Synthesis and application of gold nanocluster microspheres

Abstract

The invention discloses synthesis and application of gold nanocluster microspheres, and relates to the technical field of medicines. Taking the gold nanocluster, poloxamer and water as an internal water phase, and placing at 4 DEG C; the preparation method comprises the following steps: dissolving a polylactic acid-glycolic acid copolymer in dichloromethane to form an oil phase, dissolving polyvinyl alcohol and sodium chloride in water to form an outer water phase, mixing the inner water phase and the oil phase through a cell crusher to obtain a primary emulsion W1 / O, mixing the W1 / 0 with the outer water phase through stirring to obtain a multiple emulsion W1 / O / W2, and finally stirring the volatile oil phase to obtain cured microspheres. And centrifuging, discarding the supernatant, washing with deionized water for 3-4 times, freezing at-80 DEG C for 2 hours, and drying with a vacuum drying instrument. GA can be continuously released, the toxic and side effects on normal organism cells are small, and the safety is high. And the slow release property is good, and the slow-release drug is especially suitable for long-acting slow-release delivery in locally-closed cavities such as articular cavities.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a sustained-release microsphere for loading glutathione-coated gold nanoclusters (GSH-AuNCs), its preparation method, and its application, for the preparation of drugs for treating local chronic inflammatory or painful diseases such as joint cavities. Background Technology

[0002] Glutathione-coated gold nanoclusters (GSH-AuNCs) have attracted widespread attention in the treatment of local chronic inflammation in recent years due to their good biocompatibility, low toxicity, and potential anti-inflammatory activity. However, GSH-AuNCs have poor in vivo stability, short half-life, and are prone to aggregation, which limits their therapeutic efficacy, requires frequent dosing, and leads to poor patient compliance.

[0003] To address the aforementioned issues, there is an urgent need to develop a delivery system that can improve the stability of gold nanoclusters, achieve sustained release, and reduce the frequency of drug administration. Microspheres, as a mature long-acting drug delivery carrier, possess excellent biodegradability and controllable release properties, making them particularly suitable for the delivery of peptides, proteins, and nanomedicines.

[0004] Polylactic acid-glycolic acid copolymer (PLGA) has been approved by the FDA for drug delivery, exhibiting good biocompatibility and a controllable degradation rate. Therefore, using PLGA microspheres as carriers for gold nanoclusters holds promise for achieving long-term sustained release in sites such as joint cavities, reducing the number of injections, and minimizing injection pain, thus providing a new treatment strategy for localized chronic inflammatory or painful diseases.

[0005] Based on this, we aim to invent a PLGA microsphere capable of encapsulating peptide-gold nanoclusters, thereby improving the stability of the gold nanoclusters and enabling their slow release. Summary of the Invention

[0006] The purpose of this invention is to provide a PLGA microsphere encapsulating gold nanoclusters and its preparation method, which solves the problems of poor stability and rapid release of gold nanoclusters in the prior art, and realizes sustained release and long-term therapeutic effect after intra-articular injection.

[0007] This invention employs a dual-emulsion solvent evaporation method (W1 / O / W2) to encapsulate gold nanoclusters within PLGA microspheres, specifically including the following steps:

[0008] (1) Preparation of internal aqueous phase W1: Gold nanoclusters GA and poloxamer were dissolved in deionized water and allowed to stand at low temperature to form a uniformly dispersed phase;

[0009] (2) Preparation of oil phase O: Polylactic acid-glycolic acid copolymer PLGA was dissolved in dichloromethane;

[0010] (3) Preparation of external aqueous phase W2: Polyvinyl alcohol (PVA) and NaCl are dissolved in water to form a stabilizer solution;

[0011] (4) Preparation of colostrum W1 / O: Under ice bath conditions, the aqueous phase W1 from step (1) and the oil phase O from step (2) are mixed and ultrasonically broken to form colostrum;

[0012] (5) Preparation of double emulsion W1 / O / W2: Pour the primary emulsion from step (4) into the external aqueous phase W2 from step (3) and stir mechanically to form a double emulsion;

[0013] (6) Solvent evaporation and microsphere curing: Stir the polyemulsion obtained in step (5) to evaporate the organic solvent and form microspheres;

[0014] (7) Cleaning and freeze-drying: After centrifugation and cleaning, freeze-drying was performed to obtain yellow loose microsphere powder.

[0015] The gold nanoclusters mentioned above are glutathione gold nanoclusters with a "peptide shell" structure, specifically peptide gold nanoclusters (GA).

[0016] Meanwhile, the poloxamer, polylactic acid-glycolic acid, dichloromethane, and polyvinyl alcohol used are PL188, PLGA, DCM, and PVA, respectively.

[0017] In step (1), the concentration of gold nanoclusters (GA) in the internal aqueous phase is 5.5 mg·mL. -1 ~22mg·mL -1 ; Preferably 22 mg / mL -1 The concentration of poloxamer ranged from 4.4 to 17.6 mg / mL. -1 The preferred dosage is 8.85 mg / mL. -1 .

[0018] In step (2), the concentration of polylactic acid-glycolic acid copolymer (PLGA) in the oil phase is 10–75 mg / mL. -1 The preferred concentration of polylactic acid-glycolic acid copolymer (PLGA) is 75 mg / mL. -1 The molar ratio of LA / GA in polylactic acid-glycolic acid copolymer (PLGA) is 50:50-75:25, for example, 50:50 and 75:25, with Mw of 24000-38000 and 38000-54000 respectively, preferably 50:50, with Mw of 38000-54000.

[0019] In step (3), the mass percentage concentration of polyvinyl alcohol in the external aqueous phase W2 is 0.1-2%, preferably 1%; and the mass percentage concentration of NaCl is 0-5%, preferably 5%.

[0020] In step (4), the volume ratio of the internal aqueous phase W1 to the oil phase O is 1:5 to 20, preferably 1:10.

[0021] In step (5), the volume ratio of oil phase O to external aqueous phase W2 in the re-emulsion W1 / O / W2 is 1:20 to 80, preferably 1:40.

[0022] Furthermore, gold nanoclusters, poloxamer, and water were placed at 4°C as the inner aqueous phase. Polylactic acid-glycolic acid copolymer was dissolved in dichloromethane to form the oil phase, while polyvinyl alcohol and sodium chloride were dissolved in water to form the outer aqueous phase. The inner and oil phases were mixed using a cell disruptor to obtain the primary emulsion W1 / O. W1 / O was then mixed with the outer aqueous phase by stirring to obtain the secondary emulsion W1 / O / W2. Finally, the oil phase was evaporated by stirring to obtain solidified microspheres. The supernatant was then discarded by centrifugation, and the microspheres were washed 3-4 times with deionized water, frozen at -80°C for 2 hours, and dried using a vacuum dryer. The optimal synthesis method was determined based on actual conditions.

[0023] The power range of the cell disruptor during the synthesis of the colostrum is 50-500W, and the time is 30s-5min, preferably 200W / 1min. The stirring speed for the re-emulsion is 400rpm-1200rpm, preferably 1000rpm. The stirring speed for the volatile oil phase is 200-400rpm, preferably 400rpm.

[0024] The beneficial results of this invention are:

[0025] The microspheres synthesized in this invention have good biocompatibility, are non-toxic, and have good drug carrying capacity. The preparation steps are simple, the cost is low, and the amount of reagents required is small.

[0026] This invention can improve the stability of gold nanoclusters of peptide drugs. PLGA can encapsulate GA, preventing its rapid inactivation in the in vivo environment.

[0027] Sustained-release microspheres of gold nanoclusters containing polypeptide drugs were obtained. GA can be continuously released with minimal toxicity to normal cells and high safety. Its excellent sustained-release properties make it particularly suitable for long-term sustained-release delivery into locally closed cavities such as joint cavities. Attached Figure Description

[0028] Table 1 shows the encapsulation efficiency values ​​in the embodiments of the present invention.

[0029] Figure 1a This is a photograph of the yellow, loose solid powder obtained after drying in Example 17 of the present invention.

[0030] Figure 1b Microscopic image of the gold cluster microspheres in Test Example 2;

[0031] Figure 1cThe figure shows the presence of the gold nanocluster microspheres in aqueous solution obtained after the volatilization of dichloromethane in Example 17 of this invention.

[0032] Figure 2a This is a comparison image of gold nanocluster microspheres and blank microspheres under 365nm ultraviolet light in Test Example 3.

[0033] Figure 2b For example 3 GA solutions, GA-MS PLGA-MS fluorescence comparison image.

[0034] Figure 2c Test Example 3: GA-MS laser confocal image

[0035] Figure 3a Test Example 4: GA-MS Scanning Electron Microscope Image

[0036] Figure 3b To test 4 PLGA-MS scanning electron microscope images

[0037] Figure 4 Test Example 5: GA-MS in vitro release diagram

[0038] Figure 5a Figure showing the weight change of rat in test case 6.

[0039] Figure 5b Test Example 6: GA-MS drug concentration changes in rats

[0040] Figure 5c Test Case 6: Retention of rat knee joints by GA-MS after one week

[0041] Figure 5d HE staining image of rat knee joint in Test Example 6

[0042] Figure 5e The accumulation of gold content in rat organs after 1 week and 4 weeks.

[0043] Figure 5f S&F staining image of the knee joint of rat in Test Example 6. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can understand and implement the present invention, and further recognize its advantages.

[0045] Unless otherwise defined in this specification, all technical terms herein are used according to their conventional definitions as commonly used and understood by one of ordinary skill in the art. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; and the reagents and materials described are commercially available unless otherwise specified.

[0046] This invention provides a method for preparing gold nanocluster microspheres, which is essentially a material engineering strategy that integrates nanoscale functional units (gold nanoclusters) into micron-scale carriers through microencapsulation technology to overcome their inherent defects and achieve performance enhancement and macroscopic controllability.

[0047] Example 1

[0048] This embodiment provides a method for preparing PLGA microspheres encapsulated with gold nanoclusters that can provide a sustained-release effect:

[0049] Example 1

[0050] (1) Preparation of inner aqueous phase W1: Gold nanoclusters GA and poloxamer PL188 were dissolved in 200uL of deionized water. The concentration of gold nanoclusters in the inner aqueous phase was 5.5mg / ml and the concentration of PL188 was 4.4mg / mL. The mixture was allowed to stand at 4℃ for 5h to form a uniformly dispersed inner aqueous phase W1 for later use.

[0051] (2) Preparation of oil phase O: Dissolve PLGA (10 mg·mL⁻¹, molecular weight 24000-38000, LA:GA=75:25) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use (the ratio of the aqueous phase to the oil phase is 1:5);

[0052] (3) Preparation of external aqueous phase (W2): Dissolve 0.2g of polyvinyl alcohol (PVA, 0.1% w / v) in 20ml of deionized water, stir overnight, and swell at 4℃ for later use.

[0053] (4) Pour the aqueous phase into the oil phase, and then break it up with a cell disruptor at 50W / 2min to obtain a uniform W1 / O colostrum, which is then placed in an ice bath for later use.

[0054] (5) Formation of double emulsion (W1 / O / W2): The primary emulsion is quickly poured into the pre-cooled external aqueous phase (W2), with a volume ratio of oil phase to external aqueous phase of 1:20. The mixture is mechanically stirred at 800 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0055] (6) Finally, the oil phase was evaporated by stirring at 200 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0056] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate and wash it with pure water 3 times, freeze it at -80℃ for 2 h, and dry it in a freeze dryer for 48 h to obtain a yellow loose powder solid.

[0057] Example 2

[0058] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Embodiment 1 is that in step (1), the concentration of GA in the internal aqueous phase is 22 mg / mL, while all other conditions remain unchanged.

[0059] Example 3

[0060] (1) Dissolve gold nanoclusters GA and poloxamer PL188 in 100uL of deionized water. The concentration of gold nanoclusters in the inner aqueous phase is 22mg / ml, and the concentration of PL188 in the inner aqueous phase is 8.8mg / mL. Let stand at 4℃ for 5h to form a uniformly dispersed inner aqueous phase W1 for later use.

[0061] (2) Preparation of oil phase O: Dissolve PLGA (50 mg·mL⁻¹, preferably molecular weight 2.4-3.8 kDa, LA:GA=75:25) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use. The ratio of the aqueous phase to the oil phase is 1:10.

[0062] (3) Preparation of external aqueous phase (W2): Dissolve 0.2g of polyvinyl alcohol (PVA, 0.1% w / v) in 20ml of deionized water, stir overnight, and swell at 4℃ for later use.

[0063] (4) Under ice bath conditions, the inner aqueous phase was poured into the oil phase, and then the cell disruptor was used to disrupt the cell under ice bath conditions for 200W / 2min to obtain a uniform W1 / O colostrum.

[0064] (5) Formation of double emulsion (W1 / O / W2): The primary emulsion is quickly poured into the pre-cooled external aqueous phase (W2), with a volume ratio of oil phase to external aqueous phase of 1:20. The mixture is mechanically stirred at 800 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0065] (6) Finally, the oil phase was evaporated by stirring at 200 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0066] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate, wash it three times with pure water, freeze it at -80℃ for 2 h, and then freeze it in a freeze dryer for 48 h to obtain a yellow, loose powder solid.

[0067] Example 4

[0068] (1) Preparation of inner aqueous phase W1: Gold nanoclusters GA and poloxamer PL188 were dissolved in 100uL of deionized water and allowed to stand at 4℃ for 5h to form a uniformly dispersed inner aqueous phase W1. The concentration of gold nanoclusters in the inner aqueous phase was 22mg / ml and the concentration of PL188 in the inner aqueous phase was 8.8mg / mL.

[0069] (2) Preparation of oil phase O: Dissolve PLGA (75 mg·mL⁻¹, molecular weight 38000–54000, LA:GA=50:50) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use.

[0070] (3) Preparation of external aqueous phase (W2): Dissolve 0.2g polyvinyl alcohol (PVA, 0.5% w / v) and 0.1g NaCl (0.5% w / v) in 20ml deionized water, stir overnight, and swell at 4℃ for later use.

[0071] (4) Under ice bath conditions, pour the inner aqueous phase into the oil phase, and then continue to use a cell disruptor under ice bath conditions for 200W / 2min to obtain a uniform W1 / O colostrum, which is then placed under ice bath conditions for later use.

[0072] (5) Formation of double emulsion (W1 / O / W2): The colostrum is quickly poured into the pre-cooled external aqueous phase (W2) at a volume ratio of 1:20, and mechanically stirred at 800 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0073] (6) Solvent evaporation and microsphere solidification: Finally, the oil phase was evaporated by stirring at 400 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0074] (7) Microsphere cleaning and freeze drying

[0075] The above solution was centrifuged at 3000 rpm for 10 min. The precipitate was washed three times with pure water, frozen at -80℃ for 2 h, and then dried in a freeze dryer for 48 h to obtain a yellow, loose powder solid.

[0076] Example 5

[0077] (1) Dissolve gold nanoclusters GA and poloxamer PL188 in 100uL of deionized water and let stand at 4℃ for 5h to form a uniformly dispersed internal aqueous phase W1. The concentration of gold nanoclusters in the gold internal aqueous phase is 5.5mg / ml and the concentration of PL188 is 17.8mg / mL.

[0078] (2) Preparation of oil phase O: Dissolve PLGA (75 mg·mL⁻¹, preferably molecular weight 38000-54000, LA:GA=50:50) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use.

[0079] (3) Preparation of external aqueous phase (W2): Dissolve 0.2g of polyvinyl alcohol (PVA, 0.5% w / v) in 40ml of deionized water, stir overnight, and swell at 4℃ for later use.

[0080] (4) Under ice bath conditions, the inner aqueous phase was poured into the oil phase, and then the cell disruptor was used to disrupt the cell under ice bath conditions for 200W / 2min to obtain a uniform W1 / O colostrum.

[0081] (5) Formation of double emulsion (W1 / O / W2): The primary emulsion is quickly poured into the pre-cooled external aqueous phase (W2), with a volume ratio of oil phase to external aqueous phase of 1:20. The mixture is mechanically stirred at 800 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0082] (6) Finally, the oil phase was evaporated by stirring at 200 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0083] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate and wash it with pure water 3 times, freeze it at -80℃ for 2 h, and dry it in a freeze dryer for 48 h to obtain a yellow loose powder solid.

[0084] Example 6

[0085] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the external aqueous phase in step (3) is 0.4g of PVA solution, while all other conditions remain unchanged.

[0086] Example 7

[0087] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the external aqueous phase in step (3) is 0.8g of PVA solution, while all other conditions remain unchanged.

[0088] Example 8

[0089] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the cell disruption time in step (3) is 200W / 1min, while all other conditions remain unchanged.

[0090] Example 9

[0091] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that 0.4g of NaCl is added to the external aqueous phase in step (3), while all other conditions remain unchanged.

[0092] Example 10

[0093] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that 2g of NaCl is added to the external aqueous phase in step (3), while all other conditions remain unchanged.

[0094] Example 11

[0095] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the power of the cell disruptor in step (4) is 300W / 1min, while all other conditions remain unchanged.

[0096] Example 12

[0097] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the speed of the magnetic stirrer in step (5) is 1000 rpm, while all other conditions remain unchanged.

[0098] Example 13

[0099] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the speed of the magnetic stirrer in step (5) is 1200 rpm, while all other conditions remain unchanged.

[0100] Example 14

[0101] This embodiment provides a method for preparing gold cluster microspheres. The only difference between this method and Example 5 is that the power of the cell disruptor in step (4) is 100W / 1min, while all other conditions remain unchanged.

[0102] Example 15

[0103] (1) Dissolve 50uL (22mg / ml) gold nanoclusters GA and 8.8mg poloxamer PL188 in 50uL of deionized water, let stand at 4℃ for 5h to form a uniformly dispersed internal aqueous phase W1 for later use.

[0104] (2) Preparation of oil phase O: Dissolve PLGA (75 mg·mL⁻¹, preferably molecular weight 38000-54000, LA:GA=50:50) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use.

[0105] (3) Preparation of external aqueous phase (W2): Dissolve 0.4g polyvinyl alcohol (PVA, 1% w / v) and 0.4g NaCl (5%) in 40ml deionized water, stir overnight, and swell at 4℃ for later use.

[0106] (4) Under ice bath conditions, the inner aqueous phase was poured into the oil phase, and then the cell disruptor was used to disrupt the cell under ice bath conditions for 200W / 2min to obtain a uniform W1 / O colostrum.

[0107] (5) Formation of double emulsion (W1 / O / W2): The colostrum is quickly poured into the pre-cooled external aqueous phase (W2) at a volume ratio of 1:40, and mechanically stirred at 1000 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0108] (6) Finally, the oil phase was evaporated by stirring at 400 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0109] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate and wash it with pure water 3 times, freeze it at -80℃ for 2 h, and dry it in a freeze dryer for 48 h to obtain a yellow loose powder solid.

[0110] Example 16

[0111] (1) Dissolve gold nanoclusters GA and poloxamer PL188 in 100uL of deionized water. In the inner aqueous phase, the concentration of gold nanoclusters is 22mg / ml and the concentration of PL188 is 8.8mg / mL. Let stand at 4℃ for 5h to form a uniformly dispersed inner aqueous phase W1 for later use.

[0112] (2) Preparation of oil phase O: Dissolve PLGA (75 mg·mL⁻¹, preferably molecular weight 3.8-5.4 kDa, LA:GA=50:50) in 2 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use. The ratio of the aqueous phase to the oil phase is 1:20.

[0113] (3) Preparation of external aqueous phase (W2): Dissolve 0.4g polyvinyl alcohol (PVA, 1% w / v) and 0.4g NaCl (5%) in 40ml deionized water, stir overnight, and swell at 4℃ for later use.

[0114] (4) Under ice bath conditions, the inner aqueous phase was poured into the oil phase, and then the cell disruptor was used to disrupt the cell under ice bath conditions for 200W / 2min to obtain a uniform W1 / O colostrum.

[0115] (5) Formation of double emulsion (W1 / O / W2): The colostrum is quickly poured into the pre-cooled external aqueous phase (W2) at a volume ratio of 1:40, and mechanically stirred at 1000 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0116] (6) Finally, the oil phase was evaporated by stirring at 400 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0117] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate and wash it with pure water 3 times, freeze it at -80℃ for 2 h, and dry it in a freeze dryer for 48 h to obtain a yellow loose powder solid.

[0118] Example 17

[0119] Gold nanoclusters GA and poloxamer PL188 were mixed in 50 μL of water and allowed to stand at 4°C for 5 h to form a uniformly dispersed internal aqueous phase W1. The concentration of gold nanoclusters in the internal aqueous phase was 5.5 mg / mL and the concentration of PL188 was 17.8 mg / mL. The remaining steps were the same as in Example 11.

[0120] Example 18

[0121] (1) Dissolve 50 μL of gold nanoclusters GA and poloxamer PL188 in 50 μL of deionized water. The concentration of gold nanoclusters in the aqueous phase is 22 mg / mL, and the concentration of PL188 in the aqueous phase is 8.8 mg / mL. Let stand at 4℃ for 5 h to form a uniformly dispersed aqueous phase W1 for later use.

[0122] (2) Preparation of oil phase O: Dissolve PLGA (75 mg·mL⁻¹, preferably molecular weight 38000-54000, LA:GA=50:50) in 1 ml of dichloromethane (DCM), stir in the dark until completely dissolved, and store at 4℃ for later use.

[0123] (3) Preparation of external aqueous phase (W2): Dissolve 0.4g polyvinyl alcohol (PVA, 1% w / v) and 0.4g NaCl (5%) in 40ml deionized water, stir overnight, and swell at 4℃ for later use.

[0124] (4) Under ice bath conditions, the inner aqueous phase was poured into the oil phase, and then the cell disruptor was used to disrupt the cell under ice bath conditions for 200W / 1min to obtain a uniform W1 / O colostrum.

[0125] (5) Formation of double emulsion (W1 / O / W2): The colostrum is quickly poured into the pre-cooled external aqueous phase (W2) at a volume ratio of 1:40, and mechanically stirred at 1000 rpm for 2 min to form a W1 / O / W2 double emulsion.

[0126] (6) Finally, the oil phase was evaporated by stirring at 400 rpm for 2 hours with a magnetic stirrer to obtain solidified microspheres.

[0127] (7) Centrifuge the above solution at 3000 rpm for 10 min, take the precipitate and wash it with pure water 3 times, freeze it at -80℃ for 2 h, and dry it in a freeze dryer for 48 h to obtain a yellow loose powder solid.

[0128] Comparative Example 1

[0129] The internal aqueous phase consisted of 100 μL of water and 8.8 mg of PL188. The remaining steps were the same as in Example 18, and blank microspheres (PLGA-MS) were obtained.

[0130] Test Example 1

[0131] The gold content was measured by ICP-MS, specifically the content of free (unencapsulated) drug in solution and the total drug content in the microspheres. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = Total gold clusters in microspheres / Total gold clusters added × 100%. Finally, the amount of GA in GA-MS was measured, and the encapsulation efficiency was calculated. Table 1 shows the encapsulation efficiencies under different conditions.

[0132] Table 1

[0133] Group Encapsulation efficiency of gold cluster microspheres Example 1 2.79% Example 2 7.12% Example 3 9.40% Example 4 8.61% Example 5 12.77% Example 6 19.24% Example 7 15.10% Example 8 14.81% Example 9 17.04% Example 10 19.51% Example 11 9.27% Example 12 17.38% Example 13 14.51% Example 14 11.35% Example 15 22.09% Example 16 13.82% Example 17 8.08% Example 18 40.52%

[0134] Test Example 2

[0135] The microspheres prepared in Example 18 were observed using an inverted fluorescence microscope via GA-MS. Figure 1a The microsphere size was statistically analyzed using ImageJ, and the diameter of the gold cluster microspheres was found to be around 100 μm.

[0136] Test Example 3

[0137] The prepared gold cluster microspheres (GA-MS) were observed using laser confocal microscopy. Figure 2c The results showed that the GA microspheres were elliptical in shape and about 100 μm in size, with a uniform red base and fine texture. The GA was distributed continuously within the microspheres, and the fluorescence signal covered the entire microsphere, proving that the GA was loaded within the microspheres. Therefore, the gold nanoclusters GA were loaded within the microspheres and had good fluorescence performance, indicating that the carrier had an ideal embedding effect on GA.

[0138] And observe the results using GA-MS and PLGA-MS under 365nm UV light. Figure 2a The results showed that GA-MS emitted red light, while PLGA-MS did not emit light. The fluorescence signals of the drug-loaded GA microspheres and the blank microspheres differed significantly. The red light marker of the drug-loaded microspheres was clear and well-dispersed, resulting in ideal visualization and differentiation of the system. A suitable amount of GA solution was taken, and GA-MS and PLGA-MS were dispersed in 100 μL of aqueous solution, respectively. The fluorescence was detected using a fluorescence spectrometer, and the corresponding fluorescence patterns were obtained. Figure 2b The GA sample showed obvious fluorescence signals, and the fluorescence intensity of GA microspheres was significantly higher than that of GA aqueous solution, indicating that microsphere loading enhanced the fluorescence performance of GA.

[0139] Test Example 4

[0140] The microsphere samples (GA-MS Example 18, PLGA-MS Comparative Example 1) were fixed onto a plate with double-sided tape, sputter-coated with gold under vacuum, and then the surface morphology of the microspheres was observed by scanning electron microscopy. Figure 3a , Figure 3bScanning electron microscopy results showed that the GA-MS microspheres were well-formed, porous, and uniform in size, consistent with the typical characteristics of drug / functional molecule carriers. The surface exhibited a porous and rough morphology, a typical porous structure that facilitates the release of active substances. In contrast, the blank microspheres in Comparative Example 1 had a smooth, non-porous surface.

[0141] Test Example 5

[0142] Accurately weigh an appropriate amount of gold cluster microsphere sample (from Example 18) into a 20 mL stoppered centrifuge tube, add 9 mL of pH 7.4 phosphate buffer release medium, vortex for 1 min, and then place it in a constant temperature water bath shaker at (37 ± 0.5) ℃ at (50 ± 3) r·min. － 1 The centrifuge tubes were centrifuged at 3600 r·min⁻¹ for 15 min at 5–8 °C. After centrifugation, 1 ml of the supernatant was collected as the test solution, and 1 ml of isothermal release medium was added. The solution was vortexed for 1 min and then returned to the water bath shaker. The in vitro release amount was measured according to the method in Test Example 1, and an in vitro release graph was plotted. Figure 4 The results showed that the curve exhibited a continuously increasing sustained-release characteristic: from day 0 to approximately 40 days, the cumulative release rate gradually increased to around 80%, indicating that the microspheres had a sustained-release effect. In the early stage (0-15 days): the release rate was relatively slow, with the cumulative release rate increasing from 0 to approximately 45%, representing a slow release phase (GA diffuses slowly through the pores inside the microspheres). In the later stage (15-40 days): the release rate accelerated, with the cumulative release rate increasing from 45% to 80%, representing an accelerated release phase (possibly due to the gradual swelling / degradation of the microsphere structure, and the expansion of pores promoting GA release). This long-term sustained-release behavior (continuous release for over 30 days) indicates that the microsphere carrier can effectively delay the release of GA, avoiding the "burst release effect" (no large-scale rapid release in the initial stage), meeting the design requirements of long-acting drug carriers. Therefore, gold nanoclusters of GA microspheres possess ideal in vitro sustained-release performance, with a stable and long-lasting release process, making them suitable for applications requiring long-term drug delivery.

[0143] Test Example 6

[0144] Three 6-week-old rats were used. An appropriate amount of gold microspheres (Example 18) was added to CMC-Na solution to prepare a suspension (2 mg / 100 μL), which was then administered via intra-articular injection. Blood samples of 0.25 mL were collected from the retroocular venous plexus of the rats before administration and at 1, 6, and 12 h and 1, 2, 4, 6, 8, 10, 12, and 14 days after administration. The blood samples were then placed in heparinized test tubes and throttled at 3500 rpm.－ 1 Centrifuge for 10 min to separate plasma, and perform ICP-MS on 10 μL of plasma according to Test 1 to measure the gold content in serum. Plot an in vivo gold content curve. Figure 5b The rats were weighed daily, and their body weight was measured before and after injection. A graph showing the change in rat body weight was then plotted. Figure 5a The rats gradually gained weight and maintained good mental condition. On days 7, 14, and 28, one rat was sacrificed, and its knee joint was dissected and irradiated with 365nm ultraviolet light to observe drug retention. Figure 5c Only after 21 days of gestation were the rats sacrificed and dissected, and the knee joints were stained with hematoxylin and eosin (HE) for sectioning. Figure 5d ) and safranin-fast green stained S&F sections ( Figure 5f Hearts, livers, spleens, lungs, and kidneys of rats at 1, 2, and 4 weeks were digested and subjected to ICP-MS to observe the accumulation of gold content in each organ. Figure 5e First, the serum concentration change graph shows that some of the gold components of the gold cluster microspheres injected into the joint enter the bloodstream, with the blood drug concentration reaching its peak on the eighth day. This is consistent with the in vivo release pattern of microsphere formulations (local slow release + a small amount of systemic absorption). After intra-articular injection, the serum gold content of the gold cluster microspheres gradually reaches its peak, indicating that the gold cluster microspheres have a slow release characteristic. After reaching the peak, the blood drug concentration gradually decreases without long-term accumulation, demonstrating the rationality of the formulation's in vivo metabolism.

[0145] ICP-MS results showed that the liver was the main organ for the accumulation of gold cluster microspheres in the body (consistent with the physiological characteristics of the liver as a metabolic organ); the gold content in the heart, spleen, lungs, and kidneys was significantly lower, especially the gold content in the heart, which remained at a very low level. From 1W to 4W, the gold content in all organs showed an overall decreasing trend, indicating that gold is gradually metabolized / excreted in the body over time without continuous accumulation. The gold cluster microspheres mainly accumulated in the liver, but were gradually cleared over time. The accumulation in other organs was low and continuously decreasing, reflecting the good in vivo metabolic characteristics of the formulation and a low risk of systemic accumulation. Figure 5c The results showed that after one week, the GA gold nanocluster microspheres still remained in the knee joint, demonstrating their good retention properties in the knee joint. HE sections of the knee joint (…) Figure 5d The results showed that the joint tissue structure was clear, the joint cavity was clean, the articular cartilage surface was smooth, the chondrocyte morphology and structure were normal, the synovial cells were arranged regularly, no obvious hyperplasia was observed in the synovial connective tissue, the meniscus structure was clear, no obvious bone erosion was observed, no obvious inflammatory cell infiltration was observed, and Safranin O and Fast Green staining (…) Figure 5f The cartilage was stained red, and the osteoblasts were stained blue-green. The chondrocytes were arranged regularly, and the flaky cartilage showed lighter staining. This demonstrates that the microspheres have good biocompatibility in the knee joint.

Claims

1. A method for synthesizing gold nanoclusters, characterized in that, The gold nanoclusters were encapsulated in PLGA microspheres using a dual-emulsion solvent evaporation method, specifically including the following steps: (1) Preparation of internal aqueous phase W1: Gold nanoclusters GA and poloxamer were dissolved in deionized water and allowed to stand at low temperature to form a uniformly dispersed phase; (2) Preparation of oil phase O: Polylactic acid-glycolic acid copolymer PLGA was dissolved in dichloromethane; (3) Preparation of external aqueous phase W2: Polyvinyl alcohol (PVA) and NaCl are dissolved in water to form a stabilizer solution; (4) Preparation of colostrum W1 / O: Under ice bath conditions, the aqueous phase W1 from step (1) and the oil phase O from step (2) are mixed and ultrasonically broken to form colostrum; (5) Preparation of double emulsion W1 / O / W2: Pour the primary emulsion from step (4) into the external aqueous phase W2 from step (3) and stir mechanically to form a double emulsion; (6) Solvent evaporation and microsphere curing: Stir the polyemulsion obtained in step (5) to evaporate the organic solvent and form microspheres; (7) Cleaning and freeze-drying: After centrifugation and cleaning, freeze-drying was performed to obtain yellow loose microsphere powder.

2. The method according to claim 1, characterized in that, The gold nanoclusters mentioned above are glutathione gold nanoclusters with a "peptide shell" structure, specifically GA. The poloxamer, polylactic acid-glycolic acid, dichloromethane, and polyvinyl alcohol used are PL188, PLGA, DCM, and PVA, respectively.

3. The method according to claim 1, characterized in that, In step (1), the concentration of gold nanoclusters (GA) in the internal aqueous phase is 5.5 mg·mL. -1 ~22mg·mL -1 ; Preferably 22 mg / mL -1 The concentration of poloxamer ranged from 4.4 to 17.6 mg / mL. -1 The preferred dosage is 8.85 mg / mL. -1 .

4. The method according to claim 1, characterized in that, In step (2), the concentration of polylactic acid-glycolic acid copolymer (PLGA) in the oil phase is 10–75 mg / mL. -1 The preferred concentration of polylactic acid-glycolic acid copolymer (PLGA) is 75 mg / mL. -1 The molar ratio of LA / GA in polylactic acid-glycolic acid copolymer (PLGA) is 50:50-75:25, for example, 50:50 and 75:25, with Mw of 24000-38000 and 38000-54000 respectively, preferably 50:50, with Mw of 38000-54000.

5. The method according to claim 1, characterized in that, In step (3), the mass percentage concentration of polyvinyl alcohol in the external aqueous phase W2 is 0.1-2%, preferably 1%; and the mass percentage concentration of NaCl is 0-5%, preferably 5%.

6. The method according to claim 1, characterized in that, In step (4), the volume ratio of the internal aqueous phase W1 to the oil phase O is 1:5 to 20, preferably 1:

10.

7. The method according to claim 1, characterized in that, In step (5), the volume ratio of oil phase O to external aqueous phase W2 in the re-emulsion W1 / O / W2 is 1:20 to 80, preferably 1:

40.

8. The method according to claim 1, characterized in that, The power range of the cell disruptor during the synthesis of the colostrum is 50-500W, and the time is 30s-5min, preferably 200W / 1min. The stirring speed for the re-emulsion is 400rpm-1200rpm, preferably 1000rpm. The stirring speed for the volatile oil phase is 200-400rpm, preferably 400rpm.

9. Gold nanoclusters prepared according to any one of claims 1-8.

10. The application of the gold nanocluster microspheres prepared according to any one of claims 1-8 for the preparation of drugs for treating local chronic inflammation or painful diseases of the joint cavity.

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