precipitate, carrying out heavy suspension by using sufficient PBS again, centrifuging for 110000g and 70 minutes again, and collecting relatively pure sEV;
(2) Preparation of PEDF @ sEV nanocomposite
Preparation of PEDF solution and sEV solution
The solution is a balanced solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of PEDF solution is 1-100 mug/mL, the concentration of sEV solution is 0.05-100mg/mL, the balanced solution of the salts is PBS balanced salt solution, and the mass ratio of sEV to PEDF is 1-1000:1;
b. preparation of PEDF @ sEV complex by ultrasonic method
Carrying out ultrasonic treatment on a mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, for: 1-30 minutes, pulses are: 1-60 seconds on/1-60 seconds off;
repair of PEDF @ sEV complex
After the ultrasound is finished, putting the mixed solution in an environment of 25-37 ℃ for incubation for 30-60 minutes to recover the structure of the sEV;
purification of PEDF @ sEV Complex
After the repair of the PEDF @ sEV complex is finished, centrifuging, cleaning and purifying 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 the 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 the complex is 2-10:1; when in recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
The application of the nano-complex for treating the ocular neovascular diseases in the aspect of serving as and/or preparing a medicament for treating the ocular neovascular diseases.
Specifically, the preparation and detection examples are as follows:
example 1:
a nano-complex for treating ocular neovascular diseases is prepared by the following steps:
1. separation and purification of sEV
Collecting sufficient cell conditioned medium supernatant by primary culture of umbilical cord mesenchymal stem cells. After centrifugation of a gradient of 300g (10 min), 2000g (10-15 min), 10000g (30 min) and 110000g (70 min) in sequence, a precipitate of sEV was obtained, which was then resuspended in sufficient PBS and centrifuged again at 110000g (70 min) to collect relatively pure sEV.
2. Preparation of PEDF @ sEV
Preparing solutions of PEDF and sEV by using PBS (balanced salt solution), wherein the concentration of the solution of PEDF is 5ug/ml, the concentration of the solution of sEV is 0.1mg/ml, and the mass ratio of the sEV to the PEDF is 20:1 mixing, the total volume of the solution is 0.5ml.
The power of the ultrasound was set at 20%, the ultrasound time was 3 minutes, and the pulse parameters were 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 at 37 ℃ for 60 minutes to recover the structure of the sEV.
4. Purification of PEDF @ sEV Complex
After the end of the repair of the PEDF @ sEV complex, centrifugation was performed using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa at 4000g for 10 minutes, followed by washing 2 times with PBS. At the time of recovery, 1000g was centrifuged for 2 minutes to recover the concentrated PEDF @ sEV nanocomposite.
Example 2:
a nano-complex for treating ocular neovascular diseases is prepared by the following steps:
1. separation and purification of sEV
And collecting sufficient cell conditioned medium supernatant through primary culture of umbilical cord mesenchymal stem cells. After centrifugation through a gradient of 300g (10 min), 2000g (10-15 min), 10000g (30 min) and 110000g (70 min) in this order, a precipitate of sEV was obtained, which was then resuspended in sufficient PBS and centrifuged again at 110000g (70 min) to collect relatively pure sEV.
2. Preparation of PEDF @ sEV
Preparing solutions of PEDF and sEV by using PBS (balanced salt solution), wherein the concentration of the solution of PEDF is 10ug/ml, the concentration of the solution of sEV is 0.1mg/ml, and the mass ratio of the sEV to the PEDF is 10:1 mixing, the total volume of the solution is 0.5ml.
Setting the ultrasonic power at 25%, ultrasonic time at 2 min and pulse parameter at 2s/2s. The mixture was placed on ice and sonicated.
3. Repair of PEDF @ sEV complexes
After the ultrasound was completed, the mixture was incubated at 37 ℃ for 30 minutes to restore the structure of the sEV.
4. Purification of PEDF @ sEV Complex
After the repair of the PEDF @ sEV complex was completed, centrifugation was carried out using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa at 4000g for 10 minutes, followed by washing 2 times with PBS. At the time of recovery, 1000g was centrifuged for 2 minutes to recover the concentrated PEDF @ sEV nanocomposite.
Example 3:
a nano-complex for treating ocular neovascular diseases is prepared by the following steps:
1. separation and purification of sEV
And collecting sufficient cell conditioned medium supernatant through primary culture of umbilical cord mesenchymal stem cells. After centrifugation of a gradient of 300g (10 min), 2000g (10-15 min), 10000g (30 min) and 110000g (70 min) in sequence, a precipitate of sEV was obtained, which was then resuspended in sufficient PBS and centrifuged again at 110000g (70 min) to collect relatively pure sEV.
2. Preparation of PEDF @ sEV
Preparing solutions of PEDF and sEV by using PBS (balanced salt solution), wherein the concentration of the solution of PEDF is 5ug/ml, the concentration of the solution of sEV is 0.1mg/ml, and the mass ratio of the sEV to the PEDF is 20:1 mixing, the total volume of the solution is 1ml.
The power of the ultrasound was set at 30%, the ultrasound time was 2 minutes, and the pulse parameters were 2s/2s. The mixture was placed on ice and sonicated.
3. Repair of PEDF @ sEV complexes
After the ultrasound is finished, the mixed solution is incubated in an environment at 37 ℃ for 60 minutes to restore the structure of the sEV.
4. Purification of PEDF @ sEV Complex
After the repair of the PEDF @ sEV complex was completed, centrifugation was carried out using an ultrafiltration tube (concentration tube) having a molecular weight cut-off of 100kDa at 4000g for 10 minutes, followed by washing 2 times with PBS. At the time of recovery, 1000g was centrifuged for 2 minutes to recover the concentrated PED F @ sEV nanocomposite.
The correlation test of the present invention is as follows:
1. the detection method adopted is Transmission Electron Microscope (TEM). The sample preparation method comprises the following steps: and (3) dropwise adding 30ul of PEDF @ sEV nano-composite solution with a proper concentration on a carbon support membrane, drying at room temperature for 2 minutes, dropwise adding phosphotungstic acid staining solution, standing for 2 minutes, drying under a mercury lamp for 5 minutes, and detecting by using a transmission electron microscope. The results are shown in fig. 1, 7 and 8, and it can be seen that ultrasound loading did not significantly alter the relatively intact vesicle structure of the sEV.
2. The particle size and distribution of the PEDF @ sEV nano complex are detected by a nano particle analyzer (Naonosight, NTA for short). The detection result is shown in fig. 2, the particle size distribution of the PEDF @ sEV nano complex is mainly concentrated between 100 nm and 200nm, and it can be seen that the particle size distribution of the sEV is not obviously changed by ultrasonic medicine loading.
3. And (3) labeling sEV by using a lipophilic dye pKH26 with red fluorescence, and then constructing a nano complex. The red fluorescent labeled nano-complexes of pedf @ smev were co-cultured with the cells. Cell fixation and staining were performed at 24 hours and 48 hours, respectively, and uptake of the nanocomposite into cells was observed using a fluorescence microscope. Blue is the nucleus and red is the nanocomplex. As a result, as shown in FIG. 3, it can be seen that the PEDF @ sEV nanocomplex can be efficiently taken up by the cell and entered into the cell.
4. The up-regulation of the level of Vascular Endothelial Growth Factor (VEGF) is an important factor in the pathogenesis of neovascular eye diseases. Therefore, VEGF is adopted in cell experiments to promote the proliferation of human retinal vascular endothelial cells (HREC) as an in vitro experiment model. As a result, as shown in fig. 4, at the same concentration, the PEDF @ smev nanocomposite proliferates endothelial cells more significantly than pure PEDF and smev, and the PEDF @ smev nanocomposite has a stronger ability to inhibit cell proliferation. This suggests that the treatment effect of PEDF on proliferation inhibition is enhanced after PEDF is loaded on sEV, and it can also be seen that PEDF and sEV have a synergistic effect in PEDF @ sEV nano complex.
5. An Oxygen-Induced Retinopathy (OIR) model is a widely applied model of ocular neovascularization animals. The OIR model is to place the 7-day-old young mice into an oxygen chamber (high oxygen environment) for 5 days, and then return to the normal environment for further 5 days. Retinal blood vessels in mice begin to develop after birth, and the OIR model has two pathological features: 1. after the mice are fed in an oxygen chamber on the 7 th day, the microvasculature at the central part of the retina of the mice degenerates under the stimulation of a high-oxygen environment to form a non-perfusion area (a non-vascular area); 2. when fed back to normal conditions, the central non-perfused area becomes severely hypoxic, stimulating the retina to form a large number of neovessels with incomplete structure and morphology around the non-perfused area, peaking at postnatal day 17. Therefore, for the above 2 pathological features, the ideal therapeutic drug should have 2-point effect, 1: effectively reducing the area of the non-perfusion area, namely recovering the remodeling of the non-perfusion blood vessel; 2: effectively inhibit the proliferation of pathological new vessels.
As a result, as shown in fig. 5 and fig. 6, the PEDF @ sEV nanocomposite is more effective in inhibiting angiogenesis in the OIR animal model than pure PEDF and sEV, the PEDF @ sEV nanocomposite is more effective in inhibiting the area of the non-perfusion region in the OIR animal model than pure PEDF and sEV, and the PEDF @ sEV nanocomposite is more effective in inhibiting angiogenesis in pathological conditions and reducing non-perfusion than pure PEDF, in which PEDF and sEV in the PEDF @ sEV nanocomposite have a synergistic effect. This advantageously suggests that strev loading with PEDF strongly enhances the therapeutic efficacy of PEDF.
Although the 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 embodiments disclosed.
Claims (10)
1. A nanocomplex for the treatment of ocular neovascular diseases, characterized by: the nano-complex is prepared by loading pigment epithelium derived factor PEDF onto a small extracellular vesicle sEV derived from umbilical cord mesenchymal stem cells by using an ultrasonic method.
2. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the nanocomposite comprises:
the characteristic A is as follows: the sEV is used as a carrier, so that a complex is endowed with good cross-barrier property;
and (B) is as follows: PEDF is used as a therapeutic drug, so that the complex has better long-term safety in the aspect of inhibiting the neovascularization;
c, characteristic: PEDF was loaded onto the sEV vector by means of sonication to construct the nanocomplex.
3. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: cord mesenchymal stem cell-derived sEV is used as a vector.
4. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the small extracellular vesicle sEV is obtained by a gradient centrifugation method, namely an extracellular vesicle which is secreted by cells and has the size of 50-200 nm.
5. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the final concentration of sEV is 0.05-100mg/ml when preparing the nano-composite by ultrasonic wave, and the final concentration of PEDF is 1-100ug/ml when preparing the nano-composite by ultrasonic wave.
6. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the mass ratio of sEV to PEDF in ultrasound is 1-1000:1.
7. the nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the particle size distribution of the PEDF @ sEV nano-composite is between 100 and 200 nm.
8. The nanocomplex for the treatment of ocular neovascular diseases according to claim 1, wherein: the ultrasonic power in ultrasonic is 20-50%, and the time is as follows: 1-30 minutes, pulses are: 1-60 seconds on/1-60 seconds off, and the temperature of the mixed solution is controlled at 0-4 ℃ during ultrasonic treatment.
9. The nanocomplex for the treatment of ocular neovascular diseases according to any one of claims 1 to 8, characterized in that: the preparation method comprises the following specific steps:
(1) Preparation and isolation of sEV
Carrying out primary culture on umbilical cord mesenchymal stem cells, collecting cell conditioned medium supernatant, carrying out gradient centrifugation for 300g, 10 minutes, 2000g, 10-15 minutes, 10000g, 30 minutes, 110000g and 70 minutes in sequence to obtain sEV precipitate, carrying out heavy suspension by using sufficient PBS again, centrifuging for 110000g and 70 minutes again, and collecting relatively pure sEV;
(2) Preparation of PEDF @ sEV nanocomposite
Preparation of PEDF solution and sEV solution
The solution is a balanced solution of salt, and the two solutions are uniformly mixed according to the proportion;
the concentration of PEDF solution is 1-100 mug/mL, the concentration of sEV solution is 0.05-100mg/mL, the balanced solution of the salts is PBS balanced salt solution, the mass ratio of sEV to PEDF is 1-1000:1;
b. preparation of PEDF @ sEV complex by ultrasonic method
Carrying out ultrasonic treatment on the mixed solution of PEDF and sEV on ice, and setting 3 parameters of ultrasonic power, ultrasonic time and ultrasonic pulse;
the ultrasonic power is as follows: 20-50%, for: 1-30 minutes, pulses are: 1-60 seconds on/1-60 seconds off;
repair of PEDF @ sEV complexes
After the ultrasonic treatment is finished, putting the mixed solution into an environment of 25-37 ℃ for incubation for 30-60 minutes to recover the structure of the sEV;
purification of PEDF @ sEV Complex
After the repair of the PEDF @ sEV complex is finished, an ultrafiltration tube with the molecular weight cutoff of 100-150kDa is used for centrifuging, cleaning and purifying for 2-3 times, PBS is used as a cleaning solution, and the recovery of the nano complex is finished, so that the nano complex for treating the ocular neovascular diseases is obtained;
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; when in recovery, the centrifugal speed is 1000-4000g, and the centrifugal time is 1-30 minutes.
10. Use of the nanocomplex for the treatment of ocular neovascular diseases according to any one of claims 1 to 9 as a medicament and/or for the preparation of a medicament for the treatment of ocular neovascular diseases.
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