Nanoparticles for targeted removal of senescent cells in diabetic wounds and preparation method thereof

Abstract

This invention relates to nanospheres for targeted removal of senescent cells in diabetic wounds and their preparation method. The nanospheres of this invention are galactose-modified PLGA-loaded Fe3O4 nanospheres. Polylactic acid (GAL) is grafted onto the surface of polylactic acid-glycolic acid copolymer (PLGA) microspheres to form a GAL-PLGA complex. Fe3O4 is encapsulated within the complex to form Fe3O4-loaded nanospheres. When the microspheres are engulfed and enter senescent cells in the diabetic wound, they enter lysosomes. Utilizing the increased β-galactosidase activity in senescent cells, the surface GAL can be degraded by the increased activity of β-galactosidase, selectively and slowly releasing the Fe3O4 encapsulated within the microspheres into the senescent cells. The free iron ions generated in the acidic microenvironment of the lysosome induce ferroptosis in the senescent cells, thereby achieving the goal of targeted removal of senescent cells in diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the field of promoting the healing of diabetic wounds, and relates to a nanosphere with a targeting function, specifically a nanosphere for targeted removal of senescent cells in diabetic wounds and its preparation method. Background Technology

[0002] Chronic diabetic wounds often fail to heal, and severe ulcers can even lead to amputation. Current research has not fully elucidated the fundamental pathophysiological mechanisms of diabetic wounds, necessitating more precise treatment methods. Recent studies have shown that a large number of senescent cells in diabetic wounds affect wound healing. However, there is a lack of targeted research on eliminating senescent cells and reducing their side effects to promote wound healing in diabetic wounds. Therefore, new treatment methods are needed to reduce cellular senescence in diabetic wounds and thus promote healing. Senescent cells may be an ideal local target for treating diabetic ulcers and preventing wound chronicity and acute recurrence.

[0003] Cellular senescence is an irreversible state of cell cycle arrest. With the accumulation of senescent cells under pathological conditions, iron homeostasis is disrupted in systemic or local tissues. Iron ions accumulate in senescent cells, and ferritin, which stores iron, also accumulates extensively within the cells, leading to a decrease in free iron ions. This results in insensitivity to ferroptosis, a programmed cell death regulated by iron ions. Therefore, increasing the amount of free iron ions within cells can induce ferroptosis in senescent cells, clearing senescent cells from wounds and promoting wound healing.

[0004] The most common characteristic of senescent cells is elevated lysosomal β-galactosidase activity, which has been used as a marker of aging. In addition to β-galactosidase, the activities of other lysosomal hydrolases are also elevated in senescent cells. In fact, senescent cells exhibit significant lysosomal accumulation, as well as abnormal endosome transport and autophagy. Furthermore, studies have shown that damaged or diseased tissues contain β-galactosidase-positive cells, while cells in normal healthy tissues are negative. Therefore, using β-galactosidase as a unique marker of senescent cells could be used to preferentially deliver drugs to tissues containing a large number of senescent cells.

[0005] As a synthetic polymer, poly(lactic-co-glycolic acid) copolymer (PLGA) exhibits good biocompatibility and biodegradability. The degradation products of PLGA are non-toxic because the two constituent monomers (lactic acid and glycolic acid) are endogenous and readily metabolized by the body through the citric acid cycle. Furthermore, due to its slow degradation in vivo, PLGA microspheres are frequently used as an ideal carrier for sustained-release drugs, serving as a preferred drug delivery system. Summary of the Invention

[0006] In view of the aforementioned clinical problems, this invention provides nanospheres for targeted removal of senescent cells in diabetic wounds.

[0007] The nanospheres for targeted removal of senescent cells in diabetic wounds described in this invention are galactose-modified PLGA-loaded Fe3O4 nanospheres. Galactose (GAL) is grafted onto the surface of the polylactic acid glycolic acid copolymer (PLGA) microspheres to form a GAL-PLGA complex. Fe3O4 is encapsulated inside the complex to form Fe3O4-loaded nanospheres.

[0008] The present invention also provides a method for preparing the aforementioned nanospheres, comprising the following steps:

[0009] A. Galactose (GAL) and polylactic acid glycolic acid copolymer (PLGA) are dissolved in N,N-dimethylformamide (DMF), and GAL and PLGA are grafted together through esterification to form a GAL-PLGA complex.

[0010] B. Add cold deionized water to the DMF solution of GAL-PLGA to precipitate GAL-PLGA; centrifuge, filter, wash with water, and freeze dry to obtain GAL-PLGA complex particles.

[0011] C. GAL-PLGA and oleic acid (OA) modified Fe3O4 (OA-Fe3O4) were dissolved in the organic solvent dichloromethane to form an organic phase; the organic phase was then added to the aqueous phase and emulsified using an ultrasonic disruptor to obtain a suspension of GAL-PLGA@Fe3O4 nanospheres.

[0012] D. Stir the GAL-PLGA@Fe3O4 nanosphere suspension overnight to allow the organic solvent dichloromethane to fully evaporate;

[0013] E. Centrifuge at high speed to separate the supernatant from the precipitate, and resuspend the precipitate in deionized water;

[0014] F. Freeze-dry the suspension to obtain GAL-PLGA@Fe3O4 nanospheres.

[0015] Preferably, in step A, the concentration of PLGA is 20 mg / ml.

[0016] Preferably, in step A, the mass ratio of GAL to PLGA is 1:20, and the reaction conditions are: methanesulfonic acid as catalyst, heating in a water bath at 70°C for 24 hours.

[0017] Preferably, in step C, the concentration of the GAL-PLGA complex is 100 mg / ml, and the concentration of OA-Fe3O4 is 100 μg / ml.

[0018] Preferably, in step C, the aqueous phase is a 1% PVA solution.

[0019] Preferably, in step C, the power of the ultrasound is 200W-500W, each session lasts 4 seconds, with a 4-second interval, for a total of 10-15 minutes.

[0020] Preferably, in step D, the overnight stirring refers to stirring at 300 rpm / min for 12-16 hours under ice-water bath conditions.

[0021] Preferably, in step F, the centrifugation speed is 20,000 rpm / min, each time for 10 minutes, for a total of 3-5 times.

[0022] Preferably, except for step A, all other steps are performed in an ice-water bath.

[0023] The nanospheres described in this invention for targeted removal of senescent cells in diabetic wounds are GAL-PLGA@Fe3O4 nanospheres. When the nanospheres are engulfed and enter senescent cells in the diabetic wound, they enter the lysosome. Taking advantage of the increased β-galactosidase activity in senescent cells, the GAL on the surface can be degraded by the increased β-galactosidase activity, causing the Fe3O4 encapsulated in the nanospheres to be selectively and slowly released from the senescent cells. In the acidic microenvironment of the lysosome, free iron ions are generated, inducing ferroptosis in senescent cells, thereby achieving the purpose of targeted removal of senescent cells in diabetic wounds.

[0024] The preparation method of the nanospheres described in this invention has the following characteristics and advantages: First, GAL and PLGA are grafted together to form a GAL-PLGA complex. Then, Fe3O4 is encapsulated in the GAL-modified PLGA nanospheres using a double emulsion method. The nanospheres are phagocytosed into cells and then taken up by lysosomes. The GAL on their surface can be degraded by β-galactosidase with increased activity in senescent cells, causing the encapsulated Fe3O4 to be slowly released within the senescent cells. Due to the acidic microenvironment of the lysosomes, Fe3O4 is decomposed to produce a large amount of iron ions, inducing ferroptosis in senescent cells. In normal cells, the GAL on the surface of the nanospheres cannot be degraded, and Fe3O4 cannot be released. Ultimately, the nanospheres are expelled from the cell via exocytosis. Therefore, through the above mechanism, these nanospheres can selectively release drugs in senescent cells, achieving targeted clearance. Attached Figure Description

[0025] Figure 1This is a schematic diagram illustrating the preparation and mechanism of action of the galactose-modified PLGA-loaded Fe3O4 nanospheres (GAL-PLGA@Fe3O4 nanospheres) used for targeted removal of senescent cells in diabetic wounds, as described in this invention.

[0026] Figure 2 Scanning electron microscope image of GAL-PLGA@Fe3O4 nanospheres.

[0027] Figure 3 The particle size distribution of GAL-PLGA@Fe3O4 nanospheres is shown.

[0028] Figure 4 Potentiogram of GAL-PLGA@Fe3O4 nanospheres.

[0029] Figure 5 Fluorescence image of cellular uptake of RhoB-labeled GAL-PLGA@Fe3O4 nanospheres.

[0030] Figure 6 This demonstrates the proliferative capacity of senescent fibroblasts after treatment with GAL-PLGA@Fe3O4 nanospheres.

[0031] Figure 7 This study demonstrates the viability of senescent cells treated with GAL-PLGA@Fe3O4 nanospheres and the viability of cells treated with ferroptosis inhibitors. Detailed Implementation

[0032] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings, but the content of the present invention is not limited to the specific embodiments.

[0033] The nanospheres described in this invention employ, for example... Figure 1 The preparation method described above is used to prepare the product, and through... Figure 1 The method shown achieves targeted removal of senescent cells in diabetic wounds.

[0034] Example 1: Preparation of GAL-modified PLGA-supported Fe3O4 nanospheres

[0035] The experimental materials are listed in Table 1 below:

[0036]

[0037] This embodiment describes a preferred method for preparing GAL-modified PLGA-supported Fe3O4 nanospheres, comprising the following steps:

[0038] The first step is to dissolve 25 mg of GAL in 25 ml of DMF, then add 500 mg of PLGA and dissolve it completely. Add 8 μl of mesylate as a catalyst and heat in a 70°C water bath for 24 hours.

[0039] The second step involves adding pre-cooled deionized water to the reaction system to precipitate GAL-PLGA completely; centrifuging at 10,000 rpm / min, filtering, and washing with deionized water three times; and then freeze-drying to obtain GAL-PLGA solid powder.

[0040] Thirdly, dissolve 500 mg of GAL-PLGA in 5 ml of DCM, add 500 μl of OA-Fe3O4, and slowly pour it into 60 ml of 1% PVA solution. Emulsify using an ultrasonic homogenizer at 240 W, sonicating for 4 seconds with 4-second intervals, for a total of 15 minutes, under ice-water bath conditions. This emulsifies the Fe3O4-encapsulated GAL-PLGA into spheres.

[0041] Step 4: Stir for 12-24 hours in an ice-water bath at 300 rpm / min to allow the organic solvent to evaporate slowly; centrifuge at 20,000 rpm / min for 10 minutes; filter, wash with water to remove 1% PVA, repeat 3-5 times.

[0042] Step 5: Centrifuge at high speed to separate the supernatant from the precipitate, and resuspend the precipitate in deionized water.

[0043] Step 6: Freeze-dry the suspension of GAL-PLGA nanospheres encapsulated with Fe3O4 to obtain nanosphere powder.

[0044] In the first step described above, the concentration of PLGA is preferably 20 mg / ml.

[0045] In the third step above, the concentration of the GAL-PLGA complex is 100 mg / ml, and the concentration of OA-Fe3O4 is 100 μg / ml.

[0046] In the third step above, the ultrasound power can be 200W-500W, each session lasts 4 seconds, with a 4-second interval, for a total of 10-15 minutes.

[0047] Preferably, in the fifth step above, the centrifugation speed is 20,000 rpm / min, each time for 10 minutes, for a total of 3-5 times.

[0048] Preferably, except for the first step, all other steps can be performed in an ice water bath.

[0049] Figure 2 The image shows a scanning electron microscope (SEM) image of the nanospheres prepared in Example 1. As can be seen from the image, the microspheres prepared by the emulsification-solvent evaporation method have regular morphology and smooth surface.

[0050] Example 2: Detection of particle size and zeta potential of GAL-PLGA@Fe3O4 nanospheres

[0051] 1 mg of GAL-PLGA@Fe3O4 nanospheres was accurately weighed and dispersed in 1 ml of deionized water. The solution was then diluted with deionized water to a suitable concentration so that the microsphere suspension was transparent. The particle size and zeta potential were measured using a Malvern particle size analyzer.

[0052] The results showed that the average particle size of the prepared nanospheres was 260 nm (e.g., Figure 3 As shown), the average Zeta potential is -8.9 mV (as shown). Figure 4 (As shown).

[0053] Example 3: Cell Uptake Experiment

[0054] To label GAL-PLGA@Fe3O4 nanospheres with Rhodamine B, 1 mg of GAL-PLGA@Fe3O4 nanospheres was immersed in 1 ml of Rhodamine B solution in the dark for 24 hours. Dialysis was then performed for 3-5 days to remove the Rhodamine solution.

[0055] Rhodamine B-labeled GAL-PLGA@Fe3O4 nanospheres were diluted to 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml using complete culture medium.

[0056] Microspheres were added to adherent senescent skin fibroblasts, and the uptake of microspheres by the cells was observed after 6 h, 12 h, and 24 h of treatment. The results showed (e.g.) Figure 5 As shown in the figure, nanospheres with a concentration of 100 μg / ml were clearly observed to be phagocytosed by cells after 6 hours of treatment.

[0057] Example 4: Cytotoxicity and Cell Proliferation Experiments

[0058] Senescent fibroblasts were quantified at a rate of 1×10 4 GAL-PLGA@Fe3O4 nanospheres were seeded at a density of 100 μg / ml in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. The nanospheres were then diluted to 100 μg / ml with complete culture medium. Senescent fibroblasts were added at a concentration of 100 μl / well and cultured for 1, 3, 5, and 7 days, followed by cell proliferation assays (CCK8 assay). Results are shown below. Figure 6 As shown, cell activity initially increased and then decreased, indicating that senescent cells treated with microspheres began to die significantly after 3 days.

[0059] Senescent fibroblasts were quantified at a rate of 1×10 4Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. Cells were then treated with complete medium containing 100 μg / ml GAL-PLGA@Fe3O4 nanospheres, the ferroptosis inhibitor DFO, and Fer-1. Cytotoxicity assays (CCK8 assays) were performed 24 h later. Results are as follows: Figure 7 As shown, the cell viability of the group with added DFO and Fer-1 was greater than that of the group with only GAL-PLGA@Fe3O4 nanospheres, indicating that the cell death induced by GAL-PLGA@Fe3O4 nanospheres is ferroptosis.

Claims

1. A nanosphere for targeted removal of senescent cells in diabetic wounds, characterized in that: The nanospheres are galactose-modified polylactic-co-glycolic acid (PLGA) supported Fe3O4 nanospheres. Galactose (GAL) is grafted onto the surface of the PLGA nanospheres to form a GAL-PLGA complex. Fe3O4 is encapsulated inside the complex to form Fe3O4 supported nanospheres.

2. The method for preparing nanospheres as described in claim 1, characterized in that, Includes the following steps: A. Galactose (GAL) and polylactic acid glycolic acid copolymer (PLGA) are dissolved in N,N-dimethylformamide (DMF), and GAL and PLGA are grafted together through esterification to form a GAL-PLGA complex. B. Add cold deionized water to the DMF solution of GAL-PLGA to precipitate GAL-PLGA; centrifuge, filter, wash with water, and freeze dry to obtain GAL-PLGA complex particles. C. GAL-PLGA and oleic acid (OA) modified Fe3O4 (OA-Fe3O4) were dissolved in the organic solvent dichloromethane to form an organic phase; the organic phase was then added to the aqueous phase and emulsified using an ultrasonic disruptor to obtain a suspension of GAL-PLGA@Fe3O4 nanospheres. D. Stir the GAL-PLGA@Fe3O4 nanosphere suspension overnight to allow the organic solvent dichloromethane to fully evaporate; E. Centrifuge at high speed to separate the supernatant from the precipitate, and resuspend the precipitate in deionized water; F. Freeze-dry the suspension to obtain GAL-PLGA@Fe3O4 nanospheres.

3. The preparation method according to claim 2, characterized in that: In step A, the concentration of PLGA is 20 mg / ml.

4. The preparation method according to claim 2, characterized in that: In step A, the mass ratio of GAL to PLGA is 1:20, and the reaction conditions are: methanesulfonic acid as catalyst, heating in a water bath at 70°C for 24 hours.

5. The preparation method according to claim 2, characterized in that: In step C, the concentration of the GAL-PLGA complex is 100 mg / ml, and the concentration of OA-Fe3O4 is 100 μg / ml.

6. The preparation method according to claim 2, characterized in that: In step C, the aqueous phase is a 1% PVA solution.

7. The preparation method according to claim 2, characterized in that: In step C, the power of the ultrasound is 200W-500W, each session lasts 4 seconds, with a 4-second interval, for a total of 10-15 minutes.

8. The preparation method according to claim 2, characterized in that: In step D, overnight stirring refers to stirring at 300 rpm / min for 12-16 hours under ice-water bath conditions.

9. The preparation method according to claim 2, characterized in that: In step F, the centrifugation speed is 20,000 rpm / min, each time for 10 minutes, for a total of 3-5 times.

10. The preparation method according to claim 2, characterized in that: Except for step A, all other steps are performed in an ice water bath.

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