CN116251125A - Biomimetic mineralized vesicle and preparation method and application thereof - Google Patents

Abstract

The invention discloses a biomimetic mineralized vesicle and its preparation method and application. The biomimetic mineralized vesicle includes a cell-derived vesicle with osteogenic activity, and calcium salt chimera is formed on the surface of the cell-derived vesicle with osteogenic activity. In the crystallization of bisphosphonate, the calcium salt forms a coordination chimera with the phosphono group in the bisphosphonate through calcium ions. The biomimetic mineralized vesicles constructed by the present invention are regulated by vesicles and bisphosphonates to form osteoclastocoupling, so as to achieve the effect of promoting osteogenesis, inhibiting osteoclastosis, targeted delivery, precise release, and continuing to maintain bone mass growth after drug withdrawal .

Description

Biomimetic mineralized vesicle and its preparation method and application

technical field

The present application relates to the field of biomedicine, in particular, to a biomimetic mineralized vesicle and its preparation method and application.

Background technique

Osteoporosis is a serious public health problem that threatens health. At present, the clinical medication for osteoporosis cannot be coordinated to regulate osteoclastogenesis. Although it can improve the symptoms of osteoporosis to a certain extent, there are frequent dosing times, poor compliance, and multi-drug combination. With problems such as side effects accumulating, the therapeutic effect is not very satisfactory. Therefore, there is an urgent need to develop a new drug dosage form that can synergistically regulate bone formation and bone resorption, reduce side effects, and enhance targeting.

Studies have shown that stem cell-derived vesicles have similar tissue repair capabilities to stem cells, can promote osteogenesis and regulate bone metabolism, and are good drugs for the treatment of osteoporosis. However, their own characteristics lead to poor bone targeting and imprecise precision. reach the osteoporotic lesion. Moreover, the vesicles derived from stem cells mainly promote osteogenesis, and do not regulate bone balance well; they do not play a good role in improving the increase of osteoclast activity in the process of osteoporosis. Therefore, how to engineer vesicles has become a new research direction for vesicle therapy.

The phosphate radical in bisphosphonates can specifically bind to hydroxyapatite, thereby playing a targeting role. Previous studies have used bisphosphonates as bone-targeting factors to construct drug carriers for bone-targeted delivery. On the other hand, bisphosphonates have the functions of inhibiting osteoclast activity and promoting osteoclast apoptosis, so they are also the first-line drugs for the clinical treatment of osteoporosis. Patent application CN112717142A discloses a vesicular drug delivery system targeting osteoclasts. In this delivery system, the specific polypeptide TRAP is used as the targeting polypeptide of the vesicles, and bisphosphonates are encapsulated in the vesicles to resist osteoporosis. Drugs can maintain the activity of osteoclasts and treat osteoporosis by releasing internally loaded drugs. However, the delivery system still has technical problems such as insufficient targeting and lack of precise release.

Biomineralization is a common process in organisms that regulates the production of minerals from bioorganic molecules. Unlike mineralization in nature, biomineralization requires elements such as biomolecules, cells, and organic matrices, such as the formation of hydroxyapatite in human bone. The biomimetic mineralization method is a method of simulating biomineralization in vivo by using a mild environment in vitro, and combining inorganic and organic substances. In order to enhance the targeting and precise release of vesicles, regulate osteoclast coupling and treat osteoporosis, it is urgent to develop a new drug formulation.

Contents of the invention

The invention provides a biomimetic mineralized vesicle, the biomimetic mineralized vesicle comprises a cell-derived vesicle with osteogenic activity, and a calcium salt chimeric bisphosphine is formed on the surface of the osteogenic cell-derived vesicle The calcium salt is crystallized, and the calcium salt forms a coordination chimera with the phosphono group in the bisphosphonate through calcium ions.

Specifically, the cells with osteogenic activity are mesenchymal stem cells or induced pluripotent stem cells.

The particle size of the biomimetic mineralization vesicle is 30-1000nm.

The present invention also provides a method for preparing the aforementioned biomimetic mineralized vesicles. The preparation method includes mixing and reacting cell-derived vesicles with osteogenic activity, calcium ion solution and bisphosphonate solution, and centrifuging after the reaction is completed. precipitation.

Specifically, (1) the preparation of the biomimetic mineralized vesicles is carried out in a neutral buffer;

(2) the calcium ion is derived from a soluble calcium salt solution;

(3) the bisphosphonate has at least one phosphono group;

(4) The reaction temperature does not exceed 42°C;

(5) The centrifugation condition is more than 1000G, and the centrifugation time is more than 5min;

(6) The reaction time is more than 0.5h.

Preferably, the reaction temperature is 37°C; the centrifugation condition is 20000G for 10min;

The reaction time is 1 h.

More specifically, (1) the soluble calcium salt solution is a calcium chloride solution;

(2) The bisphosphonate is alendronate sodium or zoledronic acid;

(3) The neutral buffer is selected from D-PBS, PBS, physiological saline, α-MEM or DMEM.

More specifically, add mesenchymal stem cell-derived vesicles to phosphate buffer solution to obtain solution A with a final protein concentration of 1 mg/ml; add 20 ul of ₅₀₀ mM CaCl solution and 50 mM zoledronic acid solution to each ml of solution A 10ul, 37°C, continuously stirred for 1h, centrifuged at 20000G for 10min to collect the precipitate, washed twice with D-PBS to obtain biomimetic mineralized vesicles.

The present invention also provides the application of the aforementioned biomimetic mineralized vesicle and the preparation method in the preparation, treatment or prevention of osteoporosis or bone repair and other diseases involving osteoblast and osteoclast regulation imbalance.

The beneficial effects of the present invention include:

(1) The present invention utilizes the method of biomimetic mineralization to induce the crystallization of calcium phosphate on the surface of stem cell-derived vesicles, and further utilizes the coordination between a phosphono group in the bisphosphonate and calcium ions to insert the bisphosphonate into the In the mineralization shell, because the other phosphono group of the bisphosphonate is in a free state, it still has bone-targeting ability, thus constructing a biomimetic mineralization vesicle with bone-targeting ability, through vesicles and bisphosphonate Salt regulates osteoclast coupling to achieve the purpose of promoting bone formation and inhibiting osteoclastosis.

(2) The present invention utilizes the bone-targeting ability of bisphosphonates, preferably zoledronic acid and alendronate sodium, and the bone-targeting ability is significantly better than various polypeptide-binding drugs. Moreover, the biomimetic mineralized vesicles of the present invention can maintain a stable existence in serum, and the surface crystallization of the biomimetic mineralized vesicles can quickly disintegrate to expose the vesicles under the local acidic conditions of osteoporosis. Biomimetic mineralized vesicles can achieve targeted delivery, precise release, and coordinated regulation.

(3) The biomimetic mineralization vesicles constructed by the present invention can continue to maintain bone mass growth after drug withdrawal, which makes up for the side effects of rapid loss of bone mass after clinical drug withdrawal.

Description of drawings

Figure 1 is a schematic diagram of the preparation of biomimetic mineralized vesicles;

Figure 2 is the electron micrograph of cell-derived vesicles and biomimetic mineralized vesicles prepared in Example 1 of the present invention, wherein A is the transmission electron micrograph of cell-derived vesicles, B is the transmission electron micrograph of biomimetic mineralized vesicles, and C is the biomimetic mineralization Scanning electron micrograph of vesicles;

Fig. 3 is the time-varying curve of the particle size and potential of the biomimetic mineralized vesicle prepared in Example 1 of the present invention in serum, wherein A is the particle size change curve, and B is the potential change curve;

Figure 4 is the release curve of the biomimetic mineralized vesicles prepared in Example 1 of the present invention under different pH conditions; wherein, A is the release curve of zoledronic acid in the biomimetic mineralized vesicles under different pH conditions, and B is the biomimetic mineralized vesicles Calcium ions in the vesicles are released under different pH conditions;

Fig. 5 is the in vivo bone targeting observation pictures of each fluorescently labeled vesicle treatment group at different times;

Fig. 6 is the bone-targeted in vitro observation pictures of fluorescently labeled vesicle treatment groups;

Figure 7 is a model diagram of osteoporosis mice treated with bionic mineralized vesicles;

Fig. 8 is the Micro-CT 3D reconstruction figure of each treatment treatment osteoporosis mouse in embodiment 4;

Fig. 9 is each treatment in embodiment 4 to the Micro-CT quantitative analysis result of osteoporosis mouse, wherein, A is the bone volume fraction (BV/TV) comparison of each group, B is the bone mineral density (BMD) comparison of each group , C is the comparison of trabecular bone separation (Tb.Sp) of each group, D is the comparison of trabecular bone number (Tb.N) of each group;

Fig. 10 is the new bone situation figure observed by bone histomorphometry;

Figure 11 is a quantitative map of bone histomorphometry, where A is the comparison of mineralization deposition rates of each group, and B is the comparison of bone formation rates of each group;

Figure 12 is a model diagram of observing the bone mass maintenance of biomimetic mineralized vesicles after drug withdrawal;

Figure 13 is the micro-CT comparison chart before and after drug withdrawal in each group;

Figure 14 is the quantitative analysis diagram of micro-CT dynamic changes at different times in each group, where A is the dynamic change of bone volume fraction over time, B is the dynamic change of bone density over time, C is the dynamic change of bone trabecular separation over time, and D is the dynamic change of bone volume fraction over time. is the dynamic change of the number of trabecular bone with time;

Fig. 15 is a diagram showing the effect of bone destruction caused by the treatment of osteomyelitis with bionic mineralized vesicles.

Detailed ways

The present invention will be further illustrated and described below in conjunction with the embodiments, but the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation of a kind of biomimetic mineralization vesicle

The biomimetic mineralization method was used to induce calcium phosphate crystallization on the surface of vesicles, as shown in Figure 1, a one-pot method was used to add osteogenic cell-derived vesicles, calcium ions, bisphosphonates and neutral buffer (D- PBS) and mix well, and the calcium ions are derived from soluble calcium salts. Calcium ions combine with phosphate in neutral buffer to generate calcium phosphate, and the cell-derived vesicle membrane with osteogenic activity contains negatively charged groups, which can adsorb calcium ions and provide nucleation for calcium phosphate crystallization on the vesicle surface A phosphono group of the bisphosphonate can specifically bind calcium ions, so as the reaction time prolongs, crystal grains containing calcium phosphate chimeric bisphosphonate gradually form on the surface of the vesicles.

In this example, the cell-derived vesicles with osteogenic activity are mesenchymal stem cell (MSC)-derived vesicles, the bisphosphonate is zoledronic acid (ZOL), the calcium salt is calcium chloride (CaCl ₂ ), phosphoric acid D-PBS buffer solution (Duchener's phosphate buffered saline) was selected as the buffer solution.

Add the MSC-derived vesicles to the D-PBS buffer solution to ensure that the final concentration is 1mg/ml (protein quantification) to obtain the D-PBS solution of the vesicles; add 20ul of 500mM _CaCl2 solution and 10ul of 50mM ZOL solution to 1ml of this solution, Stir continuously at 37°C for 1 h, centrifuge at 20,000 G for 10 min to collect the precipitate, and wash twice with D-PBS to obtain biomimetic mineralized vesicles. The particle size of the obtained biomimetic mineralized vesicles reaches about 150 nm (as shown in B in FIG. 2 ).

Example 2. Detection of Stability and Acid Sensitivity of Biomimetic Mineralized Vesicles

(1) Put the biomimetic mineralized vesicles constructed in Example 1 into the serum with pH=7.4, and observe the changes of particle size and Zeta potential within one week. As shown in Figure 3, the particle size of the biomimetic mineralized vesicles remained stable within a week, and the potential decreased slightly, indicating that the prepared biomimetic mineralized vesicles could be used for subsequent intravenous administration.

(2) Acid sensitivity detection of biomimetic mineralized vesicles

The release of the content of biomimetic mineralized vesicles plays an important role in its drug action and delivery of biologically active substances in vivo. The biomimetic mineralized vesicle samples constructed in Example 1 were respectively placed in two different pH solutions of pH=4.5 and pH=7.5 to simulate the degradation in the local acidic microenvironment of osteoporosis and the normal physiological environment. The real-time observation of the calcium ion concentration (as shown in Figure 4) shows that the calcium ion release of the biomimetic mineralized vesicles does not exceed 20% within 24 hours under a physiological environment, indicating that the biomimetic mineralized vesicles can exist stably. However, in an acidic environment, more than 80% of calcium ions can be released within 1 hour and completely released within 2 hours. It shows that the calcium phosphate shell can be dissolved rapidly in an acidic environment, exposing the inner vesicle. The release of zoledronic acid was detected, and the results showed that in an acidic environment, zoledronic acid was completely released within 2 hours, which also indicated that the calcium phosphate shell could dissolve rapidly, exposing the vesicles, and then play a role in local regulation of osteoporosis Form osteoclast coupling and treat osteoporosis.

Example 3, distribution of biomimetic mineralized vesicles in vivo

(1) In vivo imaging results of biomimetic mineralized vesicles

In order to prove that biomimetic mineralized vesicles are targeted, vesicles, calcium phosphate@vesicles (only calcium phosphate and vesicles were prepared in one pot, excluding bisphosphonates), and biomimetic mineralized vesicles were fluorescently labeled , and performed fluorescence imaging at different times. The results are shown in Figure 5. It can be seen from the figure that the biomimetic mineralized vesicles can accurately target the lower limb bone (the most severe part of mouse osteoporosis), and the fluorescence contour is consistent with the lower limb femur and tibia.

(2) In vitro imaging results of fluorescently labeled biomimetic mineralized vesicles

The two groups of mice were collected, and important organs including the heart, liver, spleen, kidney and lower limb bones were dissected out, and then in vitro fluorescence imaging was performed to compare the fluorescence content in different organs. The results are shown in Figure 6. From the figure It can be seen that the biomimetic mineralized vesicle group has obvious accumulation in the bone, and is less distributed in other organs than the vesicle group and calcium phosphate@vesicle group.

Example 4. Biomimetic Mineralized Vesicles Treating Osteoporosis Curative Effect and Mechanism Exploration

At present, the commonly used model for evaluating the curative effect of osteoporosis is the osteoporosis mouse model, which is constructed by castration of mice to simulate the state of osteoporosis in postmenopausal women. In order to further explore the therapeutic effect of biomimetic mineralized vesicles on osteoporosis, the present invention simulates postmenopausal female osteoporosis by constructing a mouse castrated osteoporosis model.

(1) Model map of osteoporosis mice treated with bionic mineralized vesicles

As shown in FIG. 7 , in order to evaluate the curative effect of bionic mineralized vesicles, the present invention first constructed an osteoporosis mouse model, and continued to culture it for 8 weeks after ovariectomy, and the osteoporosis mouse model was successfully constructed. At the same time, mouse bone marrow stem cells were extracted and vesicles were extracted, and biomimetic mineralized vesicles were constructed according to the method in Example 1. Afterwards, intravenous administration was carried out, once a week, and tissue samples were collected and efficacy evaluation was performed after continuous administration for 8 weeks. In order to ensure the efficacy analysis of each component of this dosage form as much as possible, the grouping design of this part of the study took into account a single component group, a combination of two components, a simple mixing group of three components, and a PBS control group. Specifically divided into 10 groups, including control group (no administration); vesicle group; calcium phosphate group; zoledronic acid group; calcium phosphate@vesicle group; calcium phosphate@zoledronic acid group (no vesicle , simply use calcium phosphate and zoledronic acid for biomimetic mineralization to form microspheres); zoledronic acid + vesicle group (mixing zoledronic acid and vesicles together); calcium phosphate + vesicles + azole Ledronic acid group (calcium phosphate, zoledronic acid and vesicles were mixed together); biomimetic mineralized vesicle group; healthy group. Each set has 8 repetitions. The purpose of grouping is to evaluate the efficacy of each ingredient in detail, and to compare the efficacy of purely mixed and constructed biomimetic mineralized vesicles, in order to obtain comprehensive and systematic result data.

(2) Micro-CT results

Figure 8 shows Micro-CT cross-sectional reconstruction and sagittal plane reconstruction. The results of 10 groups of Micro-CT showed that the bone mass of different groups was significantly different. The bone mass was slightly increased; vesicles, zoledronic acid, and calcium phosphate supplementation all had a certain degree of improvement in bone mass. The two-component group and the three-component mixed group had a greater increase in bone mass, but it did not exceed that of healthy mice. Further comparative analysis of the biomimetic mineralized vesicle group showed that the cross-sectional bone trabecular number of the bionic mineralized vesicle group was the largest, which was more than that of healthy mice at the same age, indicating that its potent osteogenesis-promoting effect.

Figure 9 shows the results of Micro-CT quantitative analysis of osteoporotic mice for each treatment. It can be seen from Figure 9 that the bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular bone density of the biomimetic mineralized vesicle group The number (Tb.N) was significantly higher than that of other groups, and the degree of separation of trabecular bone (Tb.Sp) was significantly lower than that of other groups, indicating that the treatment effect was significant.

(3) Observation of new bone by bone histomorphometry

Calcein and alizarin complexes were used to observe osteometric conditions. The purpose of the study was to observe the new bone conditions within 7 days through the characteristics of calcein and alizarin complexes that can be deposited in new bone. The results of the study are shown in Figure 10. It can be seen from the figure that the new bone in the control group was the least, indicating that the osteogenesis ability decreased significantly during the process of osteoporosis, which is also an important reason for the gradual aggravation of osteoporosis. The distance between calcein and alizarin complexes in the biomimetic mineralized vesicle group was significantly higher than that in the other groups. The bone mineralization deposition rate and bone formation rate were quantified, and the results are shown in Figure 11. From Figure 11, it can be seen that the bone mineralization deposition rate and bone formation rate of the biomimetic mineralization vesicles were significantly higher than those of other groups.

Example 5. Evaluation of curative effect maintenance of biomimetic mineralized vesicles after drug withdrawal

Comparing biomimetic mineralizing vesicles with other clinical first-line drugs, and exploring the bone mass maintenance of different drugs after drug withdrawal, so as to comprehensively evaluate the efficacy and safety of biomimetic mineralizing vesicles, and provide evidence support for subsequent clinical transformation .

(1) Observation model diagram of bone mass maintenance after drug withdrawal of biomimetic mineralized vesicles

As shown in Figure 12, in order to evaluate the improvement of bone loss after drug withdrawal by bionic mineralized vesicles, the present invention first constructed an osteoporosis mouse model, and continued to culture for 8 weeks after ovariectomy, and the osteoporosis mouse model was successfully constructed . At the same time, mouse bone marrow stem cells were extracted and vesicles were extracted, and biomimetic mineralized vesicles were constructed according to the method in Example 1. Afterwards, intravenous administration was carried out once a week, and the administration was continued for 8 weeks, and then the administration was stopped for 8 weeks. At the 36th week, tissue samples were collected and efficacy evaluation was performed. The research group was divided into 4 groups, including control group (n=8), zoledronic acid group (n=8), teriparatide group (n=8), and biomimetic mineralized vesicle group (n=8 ). The purpose of grouping is to compare biomimetic mineralized vesicles with two typical clinical first-line drugs in detail, in order to obtain comprehensive and systematic result data.

(2) Micro-CT results

The distribution of bone trabeculae in each group is significantly different in different periods. According to the cross-sectional reconstruction diagram of the mice in Figure 13, the changes in bone mass of the four groups at the 28th week (administration for 8 weeks) and the 36th week (drug withdrawal for 8 weeks) can be seen. It can be seen that the bone mass of the control group continued to decrease, while the bone mass of the zoledronic acid group increased after 8 weeks of drug withdrawal; the bone mass of the teriparatide group was rapidly lost after 28 weeks of drug withdrawal, and by the 36th week The bone mass at week 1 was lower than that before treatment (20th week), and lower than that of the control group at the same period. By (Figure 14) dynamic quantitative comparison of bone volume fraction (BV/TV), bone density (BMD), trabecular bone number (Tb.N), and bone trabecular separation (Tb.Sp), it can be seen in 12 There was no difference between the groups, but after the osteoporosis model was completed, the bone volume fraction, the number of trabeculae, and bone density were all significantly reduced, indicating that the model was successfully constructed. After administration, the results showed that at 8 weeks of administration, the biomimetic mineralized vesicles could significantly increase bone mass, and all four micro-CT indicators were improved. The efficacy of zoledronic acid and teriparatide group was similar. However, after 8 weeks of drug withdrawal, the biomimetic mineralized vesicles could maintain and further increase bone mass, and the bone mass of the zoledronic acid group also increased slightly, while the bone mass of the teriparatide group decreased rapidly, and its bone volume fraction returned to At the 20-week level, the number of trabecular bone, bone density and bone volume fraction were less than those at 20 weeks.

Example 6. Evaluation of Bone Destruction Caused by Osteomyelitis Treated by Biomimetic Mineralized Vesicles

By comparing the biomimetic mineralized vesicles with other clinical drugs, and by exploring the treatment of different drugs on bone destruction caused by osteomyelitis, the curative effect of the biomimetic mineralized vesicles was comprehensively evaluated, and provided evidence support for subsequent clinical transformation.

In order to evaluate the bone loss caused by bionic mineralized vesicles in the treatment of osteomyelitis, the present invention firstly constructed a mouse model of osteomyelitis bone destruction, and we constructed the osteomyelitis bone destruction model by adding inactivated bacteria and other inflammatory factors through bone marrow cavity puncture , into a mold two weeks later. At the same time, mouse bone marrow stem cells were extracted and vesicles were extracted, and biomimetic mineralized vesicles were constructed according to the method in Example 1. Afterwards, intravenous administration was performed once a week, and tissue samples were collected and curative effect evaluation was performed after 4 weeks of continuous administration. The research group was divided into 4 groups, including control group (n=6), zoledronic acid group (n=6), teriparatide group (n=6), and biomimetic mineralized vesicle group (n=6 ). The biomimetic mineralized vesicles were compared with two typical clinical first-line drugs to provide reference for future clinical drugs. As shown in Figure 15, it can be seen that the bone volume fraction (BV/TV), bone mineral density (BMD), and bone trabecular number (Tb.N) of the bionic mineralized vesicle group were significantly higher than those of the control group and clinical medication In the treatment group, the degree of trabecular separation (Tb.Sp) was lower than that in the other groups, indicating that it has a significant therapeutic effect on bone destruction caused by osteomyelitis.

Claims (8)

1. A biomimetic mineralized vesicle, characterized in that the biomimetic mineralized vesicle source comprises a cell-source vesicle with osteogenic activity, the surface of the cell-source vesicle with osteogenic activity is formed with crystals of calcium salt chimeric bisphosphonate, and the calcium salt forms coordination chimeric with a phosphono group in the bisphosphonate through calcium ions.

2. The biomimetic mineralized vesicle of claim 1, wherein the cells with osteogenic activity are mesenchymal stem cells or induced pluripotent stem cells.

3. The biomimetic mineralized vesicle of claim 1, wherein the particle size of the biomimetic mineralized vesicle is 30-1000nm.

4. A method for preparing a biomimetic mineralized vesicle according to any one of claims 1 to 3, comprising uniformly mixing and reacting a cell-derived vesicle having osteogenic activity, a calcium ion solution and a bisphosphonate solution, and centrifuging to obtain a precipitate after the reaction is completed.

5. The method of claim 4, further comprising one or more of the following (1) - (6):

(1) The biomimetic mineralized vesicles are prepared in neutral buffer;

(2) The calcium ions are derived from a soluble calcium salt solution;

(3) The bisphosphonate has at least one phosphono group;

(4) The reaction temperature is not more than 42 ℃;

(5) The centrifugation condition is more than 1000G for more than 5 min;

(6) The reaction time is more than 0.5 h.

6. The method of claim 5, further comprising one or more of the following (1) - (3):

(1) The soluble calcium salt solution is calcium chloride solution;

(2) The bisphosphonate is alendronate sodium or zoledronic acid;

(3) The neutral buffer is selected from D-PBS, physiological saline, alpha-MEM or DMEM.

7. The method of claim 5, further comprising one or more of the following (1) - (3):

(1) The reaction temperature is 37 ℃;

(2) The centrifugation condition is 20000G for 10min;

(3) The reaction time was 1h.

8. Use of the biomimetic mineralized vesicles according to any one of claims 1 to 3 and the preparation method according to any one of claims 4 to 7 for preparing a medicament for treating or preventing osteoblasts and osteoclast function-controlling diseases related to bone mass reduction, bone destruction and bone defects.

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