A novel hydrogel-based BMSCs-loaded azithromycin preparation and its preparation method and application

Abstract

The present invention discloses a novel hydrogel-based BMSCs-loaded azithromycin formulation, its preparation method, and application. The novel hydrogel-based BMSCs-loaded azithromycin formulation comprises albumin-azithromycin-nanoparticles sequentially encapsulated by rat bone marrow mesenchymal stem cells and chitosan hydrogel; the mass ratio of the albumin-azithromycin-nanoparticles, rat bone marrow mesenchymal stem cells, and chitosan hydrogel is 1:1-2:1-2; the albumin-azithromycin-nanoparticles are nanoparticles formed by encapsulating azithromycin in albumin, and the mass ratio of the azithromycin to the albumin is 1:4-5. The present invention successfully prepared a novel formulation, BMSCs-TAT-AZM-NPs@Gel; this system not only exhibits anti-inflammatory, antioxidant, and promotes damaged tissue repair; at the same time, the novel formulation also reduces the toxic and side effects of AZM, improves the safety of the drug, and provides a new approach for the treatment of spinal cord injury.

Description

Technical Field

[0001] The present invention relates to the technical field of nano preparations, and in particular to a method for preparing a novel hydrogel-based BMSCs-loaded azithromycin preparation. Background Art

[0002] Spinal cord injury (SCI) is a devastating traumatic condition affecting the central nervous system. Each year, over 600,000 people lose sensory and motor function in their limbs due to SCI, and in severe cases, can lead to physical disability. Therefore, there is an urgent need to strengthen research on the treatment of SCI. SCI typically manifests in two ways: primary and secondary. Primary injury is directly caused by mechanical trauma (such as trauma). Over time, and through various physiological and pathological changes, a series of secondary events, such as neuronal damage, inflammatory responses, and spinal cord demyelination, gradually occur, leading to secondary damage and worsening the patient's neurological deficits. As an incurable neurological disease, SCI treatment research has attracted widespread attention from scientific researchers worldwide. However, in addition to drug therapy, current SCI treatments include surgical intervention and cell transplantation. However, these methods not only have limited therapeutic effects but also may damage normal tissue, thereby worsening the patient's neurological deficits. Furthermore, the complex pathological mechanisms of SCI mean that there is currently no effective cure for SCI. The strategies currently used in clinical practice often make it difficult for drugs to achieve effective therapeutic effects at the site of spinal cord injury. After systemic administration, the amount of drug accumulated at the site of injury is extremely small. Even for drugs with barrier permeability, systemic administration is usually required, coupled with high-dose treatment, to barely reach therapeutic levels at the site of injury; however, this will lead to harmful off-target effects. In summary, increasing the effective therapeutic concentration of drugs at the site of spinal cord injury, thereby better promoting neural repair and reducing the degree of spinal cord cavitation, is the key to the effective treatment of spinal cord injury.

[0003] Azithromycin (AZM), with a half-life of approximately 11–17 hours, has low toxicity and side effects and a high safety profile, making it one of the most commonly used antibiotics. Studies have shown that azithromycin can reduce gene expression in M1 macrophages while increasing gene expression in M2 macrophages, thereby exerting anti-inflammatory effects. This provides a theoretical basis for its application in SCI. AZM has been reported to be both safe and effective in treating infections in SCI patients. However, research on the therapeutic effects of AZM after SCI is very limited, likely due to azithromycin's low bioavailability and the potential for toxic side effects with high doses. Nanoformulations offer advantages such as improved drug absorption, modulated drug release rate, and enhanced drug access to tissues and capillaries. These advantages can reduce the administered dose to a certain extent, offering unique advantages for the application of poorly soluble drugs and addressing current clinical challenges. However, the various substances comprising nanoformulations can significantly impact the toxicity of the target drug delivery system.

[0004] Therefore, how to develop an azithromycin nanoparticle that can not only overcome the inherent defects of azithromycin and the limitations of clinical application, but also solve the biocompatibility and toxicity problems of nanoformulations, while improving drug delivery efficiency and enhancing efficacy has become a technical problem that needs to be solved urgently. Summary of the Invention

[0005] The purpose of the present invention is to provide a new hydrogel-based BMSCs-loaded azithromycin formulation and its preparation method and application. The new hydrogel-based BMSCs-loaded azithromycin formulation combines azithromycin, albumin, bone marrow mesenchymal stem cells and hydrogel drug delivery system with a powerful therapeutic effect. It can not only overcome the inherent defects of azithromycin and the limitations of clinical application, but also solve the biocompatibility and toxicity problems of nanoformulations, thereby improving drug delivery efficiency while enhancing therapeutic efficacy.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a novel hydrogel-based BMSCs-loaded azithromycin preparation is provided, wherein the novel hydrogel-based BMSCs-loaded azithromycin preparation comprises albumin-azithromycin-nanoparticles sequentially encapsulated by rat bone marrow mesenchymal stem cells and chitosan hydrogel; wherein the mass ratio of the albumin-azithromycin-nanoparticles, rat bone marrow mesenchymal stem cells, and chitosan hydrogel is 1:1-2:1-2; the albumin-azithromycin-nanoparticles are nanoparticles formed by albumin encapsulating azithromycin, and the mass ratio of the azithromycin to the albumin is 1:4-5.

[0008] In a second aspect of the present invention, a method for preparing a novel hydrogel-based BMSCs-loaded azithromycin formulation is provided, the method comprising:

[0009] The main drug azithromycin, excipients and chloroform are ultrasonically dissolved to obtain an oil phase; a 1% to 5% mass fraction human serum albumin solution is adjusted to a pH of 3.5 to 4.5 to serve as an aqueous phase; the oil phase and the aqueous phase are mixed and sheared to obtain milky white colostrum; the colostrum is subjected to rotary evaporation to remove chloroform to obtain albumin-azithromycin-nanoparticles, namely TAT-AZM-NPs;

[0010] The albumin-azithromycin-nanoparticles were mixed with rat bone marrow mesenchymal stem cells and incubated together in an incubator. The mixture was then digested, centrifuged, the supernatant was discarded, and fresh culture medium was added for resuspending. Chitosan hydrogel was then added, mixed, and cultured to obtain a new hydrogel-based BMSCs-loaded azithromycin preparation, namely BMSCs-TAT-AZM-NPs@Gel.

[0011] In a third aspect of the present invention, provided is the use of the novel hydrogel-based BMSCs-loaded azithromycin preparation in the preparation of a drug for treating spinal cord injury.

[0012] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0013] 1. The present invention provides a novel hydrogel-based BMSCs-loaded azithromycin formulation, which consists of a hydrophobic drug azithromycin sequentially encapsulated by an albumin carrier, a mesenchymal stem cell carrier, and a hydrogel (drug release requires a three-layer structure), thereby solving the toxic and side effects of azithromycin AZM; improving the survival rate and effectiveness of BMSCs as drug carriers and the uptake rate of AZM, and achieving the therapeutic effect on spinal cord injury in rats in vitro and in vivo, ultimately achieving the goal of efficiently treating spinal cord injury. Specifically: Although nanoformulations have the characteristics of improving drug absorption, regulating drug release rate, and making it easier for drugs to enter tissues and capillaries, they will greatly affect the toxicity of the target drug delivery system. Therefore, human serum albumin with low toxicity and good biocompatibility is selected as the material for preparing nanoparticles; the hydrogel prepared by chitosan and sodium β-glycerophosphate is a hydrogel with mild properties and almost neutral surface charge; the selection of appropriate nanoparticle preparation materials and hydrogel preparation materials can improve the stability and biocompatibility of drugs, and avoid immune rejection reactions, inflammatory reactions and aggravation of each preparation group when acting on BV2 cells and SH-SY5Y cells in subsequent in vivo experiments. neurodegenerative diseases; although azithromycin nanoparticles have anti-inflammatory, antioxidant and neuroprotective effects, spinal cord injury is a disease that is seriously destructive to the central nervous system and spinal cord tissue. Therefore, bone marrow mesenchymal stem cells are added to the drug delivery system to exert their tissue repair effect; researchers generally believe that the survival rate of bone marrow mesenchymal stem cells during transplantation and treatment is low. The present application found that adding injectable hydrogels to form tissue engineering scaffolds can protect the survival rate of stem cells, and using injectable hydrogels as tissue engineering scaffolds for bone marrow mesenchymal stem cells to carry out the efficacy of azithromycin human serum albumin nanoparticles, so that they can efficiently, stably and continuously exert therapeutic effects at the site of spinal cord injury. Experimental results showed that the constructed drug delivery system combining azithromycin, bone marrow mesenchymal stem cells and hydrogel has a powerful therapeutic effect: changes in rat BBB scores and body mass index indicated that the drug delivery system can improve the motor function of the rat hind limbs; magnetic resonance imaging results showed that the drug delivery system can promote the repair of damaged spinal cord tissue; H&E staining results showed that the drug delivery system can improve the pathological characteristics of damaged spinal cord tissue; Nissl staining and TUNNEL staining showed that the drug delivery system can reduce nerve cell apoptosis and promote neuroprotection; immunohistochemistry results showed that the drug delivery system can also promote axon survival and regeneration.

[0014] Compared with the covalent binding method, encapsulating azithromycin nanoparticles in bone marrow mesenchymal stem cells allows azithromycin and stem cells to form a whole, which not only enhances the stability of the entire drug delivery system, but also enhances the sustained-release effect of the drug.

[0015] 2. The present invention provides a method for preparing a novel hydrogel-based BMSCs-loaded azithromycin formulation. Azithromycin is prepared into human serum albumin nanoparticles (TAT-AZM-NPs), which are then loaded with bone marrow mesenchymal stem cells (BMSCs-TAT-AZM-NPs). The bone marrow mesenchymal stem cells loaded with azithromycin human serum albumin nanoparticles are then encapsulated in a chitosan hydrogel. The BMSCs-TAT-AZM-NPs@Gel drug delivery system is prepared by co-incubation and stained with a live / dead cell double staining kit for formative verification of the drug delivery system. Microscopic observation shows that the hydrogel adheres to the stem cell surface. Experimental results show that BMSCs-AZM-NPs@Gel exhibits a good sustained-release effect in PBS buffer at pH 7.4. The in vitro release behavior of each formulation group was investigated. The initial in vitro release process of AZM-NPs and TAT-AZM-NPs was in a burst release phase, which was caused by the rapid release of azithromycin adsorbed on the nanoparticle surface and distributed in the outer layer. This phase was followed by a sustained release phase. In contrast, for the BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel groups, after the rapid release of the nanoparticles, further diffusion became difficult, and the release process also depended on the transmembrane behavior of the BMSCs and the release behavior of the hydrogel. The overall release process showed that, compared with the azithromycin API, BMSCs-TAT-AZM-NPs@Gel provided a superior sustained release of the drug, enabling it to better exert its therapeutic effect.

[0016] 3. The present invention used in vitro hemolysis experiments to investigate the safety of BMSCs-TAT-AZM-NPs@Gel. The experimental results showed that all formulation groups maintained a low hemolysis rate (<5%), which is suitable for subsequent experiments. The cytotoxicity of BMSCs-TAT-AZM-NPs@Gel was investigated using MTT experiments. The experimental results showed that the survival rate of BV2 cells and SH-SY5Y cells gradually decreased with increasing dose concentration and duration. However, the survival rate of each formulation group was above 80% at different concentrations, indicating that the cytotoxicity of each formulation group was low. The anti-inflammatory and antioxidant effects of BMSCs-TAT-AZM-NPs@Gel were investigated in vitro using ELISA and Griess kits. The test results showed that BMSCs-TAT-AZM-NPs@Gel can achieve anti-inflammatory and antioxidant effects by reducing the levels of inflammatory factors (IL-6, TNF-α) and oxidative factors (NO). This project used H2O2 to stimulate SH-SY5Y cells to establish a nerve damage model, and then applied drug intervention to verify the neuroprotective effects of each formulation group. Experiments showed that BMSCs-TAT-AZM-NPs@Gel has neuroprotective effects in vitro.

[0017] 4. The present invention successfully established a chromatographic method for determining the content of azithromycin in in vivo samples using a high performance liquid chromatograph. The pharmacokinetic analysis of BMSCs-TAT-AZM-NPs@Gel showed that BMSCs-TAT-AZM-NPs@Gel could effectively delay the residence time of the drug in the body and increase the half-life of the drug. The in vivo distribution of BMSCs-TAT-AZM-NPs@Gel was investigated using a small animal in vivo imaging device. The experimental results showed that BMSCs-TAT-AZM-NPs@Gel could enhance the concentration of the drug at the lesion site. The in vivo distribution of BMSCs-TAT-DiR-NPs@Gel was investigated using a fluorescent labeling method. The results showed that BMSCs-TAT-DiR-NPs@Ge1 could be effectively distributed to the damaged area of ​​rats with spinal cord injury and its elimination rate was significantly slower than that of other groups, effectively prolonging the half-life of the drug and enhancing the drug concentration at the damaged area of ​​the spinal cord, thereby enhancing the therapeutic effect.

[0018] 5. The present invention established a spinal cord injury model in SD rats and evaluated the in vivo pharmacodynamics of BMSCs-TAT-AZM-NPs@Gel. The in vivo therapeutic efficacy of the drug delivery system was investigated using methods such as the Basso-Beattie-Bresnahan (BBB) ​​score, magnetic resonance imaging, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Griess assay, H&E staining, Nissl staining, and TUNEL staining. The results of BBB score and body mass index showed that BMSCs-TAT-AZM-NPs@Gel could improve the kinematic function of the hind limbs of SCI rats. The results of magnetic resonance imaging showed that the STIR fat suppression sequence and T1 weighted bright and dark signal distribution of the BMSCs-TAT-AZM-NPs@Gel group were uniform, the cavity area was significantly reduced, and the therapeutic effect was better than that of other groups. After administration of BMSCs-TAT-AZM_NPs@Gel to spinal cord injury rats, the concentrations of IL-6, TNF-α, NO, MPO and MDA in the spinal cord tissue were significantly reduced, and the activity of SOD enzyme was significantly enhanced, indicating that BMSCs -TAT-AZM-NPs@Gel can reduce secondary damage after spinal cord injury in rats; H&E staining, Nissl staining and TUNEL staining results show that after 5 weeks of treatment, the pathological characteristics of the BMSCs-TAT-AZM-NPs@Gel group were significantly reduced compared with other groups, and the damaged neurons and apoptotic nerve cells were also significantly reduced, indicating that BMSCs-TAT-AZM-NPs@Gel has obvious neuroprotective effects; in vivo safety results show that BMSCs-TAT-AZM-NPs@Gel significantly reduces the hepatotoxicity of the raw material AZM, and its safety is relatively high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Schematic diagram of the hydrogel-based bone marrow mesenchymal stem cell-loaded azithromycin nanoparticles for the treatment of spinal cord injury. BMSCs: bone marrow mesenchymal stem cells; TAT: cell-penetrating peptide; AZM: azithromycin; NPs: human serum albumin nanoparticles.

[0021] Figure 2 In vitro characterization results of each formulation group. (A&B) Morphology, particle size distribution, and zeta potential of Blank NPs, (C&D) AZM-NPs, and (E&F) TAT-AZM-NPs.

[0022] Figure 3 Identification of bone marrow mesenchymal stem cells. (A) Normal growth of BMSCs, (B&C&D) Cell immunohistochemistry.

[0023] Figure 4 Characterization of BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel. (A) Fluorescence image of BMSCs-TAT-AZM-NPs; (B) FTIR image of the injectable hydrogel; (C) SEM image of the injectable hydrogel; (D&E) Live / dead cell staining images of BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel.

[0024] Figure 5 Figure 2 shows the in vitro release characteristics of BMSCs-TAT-AZM-NPs@Gel. The cumulative release of BMSCs-TAT-AZM-NPs@Gel was measured in PBS at 37°C, pH 7.4. The results are expressed as mean ± SD (n = 3).

[0025] Figure 6Results of the cellular uptake experiment of BMSCs-TAT-AZM-NPs@Gel. Confocal microscopy images of BV2 cells and SH-SY5Y cells after incubation at 37°C for 2 h, 4 h, and 6 h, respectively, with (A, C) Cy5-labeled AZM-NPs, BMSCs-AZM-NPs, and BMSCs-AZM-NPs@Gel, and (B, D) FITC- and Cy5-colabeled TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel. The green color represents FITC-labeled TAT peptide, and the red color represents Cy5-labeled human serum albumin nanoparticles. Semi-quantitative analysis of the mean red fluorescence intensity of (E) BV2 cells and (F) SH-SY5Y cells was performed using Image J software.

[0026] Figure 7 This study investigates the in vitro anti-inflammatory efficacy of BMSCs-TAT-AZM-NPs@Gel. (A) TNF-α, (B) IL-6, (C) NO, and (D) cell viability in the supernatant of BV2 cell culture fluid were measured. Inflammatory cytokine concentrations were determined using ELISA and Griess assays. Results are presented as mean ± SD (n = 3).

[0027] Figure 8 In vitro neuroprotective effects of BMSCs-TAT-AZM-NPs@Gel. (A) Survival rate of SH-SY5Y cells after stimulation with different concentrations of H2O2; (B) Survival rate of SH-SY5Y cells after pretreatment with different drug concentrations and then stimulation with H2O2; (D) Live / dead cell fluorescence staining images of SH-SY5Y cells treated with different agents, and (C) viable cell counts using Image J.

[0028] Figure 9 The pharmacokinetic curves of each preparation group in plasma. The concentration-time curves of each preparation group in plasma of (A) normal rats and (B) SCI rats at different time points after administration. The results are expressed as Mean±SD (n=6).

[0029] Figure 10 In vivo fluorescence imaging of different formulations in SCI rats. Following in situ injection of DiR, DiR-NPs, TAT-DiR-NPs, BMSCs-TAT-DiR-NPs, and BMSCs-TAT-DiR-NPs@Gel, (A) the in vivo fluorescence distribution of the drugs at different time points was observed using the IVIS small animal imaging system. (B) Twelve hours later, rat organs and spinal cord tissue were isolated, and tissue fluorescence distribution was observed using the in vivo animal imaging system. (C) Semi-quantitative analysis of in vivo fluorescence and (D) tissue fluorescence intensity was performed using Image J.

[0030] Figure 11 Figure 3. BMSCs-TAT-AZM-NPs@Gel improves hindlimb kinematic function in rats with spinal cord injury. (A) Representative photographs of spinal cord tissue before and after modeling; (B) Representative photographs of spinal cord tissue in each group 5 weeks after surgery; (C) Kinematic function scores of rats in different groups within 5 weeks after surgery; (D) Body weight change curves of rats in each group within 5 weeks after surgery.

[0031] Figure 12 MRI characteristics of rats with spinal cord injury after treatment. (A) STIR- and T1-weighted imaging of spinal cord tissue was performed 24 hours and 35 days after treatment with different drug groups (a: Healthy, b: Saline, c: Blank Gel, d: AZM, e: BMSCs, f: TAT-AZM-NPs, g: BMSCs-TAT-AZM-NPs, h: BMSCs-TAT-AZM-NPs@Gel, i: Sham). The red dotted circle represents the injury site. (C) STIR and (D) T1-weighted signal intensities were semiquantitatively analyzed using a Philips MR Achieva / Intera MRI system.

[0032] Figure 13 Figure 3. Anti-inflammatory and anti-NO effects of BMSCs-TAT-AZM-NPs@Gel on rats with spinal cord injury. IL-6, TNF-α, and NO concentrations in (A, C, E) serum and (B, D, F) spinal cord tissue of rats after different treatments.

[0033] Figure 14 Figure 2. The antioxidant effect of BMSCs-TAT-AZM-NPs@Gel on rats with spinal cord injury. Changes in the concentrations and activities of MPO, MDA, and SOD in serum (A, C, E) and spinal cord tissue (B, D, F) of SCI rats after different treatments. Results are expressed as mean ± SD (n = 5).

[0034] Figure 15Figure 4: Improvement of spinal cord pathological features and neuronal survival after treatment. SCI rats were treated with different dosing groups (a: Healthy, b: Saline, c: Blank Gel, d: AZM, e: BMSCs, f: TAT-AZM-NPs, g: BMSCs-TAT-AZM-NPs, h: BMSCs-TAT-AZM-NPs@Gel, i: Sham). (A) H&E staining and (B) Nissl staining of neurons. Scale bar: 50 μm. Black arrows indicate neuronal necrosis, blue arrows indicate microglial proliferation, and yellow arrows indicate neurophagocytosis. Quantitative analysis of (C) H&E staining and (D) Nissl staining is presented as mean ± standard deviation (n = 3).

[0035] Figure 16 Figure 5. Neural repair in the spinal cord tissue of SD rats treated with different treatment groups after 5 weeks. (A) Representative images of TUNEL staining. Scale bar, 50 μm. Red arrows indicate Nissl bodies. (B) Quantitative analysis of TUNEL staining results. Mean ± SD (n = 3).

[0036] Figure 17 BMSCs-TAT-AZM-NPs@Gel exhibits neuroprotective effects after SCI and reduces glial scarring after spinal cord injury. Immunohistochemical images of spinal cord tissues from each group 5 weeks after injury (A) anti-NF200, (B) anti-MBP, and (C) anti-GFAP staining. Scale bar = 40 μm. Cell nuclei appear blue after hematoxylin staining; DAB expression appears brown. We used the Halo data analysis system to calculate the percentage of positive areas in each image. Quantitative analysis of (D) anti-NF 200 , (E) anti-MBP, (F) anti-DFAP immunostaining are expressed as mean ± SD (n = 3). DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0038] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0040] The present invention will be described in detail below with reference to the examples, comparative examples and experimental data. 2000 -Chol was synthesized in our laboratory and its preparation method has been published in the literature: Li J, Wei J, Wan Y, Du X, Bai X, Li C, Lin Y, Liu Z, Zhou M, Zhong Z. TAT-modified tetramethylpyrazine-loaded nanoparticles for targeted treatment of spinal cord jury. J Control Release. 2021, 335: 103-116.

[0041] Example 1. Preparation and characterization of a novel hydrogel-based BMSCs-loaded azithromycin formulation

[0042] 1. Preparation and Characterization of TAT-AZM-NPs

[0043] 1 Preparation of TAT-AZM-NPs

[0044] By utilizing the principle that nanoparticles can encapsulate poorly soluble drugs and combining the emulsification-dispersion method, we prepared TAT-AZM-NPs (a new hydrogel-based BMSCs-loaded azithromycin preparation) with a light blue opalescent appearance and complete structure. The specific operation is as follows: the main drug is AZM, and the excipient is TAT-PEG synthesized in our laboratory. 2000 -Chol and lecithin. Accurately weigh 5 mg of the main drug, 25 mg each of the excipients cholesterol TAT-PEG2000-Chol and lecithin, and add an appropriate amount of chloroform. After sonication, the solution after the main drug and excipients are dissolved is the oil phase. At the same time, a 2% human serum albumin (HSA) solution is used as the aqueous phase, and the pH value of the aqueous phase is adjusted to approximately 4.0. The oil and aqueous phases are mixed in a centrifuge tube and sheared using an ultrasonic disruptor to obtain a milky white solution, namely colostrum. The colostrum is removed from the oil phase by a rotary evaporator to obtain TAT-AZM-NPs with a light blue opalescence. After passing through a membrane, store for later use.

[0045] At the same time, accurately weigh the same weight of AZM, cholesterol and lecithin, and operate in the same way to obtain AZM-NPs; accurately weigh the same weight of cholesterol and lecithin, and operate in the same way to obtain Blank NPs.

[0046] 2. Determination of particle size, PDI, and Zeta potential

[0047] Combined with TME and Malvern laser particle size analyzer, the particle size distribution, PDI and Zeta potential of TAT-AZM-NPs, AZM-NPs and Blank NPs were observed to see whether they met the formulation requirements.

[0048] Depend on Figure 2 It can be seen that the particle size distribution and Zeta potential of Blank NPs, AZM-NPs and TAT-AZM-NPs were investigated using a Malvern laser particle size analyzer. The experimental results are as follows: the average particle size of Blank NPs is 91.463nm±0.186nm, PDI is 0.202±0.008, and Zeta potential is -22.181mV±5.4mV ( Figure 2 A, 2B); the average particle size of AZM-NPs was 108.832nm±0.851nm, PDI was 0.216±0.008, and Zeta potential was -20.887mV±6.17mV( Figure 2 C, 2D); the average particle size of TAT-AZM-NPs was 136.953nm±0.496nm, PDI was 0.212±0.014, and Zeta potential was -25.338mV±4.63mV( Figure 2 E, 2F). The average particle sizes of blank NPs, AZM-NPs, and TAT-AZM-NPs gradually increased, which indirectly reflects the successful preparation of nanoparticles and the uniform particle size distribution.

[0049] 3 Morphological investigation

[0050] After TAT-AZM-NPs, AZM-NPs and BlankNPs were freeze-dried, negative staining was performed, and finally the morphologies of the three were observed under a transmission electron microscope (TEM) and recorded in a timely manner.

[0051] Depend on Figure 2 As can be seen, the BlankNPs, AZM-NPs, and TAT-AZM-NPs prepared by this process are regular spherical in morphology, with normal distributions of particle size and zeta potential. Since BlankNPs, AZM-NPs, and TAT-AZM-NPs are prepared using the same method, a comparison of the morphological images clearly shows that the average particle size of the drug-loaded AZM-NPs and TAT-AZM-NPs is significantly larger than that of BlankNPs, which indirectly proves that AZM has entered the hydrophobic core of the nanoparticles and that TAT has successfully linked to the AZM-NPs.

[0052] 4 Determination of encapsulation efficiency

[0053] An appropriate amount of prepared TAT-AZM-NPs and AZM-NPs were added to an ultrafiltration centrifuge tube and centrifuged at 8,000 × g for 5 min. The lower filtrate was collected and diluted to volume with acetonitrile. After passing through the membrane, the filtrate was subjected to HPLC determination. The sample was injected three times in parallel and the peak area was recorded to obtain the free drug content (M free ).

[0054] Take appropriate amount of TAT-AZM-NPs and AZM-NPs, add equal amount of acetonitrile, vortex and centrifuge at 8,000×g for 5 minutes. After taking out the supernatant and filtering the membrane, it was subjected to HPLC determination. The sample was injected three times in parallel and the peak area was recorded to obtain the total drug content (M total ), the calculation formulas for encapsulation efficiency and drug loading are as follows:

[0055] Encapsulation efficiency (%) = (M total -M free ) / M total ×100%

[0056] Drug loading (%) = (M total -M free ) / M NPs ×100%

[0057] The results showed that the encapsulation efficiency of AZM-NPs was 94.425% ± 0.231%, and the drug loading was 14.136% ± 0.167%. The encapsulation efficiency of TAT-AZM-NPs was 91.751% ± 0.174%, and the drug loading was 14.092% ± 0.112%. These results show that both AZM-NPs and TAT-AZM-NPs have high encapsulation efficiency and drug loading.

[0058] 2. Preparation and Characterization of BMSCs-TAT-AZM-NPs

[0059] 1 Isolation and culture of BMSCs

[0060] SPF-grade Sprague-Dawley (80–100 g) rats were anesthetized with ether and then sterilized by immersion in a 75% ethanol solution for 5 minutes. The tibia and femur were quickly isolated under sterile conditions in a clean bench. After isolation, the epiphyses were gently cut with sterile scissors to expose the medullary cavity of the epiphyses. The cells were then rinsed multiple times with fresh culture medium. The rinses were then passed through a 200-mesh sieve and collected in a 15 mL sterile centrifuge tube. The resulting cell suspension was centrifuged at 80 × g for 5 minutes, the supernatant discarded, and culture medium (10% FBS + penicillin–streptomycin + α-MEM) was added to the pellet and thoroughly pipetted until a uniform single-cell suspension was formed. The cells were diluted to an appropriate density with culture medium and then inoculated into culture flasks. The flasks were then incubated in a humidified incubator with 5% CO₂ and 37°C. When changing the medium three days before culture, discard half of the culture medium and add the other half of fresh culture medium. After three days, the fresh culture medium should be changed every day, and the cells should be observed every day using an inverted microscope. When the cells grow to more than 80% to 90% of the culture flask, digest them with 0.25% trypsin + 0.02% EDTA and then passage them at a ratio of 1:3.

[0061] In vitro culture results of BMSCs: When BMSCs were first inoculated, the primary cells were round and floated in the culture flask; after 24 hours of growth in complete medium, a few cells attached to the wall and took on a spindle shape. After 48 hours of growth, most cells took on a spindle shape ( Figure 3 A).

[0062] Identification of 2BMSCs

[0063] Flow cytometry was used to identify the expression of rat bone marrow mesenchymal stem cell marker antigens. The three main antibodies used in this experiment were CD44, CD45, and CD90. BMSCs cultured to the third generation were digested, and the digested suspension was centrifuged at 80×g for 5 minutes. The supernatant was discarded and resuspended with 1mL of PBS solution containing 1% tertiary antibody and counted to make the number reach 100,000 / tube. After counting, the suspension was transferred to four EP tubes, 1mL of PBS solution was added, and the suspension was centrifuged at 80×g for 2 minutes. The supernatant was discarded, and 1:100 diluted monoclonal antibodies CD44, CD45, and CD90 were added to three EP tubes respectively. The remaining one was used as a blank tube. After incubation at 4°C for 30 minutes, the suspension was washed once with PBS solution, centrifuged at 80×g for 2 minutes, and the supernatant was discarded. The tube was identified using a totipotent cell state analyzer. The identification results of BMSCs are as follows. Figure 3 As shown by Figure 3It can be seen that the identification of BMSCs immunophenotype by flow cytometry showed that: CD44 (PE-Cy7-H channel) positive expression was 98.16%, CD45 (APC-Cy7-A channel) negative expression was 84.28%, and CD90 (APC-H channel) positive expression was 99.26%. This result is consistent with the expression of BMSCs immunophenotype.

[0064] 3 Preparation of BMSCs-TAT-AZM-NPs

[0065] After human serum albumin was labeled with red fluorescence by fluorescent dye Cy5 (5 μg / mL), AZM-NPs with red fluorescence were prepared and then combined with TAT-PEG labeled with FITC. 2000 The TAT-AZM-NPs with both red and green fluorescence were prepared by using a 5% CO2-saturated humidity incubator at 37°C for 6 h, and then photographed under a fluorescence microscope.

[0066] The preparation results of BMSCs-TAT-AZM-NPs are shown in Figure 4 As shown in A, the blue fluorescence is DAPI, representing the nucleus of BMSCs, the red fluorescence is Cy5, representing AZM-NPs, and the green fluorescence is FITC, representing TAT. As can be seen from the figure, both red and green fluorescence are distributed around the nucleus, proving that TAT-AZM-NPs are encapsulated by BMSCs and that BMSCs-TAT-AZM-NPs are successfully prepared. Figure 4 As shown in D, live / dead staining shows that after BMSCs are loaded with TAT-AZM-NPs, BMSCs still maintain good vitality and uniform distribution. The experimental results show that after 6 hours of incubation, the encapsulation rate and drug loading of BMSCs on TAT-AZM-NPs reach the maximum, which are 43.25%±5.27% and 27.45±5.27pg / cell, respectively. When the incubation time is increased, the encapsulation rate and drug loading of BMSCs on nanoparticles reach a dynamic equilibrium, and the changes are negligible. It should be noted that fluorescent labeling is used here for the convenience of observation. In other embodiments, red fluorescence and green fluorescence labeling are not necessary. Specifically, the albumin-azithromycin-nanoparticles are mixed with rat bone marrow mesenchymal stem cells and incubated together in a 5% CO2 saturated humidity incubator at 37°C to obtain BMSCs-TAT-AZM-NPs. Among them, the rat bone marrow mesenchymal stem cells adopt rat bone marrow mesenchymal stem cell suspension, and each milliliter of the cell suspension contains 10 5 ~2×10 5The mass ratio of the albumin-azithromycin-nanoparticles to the rat bone marrow mesenchymal stem cell suspension is 1:1-2, preferably 1:1.

[0067] 3. Preparation and Characterization of BMSCs-TAT-AZM-NPs@Gel

[0068] 1 Preparation of injectable hydrogel

[0069] Weigh an appropriate amount of chitosan (CS) powder and add 0.1 mol / L hydrochloric acid to prepare a solution of a desired concentration. Store the solution at 4°C until ready for use. Dissolve an appropriate amount of sodium β-glycerophosphate (β-GP) in 1 mL of deionized water. While stirring in an ice-water bath, slowly add β-GP to the previously prepared 9 mL chitosan solution. The mixed solution should be quickly placed in a 37°C water bath to prepare an injectable hydrogel.

[0070] FTIR spectrum of injectable hydrogel Figure 4 As shown, from Figure 4 B can be seen at 3000cm -1 to 3500cm -1 CS generates stretching vibration absorption bands of OH and NH2 due to its intramolecular and intramolecular hydrogen bonds; the stretching vibration absorption peak of aliphatic CH is 2874.7 cm -1 The elongation vibration absorption peak of C=O is 1646.4cm -1 The elongation vibration absorption peak of NH is 1600.5cm -1 , 1157.0cm -1 , 1084.8cm -1 , 1023.9cm -1 is the stretching vibration absorption peak of CO. In β-GP, there are hydrogen bonds between hydroxyl groups, thus forming a broad OH stretching vibration absorption band located at 3377.6 cm -1 Nearby; and 1131.4cm -1 , 1088.6cm -1 is the asymmetric stretching vibration absorption peak of the phosphate group, 975.1 cm -1 PO4 3- Symmetrical stretching vibration absorption peak, 500 cm -1 ~800cm -1 PO4 3- In the CS / β-GP spectrum, the absorption peaks of OH and NH are at 3390.3 cm -1The overlap near the CS / β-GP solution indicates that there is a hydrogen bond between the two. By comparing the above results, it can be seen that the addition of β-GP changes the intermolecular forces and conformation of CS, thereby promoting the formation of a gel in the CS / β-GP mixed solution at 37°C.

[0071] Scanning electron microscopy was used to clarify the internal morphology and structure of the hydrogel scaffold. Figure 4 C The results showed that the prepared hydrogel exhibited a porous structure, which indicated that the prepared hydrogel could provide growth space and sustained release conditions for BMSCs-TAT-AZM-NPs.

[0072] Preparation of 2BMSCs-TAT-AZM-NPs@Gel

[0073] The albumin-azithromycin-nanoparticles were mixed with rat bone marrow mesenchymal stem cells and incubated in an incubator. The cells were digested and centrifuged, and the supernatant was discarded. Fresh culture medium was added for resuspending, and the prepared injectable hydrogel was added. The cells were pipetted and mixed to obtain a BMSCs-TAT-AZM-NPs@Gel suspension. The mass ratio of the albumin-azithromycin-nanoparticles to the rat bone marrow mesenchymal stem cells and chitosan hydrogel was 1:1-2:1-2, preferably 1:1:1.

[0074] The BMSCs-TAT-AZM-NPs@Gel was incubated at 37°C with 5% CO2 until ready to use. The freeze-dried hydrogel was accurately weighed and reconstituted with an appropriate amount of PBS. After dissolution, 200 μL of cell lysis buffer was added to obtain a cell lysate. The lysate was then vortexed and extracted with 800 μL of acetonitrile for 5 minutes. After centrifugation at 8,000 × g for 5 minutes, the drug loading of the BMSCs-TAT-AZM-NPs@Gel was analyzed by HPLC.

[0075] like Figure 4 As shown in D, live / dead staining showed high cell viability and uniform cell distribution. After BMSCs-TAT-AZM-NPs were encapsulated in the injectable hydrogel for 24 h, the hydrogel widely spread on the surface, but the cells still maintained a round shape ( Figure 4 E). This demonstrates the successful preparation of BMSCs-TAT-AZM-NPs@Gel. The experimental results show that the drug loading of AZM in BMSCs-TAT-AZM-NPs@Gel is 12.14% ± 1.26%.

[0076] In vitro release study of 3BMSCs-TAT-AZM-NPs@Gel

[0077] To investigate the in vitro release behavior of BMSCs-TAT-AZM-NPs@Gel, we investigated the in vitro release characteristics of AZM from BMSCs-TAT-AZM-NPs@Gel in PBS buffer (pH 7.4). AZM, as well as 4 mL of prepared TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel, were dispersed in PBS and placed in dialysis bags (3.0 kD cutoff). Three replicates were prepared for each formulation. PBS was used as the release medium, and the mixture was stirred at 37°C and 2,000 × g. Appropriate amounts of the solution were removed from the release medium at different time points. The sample was diluted to 5 mL with acetonitrile, filtered, injected, and the drug release was calculated. All data were expressed as mean ± SD, and statistical analysis was performed using GraphPad Prism 7.0. One-way analysis of variance was used for statistical analysis. When p < 0.05, it was considered to be significantly different.

[0078] Depend on Figure 5 It can be seen that the in vitro cumulative release rate of AZM exceeded 80% at the sampling point of 12 hours, while the release rates of TAT-AZM-NPs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel groups reached more than 70% after 24 hours. After 96 hours, the cumulative release rates reached 81.75%±0.49%, 82.15%±0.66% and 79.15%±0.56%, respectively, indicating that the drug has a good sustained-release effect after being prepared into nanoparticles.

[0079] Experimental Example 1: In vitro experimental study of BMSCs-TAT-AZM-NPs@Gel

[0080] 1 Experimental cell lines

[0081] Mouse microglia (BV2) and human neuroblastoma cells (SH-SY5Y) were provided by Dr. Wu Jianming's group in the Department of Pharmacology, Southwest Medical University and stored in our laboratory.

[0082] 2 Cell culture

[0083] This experiment used immortalized mouse microglia (BV2) and human neuroblastoma (SH-SY5Y) cells as model cells. Both cells were cultured in DMEM (20% FBS and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. For the experiment, BV2 and SH-SY5Y cells with a good growth index were selected, the culture medium was discarded, and the cells were digested, resuspended, and used.

[0084] 3 Cell-related experiments

[0085] 3.1 Cellular uptake experiments

[0086] Human neuroblastoma cells and LPS-activated microglia with inflammatory characteristics were used as representative cells. After Cy5 was applied to human serum albumin materials, ligand-free nanoparticles showed red fluorescence. TAT was labeled with FITC, and TAT-AZM-NPs showed both red and green fluorescence. SH-SY5Y cell and LPS-activated BV2 cell suspensions were taken and diluted to 5×10 4 Cells were seeded at 1 mL / well in a 24-well culture plate with a cell slide placed on the bottom. 1 mL was added to each well. After incubation, appropriate amounts of 200 μg / mL of AZM-NPs, BMSCs-AZM-NPs, BMSCs-AZM-NPs@Gel, and TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel solutions were added to each well. Following incubation, the supernatant was aspirated and the plates were washed with PBS until no impurities or crystals were present. Cells were then fixed with 4% paraformaldehyde and DAPI was added. After 15 minutes, the plates were sealed and the fluorescence distribution was observed and recorded under a laser confocal microscope. Fluorescence intensity was calculated using Image J software.

[0087] After BV2 cells and SH-SY5Y cells were incubated with AZM-NPs, BMSCs-AZM-NPs, BMSCs-AZM-NPs@Gel, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel for different time periods, the results were shown in Figure 2. Figure 6 As shown in Figures A, 6B, 6C, and 6D, BV2 cells and SH-SY5Y cells can quickly take up AZM-NPs, BMSCs-AZM-NPs, BMSCs-AZM-NPs@Gel, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel. Fluorescence can be observed in the cells after 2 hours of incubation. TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel can be more efficiently taken up by BV2 and SH-SY5Y cells. As the incubation time increases, the fluorescence intensity also increases. In addition, it was observed that red and green fluorescence are mainly distributed in the same position in the cytoplasm, which indicates that the polypeptide TAT and AZM-NPs are successfully connected into a whole. This result is consistent with the results of the first chapter. Figure 6As shown in E and 6F, the blue fluorescence of the cell nucleus stained with DAPI (representing the number of cells) was used for calibration. The red fluorescence intensity was quantitatively analyzed by Image J software. It can be seen that with the increase of uptake time, the uptake rate of BV2 cells and SH-SY5Y cells for each preparation group increased, showing a clear time dependence. After adding TAT, the fluorescence uptake after the same incubation time was compared, and there was a significant difference ( **** P < 0.0001), indicating that TAT helps cells to take up nanoparticles; after adding hydrogel, the amount taken up by cells was also lower than that of other groups within the same period of time, indicating that hydrogel can serve as a drug reservoir and slowly release drugs. This advantage becomes more obvious as time goes by.

[0088] 3.2 Cellular anti-inflammatory pharmacodynamics studies

[0089] In this study, lipopolysaccharide (LPS) was used to stimulate BV2 cells to secrete inflammatory factors. The levels of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as NO were determined according to the operation methods of enzyme-linked immunosorbent assay (ELISA) and Griess kit to determine the in vitro anti-inflammatory effect of each preparation group.

[0090] BV2 cells were seeded at a density of 4 × 10 4 cells / well. After incubation for a period of time, LPS solution of appropriate concentration was added. After stimulation for 4 hours, BV2 cells became inflammatory cells. Then, AZM (final concentration of 100 μg / mL), BMSCs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel (final concentration of 200 μg / mL) were added. After incubation for one day, the upper culture medium was aspirated and stored. The actual contents of TNF-α, IL-6 and NO in the upper culture medium were determined according to the method of the kit. At the same time, the cell viability was detected at 570 nm using a microplate reader.

[0091] In this study, lipopolysaccharide (LPS) was used to stimulate BV2 cells to quantitatively investigate the anti-inflammatory pharmacodynamics of the target drug delivery system. Figure 7 .like Figure 7 As shown in A, 7B, and 7C, the inflammatory factors released by BV2 cells after LPS stimulation increased significantly ( **** P<0.0001); Compared with the Saline group, the concentrations of TNF-α, IL-6, and NO in the cell supernatant of stimulated BV2 cells were significantly decreased after co-incubation with AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel ( ****P < 0.0001), showing a significant anti-inflammatory effect. Overall, blank nanoparticles had no significant inhibitory effect on the release of inflammatory factors.

[0092] like Figure 7 As shown in Figure 3D, the cells in each intervention group maintained a high cell activity, and their cell survival rates were all above 90%, indicating that after BV2 cells were co-incubated with AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel, the decrease in TNF-αt, IL-6, and NO levels was achieved by the drugs inhibiting the inflammatory response, and had no significant effect on cell survival rate.

[0093] 3.3 Neuroprotective Effect Studies

[0094] 3.3.1 Tetrazolium colorimetry

[0095] SH-SY5Y cells were seeded in 96-well plates and incubated. Different concentrations of H2O2 solution were then added for stimulation, allowing the SH-SY5Y cells to exhibit neural cell characteristics. After incubation, MTT solution was added, and cell survival was assessed using a microplate reader. The optimal H2O2 concentration was the one that caused some damage to the cells but still maintained a good survival rate.

[0096] SH-SY5Y cells were inoculated in the same way and incubated with different concentrations of AZM, Blank Gel, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM_NPs@Gel for 0.5 h, and then stimulated with the addition of the most suitable concentration of H2O2. After stimulation, a certain concentration of MTT solution was added to each well, and the cell survival was detected under a microplate reader. The most suitable drug concentration was thus screened for subsequent studies.

[0097] To investigate the neuroprotective effects of different drugs, H2O2 was used to stimulate SH-SY5Y cells to construct a neuronal cell damage model. First, the optimal stimulation concentration of H2O2 was investigated. The results were as follows: Figure 8 As shown in Figure A, it can be found that the cell survival rate gradually decreases with the increase of the H2O2 solution stimulation concentration. The experiment found that when the H2O2 concentration was 100μM, the cell survival rate was 47.16%±3.61%, and this concentration was selected for subsequent experiments.

[0098] SH-SY5Y cells were first incubated with different concentrations of AZM, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel for 0.5 h, and then stimulated with H2O2 for 24 h ( Figure 8B), it can be found that when the concentration of AZM, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel was 200 μg / mL, the cell viability was 84.24%±2.75%, 112.64%±1.74%, 117.26%±1.2%, and 130.6%±1.91%, respectively, which was significantly increased compared with the Blank Gel group at the corresponding concentration ( **** P < 0.0001), indicating that each preparation group has a certain degree of neuroprotective effect; and within the experimental concentration range, compared with other concentration groups, the protective effect of each preparation on H2O2-induced damaged nerve cells was the strongest at a concentration of 200 μg / mL, so we chose this concentration for subsequent studies.

[0099] 3.3.2 Live / dead cell staining method

[0100] SH-SY5Y cells (1×10 4 Cells were seeded into 96-well plates (10 cells / well). After cell attachment, saline, AZM, Blank Gel, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel were added and incubated for 0.5 hours. The cells were then stimulated with H2O2 for 24 hours. 100 μL of live / dead cell staining solution was added, mixed, and incubated at 37°C, 5% CO2, and saturated humidity for 15 minutes. Cell viability was observed under a fluorescence microscope.

[0101] like Figure 8 As shown in Figure 8C and Figure 8D, at the same concentration, after the Saline, Blank Gel, AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel preparation groups acted on SH-SY5Y cells, the quantitative analysis and counting by Image J image software showed that the number of viable cells were 141, 172, 235, 246, 295, 342, and 381, respectively, indicating that compared with the Saline and Blank Gel groups, each drug preparation group had a promoting effect on the growth of SH-SY5Y cells, among which BMSCs-TAT-AZM-NPs@Gel had the strongest promoting effect.

[0102] Experimental Example 2: Pharmacokinetics and in vivo distribution of BMSCs-TAT-AZM-NPs@Gel

[0103] The experiment used SPF-grade female Sprague-Dawley rats weighing 200 g ± 20 g. These rats were purchased and housed in an SPF-grade animal room at the Laboratory Animal Center of Southwest Medical University. To ensure the legality and ethics of animal experiments, this experiment was approved by the Animal Ethics Committee (approval number: 20220060) and conducted in strict accordance with the Southwest Medical University Regulations on Laboratory Animal Care.

[0104] 1 Establishment of rat spinal cord injury model

[0105] An animal model of spinal cord injury was established using the Allen's heavy impact method. Before the experiment began, all surgical instruments (such as forceps, scissors, and scalpels) were sterilized using autoclave to minimize the risk of infection. Rats were then anesthetized with an intraperitoneal injection of 80 to 100 mg / kg of phenobarbital sodium. After the rats lost consciousness, female Sprague-Dawley rats weighing 200 ± 20 g were secured to the operating table on their backs and their dorsal hair was shaved. Hair was shaved from the dorsal surface approximately 3 cm above the level of the spinous processes between T8 and T10 using a shaver. After topical disinfection with iodine, the outer skin was cut open with a scalpel, the inner muscle was dissected, and the spinal column was opened with rongeurs to expose the spinal cord. The spinal cord was then impacted with a homemade Allen's impactor. Successful modeling was indicated by the onset of spastic tail-wagging. Postoperatively, bladder compression was performed twice daily, bedding was changed frequently to maintain a clean and hygienic environment, and 2.5% penicillin was administered daily for 7 consecutive days to prevent infection. The sham-operated group received the same surgical procedure except that the spinal cord was not struck.

[0106] The rats are quite weak after modeling. In order to ensure the smooth progress of subsequent experiments, it is necessary to ensure the hygiene and cleanliness of the animal breeding environment, press the bladder on time every day to assist urination, and carry out careful postoperative management to ensure the safety of the rats and the reliability of the experiment.

[0107] 2 Experimental animal grouping and dosing regimen

[0108] Normal rats and rats with successful modeling were randomly divided into the following groups: normal group + AZM, normal group + BMSCs, normal group + AZM-NPs, normal group + TAT-AZM-NPs, normal group + BMSCs-TAT-AZM-NPs, normal group + BMSCs-TAT-AZM-NPs@Gel, model group + AZM, model group + BMSCs, model group + AZM-NPs, model group + TAT-AZM-NPs, model group + BMSCs-TAT-AZM-NPs, model group + BMSCs-TAT-AZM-NPs@Gel. The AZM group was intravenously injected with 10 mg / kg; the TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups were intravenously injected with a dose equivalent to 10 mg / kg of AZM.

[0109] Pharmacokinetic study of 3BMSCs-TAT-AZM-NPs@Gel

[0110] At 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 24 h, 28 h, 32 h, and 36 h after administration, 0.5 mL to 1.0 mL of blood was collected from the heart of rats in the AZM, AZM-NPs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups. After rapid centrifugation at 3,000 × g for 3 min, 0.1 mL of plasma was collected. Peak area was determined by HPLC, and AZM concentrations at different time points were calculated. Pharmacokinetic data were processed using DAS ver. 2.1.1 pharmacokinetic software, and relevant pharmacokinetic parameters were calculated in the plasma of normal rats and rats with spinal cord injury after in situ injection of each drug group.

[0111] This study mainly used HPLC to quantitatively study the drug concentration of AZM in the plasma of normal rats and spinal cord injured rats at different time points. Figure 9 As shown in the results, the AZM group has the characteristics of rapid absorption, widespread distribution, and rapid elimination; after 48 hours, AZM is basically undetectable in the plasma, while the drug concentrations of the AZM-NPs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel groups in the plasma still remain at a high concentration, indicating that encapsulating AZM in human serum albumin nanoparticles can significantly prolong the half-life of the drug, reduce the number of administrations, and also improve the efficacy, which is consistent with the results of the in vitro release experiment, proving that the target drug delivery system BMSCs-TAT-AZM-NPs@Gel has good sustained-release and long-lasting effects. Compared with AZM-NPs and TAT-AZM-NPs, when the drug concentration reaches the peak, the C of TAT-AZM-NPs is higher than that of AZM-NPs. max The results were slightly lower than those of AZM-NPs because TAT increased the efficiency of nanoparticles uptake by cells in the body; however, there was no significant difference in the blood drug concentrations between the TAT-AZM-NPs and BMSCs-TAT-AZM-NPs groups at the corresponding time points, indicating that BMSCs had no significant effect on the in vivo metabolism of drug-loaded nanoparticles. Figure 9 As shown in Figures A and 9B, there was no significant difference in the drug concentrations in the plasma of normal rats and spinal cord injury rats at the corresponding time points in each drug group, indicating that there was no significant difference in the metabolism of the five preparations in normal rats and spinal cord injury model rats.

[0112] The area under the drug-time curve, half-life, and mean residence time of BMSCs-TAT-AZM-NPs@Gel were significantly greater than those of AZM, confirming that BMSCs-TAT-AZM-NPs@Gel has the characteristics of sustained release and long-lasting effect; there was no significant difference in the pharmacokinetic parameters of each drug group in the plasma of normal rats and spinal cord injury rats at the corresponding time points, indicating that the disease has no obvious effect on the metabolism of each preparation.

[0113] 4. Fluorescence labeling method to investigate the in vivo distribution of target drug delivery system

[0114] To investigate whether a novel drug delivery system can enhance the concentration of therapeutic drugs at lesion sites, this study utilized fluorescence labeling to investigate the in vivo distribution of this novel drug delivery system. Nanoparticles labeled with the fluorescent dye DiR were prepared to produce DiR-NPs, TAT-DiR-NPs, BMSCs-TAT-DiD-NPs, and BMSCs-TAT-DiD-NPs@Gel. Healthy, SCI+Saline, and SCI+DiD control groups were used. Fluorescence distribution in SD rats was observed using an IVIS imaging system at 1, 2, 4, 8, and 12 hours after drug administration, respectively, and photographs were taken. Following the experiment, the heart, liver, spleen, lung, kidney, and spinal cord tissues of each group were rapidly isolated. Fluorescence intensity in these organs and the spinal cord was observed using an IVIS imaging system 12 hours after drug administration, and photographs were taken.

[0115] Results of in vivo tissue distribution of 5BMSCs-TAT-DiR-NPs@Gel

[0116] The experimental results are as follows Figure 10 As shown in A and 10C, 2 hours after administration, the fluorescence was mainly concentrated in the spinal cord tissue, indicating that in the early stage of administration, most of the fluorescence in each preparation group was distributed in the damaged area of ​​the spinal cord injured rats. As the administration time prolonged, the fluorescence intensity of each preparation group ( * P<0.05, ** P<0.01 *** P < 0.001, **** P < 0.0001). Among them, the fluorescence intensity of the BMSCs-DiR-NPs@Gel group decreased significantly slower than that of the other groups, indicating that the preparation of the drug as a nanoformulation can significantly prolong its residence time at the lesion site. After the in vivo experiment of each group of rats was completed, the organs and spinal cord tissues in the rats were immediately isolated, and the fluorescence intensity of the isolated organs and spinal cord was observed using the IVIS imaging system. The fluorescence intensity of the living rats and tissues was semi-quantitatively analyzed using Image J. The results are as follows Figure 10As shown in B and 10D, it was found that the DiR group, DiR-NPs group, TAT-DiR-NPs, BMSCs-TAT-DiR-NPs, and BMSCs-TAT-DiR-NPs@Gel group were all distributed more in the spinal cord tissue, and the distribution of BMSCs-TAT-DiR-NPs@Gel in the spinal cord tissue was significantly higher than that of the other groups ( * P<0.05, ** P<0.01 *** P < 0.001, **** P < 0.0001). The above experimental results show that the use of hydrogel to encapsulate BMSCs-loaded nanoparticles can significantly prolong the half-life of the drug AZM, and this drug delivery system can allow AZM to stay longer at the injury site, thereby enhancing the therapeutic effect.

[0117] Experimental Example 3: In vivo pharmacodynamics study

[0118] The experiment used SPF-grade female Sprague-Dawley rats weighing 200 ± 20 g. These rats were purchased and housed in an SPF-grade animal room at the Laboratory Animal Center of Southwest Medical University. To ensure the legality and ethics of animal experiments, this experiment was approved by the Animal Ethics Committee (approval number: 20220060) and conducted in strict accordance with the Southwest Medical University Regulations for Laboratory Animal Care.

[0119] 1 Establishment of rat spinal cord injury model: see Experimental Example 2.

[0120] Improvement of 2BMSCs-TAT-AZM-NPs@Gel on the kinematic function of rats with spinal cord injury

[0121] 2.1 Experimental Animal Grouping and Dosage Scheme

[0122] The rats were divided into the following groups:

[0123] ①Normal control group: Healthy group (Healthy)

[0124] ②Negative control group: SCI+Saline (Saline)

[0125] ③Blank nanoparticle group: SCI+Blank Gel (Blank Gel)

[0126] ④ Free drug group: SCI+AZM (AZM)

[0127] ⑤Drug-loaded nanoparticle group: SCI+BMSCs (BMSCs)

[0128] ⑥TAT modified drug-loaded nanoparticles group: SCI+TAT-AZM-NPs (TAT-AZM-NPs)

[0129] ⑦BMSCs combined with TAT modified drug-loaded nanoparticles group: SCI+BMSCs-TAT-AZM-NPs (BMSCs-TAT-AZM-NPs)

[0130] ⑧ Hydrogel-encapsulated BMSCs combined with TAT-modified drug-loaded nanoparticles group: SCI+BMSCs-TAT-AZM-NPs@Gel (BMSCs-TAT-AZM-NPs@Gel)

[0131] ⑨Sham operation group: sham operation + saline (Sham)

[0132] Six hours after successful model establishment, the AZM group was administered orally at a dose of 10 mg / kg; the TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups were administered orally at a dose equivalent to 10 mg / kg of AZM. Dosing continued once daily for 7 consecutive days.

[0133] 2.2 Kinematic function scoring of rat hindlimbs

[0134] After treatment with each formulation, the Basso-Beattie-Bresnahan (BBB) ​​scoring system was used to assess the recovery of motor function in rats. Scoring was performed on days 1, 3, 7, 14, 21, 28, and 35 after surgery. Scoring was performed double-blind; rats scoring 0 to 1 in the initial scoring were considered successful models.

[0135] 2.3 Observation of general condition of rats

[0136] During the BBB scoring of rats, the weight changes of rats in each group were recorded and the health status of the rats was observed.

[0137] 3 Magnetic resonance imaging experiments

[0138] Immediately after modeling, rats were anesthetized and then placed in a prone position on a Philips MR Achieva / Intera MRI scanner. T1-weighted and STIR fat-suppressed scans of the spinal cord were performed. Similarly, rats in each group were scanned using the same method after treatment.

[0139] Inhibitory effect of 4BMSCs-TAT-AZM-NPs@Gel on secondary injury

[0140] 4.1 Experimental Animal Grouping and Dosage Scheme

[0141] Normal rats and rats with successful modeling were divided into the same groups as described above. Six hours after successful modeling, the AZM group received an in situ injection of 10 mg / kg; the TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups received an in situ injection of a dose equivalent to 10 mg / kg of AZM.

[0142] Photos of the spinal cord before and after modeling Figure 11 As shown in A, the injured area bleeds rapidly after being hit. Figure 11 Shown in B are representative photos of the spinal cord tissues of each group 5 weeks after surgery. As can be seen from the figure, compared with the Saline group, the spinal cord tissue of the BMSCs-TAT-AZM-NPs@Gel group was the most complete and had the best recovery.

[0143] In this study, the rats in the Healthy group and the Sham group were used as controls. Based on the rat BBB scoring rules and the weight changes of the rats in each group after eating, the motor function of the SCI rats was investigated after treatment with Saline, Blank Gel, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel. Figure 11 As shown in Figures C and 11D, immediately after modeling, rats in all formulation groups showed hind limb paralysis, with no significant differences in BBB scores or weight change index. Two weeks after surgery, the motor function of SCI rats in the Saline group had improved to some extent compared to the Saline group, demonstrating the therapeutic efficacy of each formulation. Furthermore, the motor function of SCI rats in the BMSCs-TAT-AZM-NPs@Gel group recovered more significantly, demonstrating that the BMSCs-TAT-AZM-NPs@Gel group had the best therapeutic effect. 2-5 weeks after surgery, the improvement of hindlimb kinematic function in the BMSCs-TAT-AZM-NPs group and the BMSCs-TAT-AZM-NPs@Gel group was more obvious than that in the other groups, especially in the BMSCs-TAT-AZM-NPs@Gel group. Compared with the Saline group, the kinematic function of the AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups was significantly improved ( **** P<0.0001); body mass index also increased significantly, increasing by 46.86g±1.28g compared with before modeling.

[0144] This experiment used Philips MR Achieva / Intera magnetic resonance scanner to scan the spinal cord of rats. The experimental results are as follows Figure 12As shown, the spinal cords of rats in the Healthy group appeared intact, with uniform STIR fat-suppressed and T1-weighted bright and dark signals. However, rats in all formulation groups showed large cavities immediately after modeling, with darker signals on T1-weighted and stronger bright signals on STIR fat-suppressed. After 35 days of treatment, the BMSCs-TAT-AZM-NPs@Gel group showed uniform STIR fat-suppressed and T1-weighted bright and dark signal distributions, and the cavitation area was significantly reduced, indicating good recovery after treatment.

[0145] 4.2 Tissue sampling

[0146] After a period of dosing in each formulation group, rats were anesthetized with an intraperitoneal injection of 80-100 mg / kg of phenobarbital sodium. Cardiac blood was placed in an EP tube containing sodium heparin and centrifuged at 3,000 × g for 3 minutes to separate red blood cells and plasma. The supernatant plasma was saved for later use. Subsequently, the rats were sacrificed and the intact spinal cord tissue was removed. After washing with physiological saline, the tissue was transferred to a standard EP tube, and three times the volume of physiological saline was added. The spinal cord was then sheared and ground using a homogenizer to form a tissue homogenate. The tissue homogenate was centrifuged at 15,000 × g for 15 minutes. The supernatant was aspirated and stored at -20°C for later use.

[0147] 4.3 Determination of inflammatory factors in serum and spinal cord tissue

[0148] In this study, enzyme-linked immunosorbent assay (ELISA) was used to quantitatively analyze the levels of TNF-α and IL-6 in serum and spinal cord tissue to investigate the in vivo anti-inflammatory efficacy of the BMSCs-TAT-AZM-NPs@Gel drug delivery system. After spinal cord injury, the release of inflammatory factors IL-6 and TNF-α increased significantly. Therefore, in order to study the anti-inflammatory effect of each preparation group, we used ELISA analysis to quantitatively analyze the content of inflammatory factors around the lesion site after spinal cord injury. The results are shown in Figure 2. Figure 13 As shown in Figure 3, compared with the Healthy group, the concentrations of IL-6 and TNF-α in the serum (A&C) and spinal cord tissue (B&D) of all SCI groups were significantly increased. Compared with the Saline group, the concentrations of IL-6 and TNF-α in the BMSCs-TAT-AZM-NPs@Gel group were significantly decreased ( #### P<0.0001), and the BMSCs-TAT-AZM-NPs@Gel group had a better anti-inflammatory effect than the other treatment groups, and there was also a significant difference ( * P<0.05, ** P < 0.01, *** P < 0.001, **** P<0.0001).

[0149] 4.4 Determination of NO content in serum and spinal cord tissue

[0150] After spinal cord injury, the NO content in the injured area increases significantly, further aggravating the secondary damage of the injured tissue. To study the anti-NO effect of each preparation group, we used the Griess analysis method to quantitatively analyze the NO content around the lesion site after spinal cord injury. Figure 13 As shown in E&F, the concentrations of NO in the serum and spinal cord tissue of the Healthy group were significantly lower than those in the Saline group, with significant differences ( #### P<0.0001); after SCI rats were treated with AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel, the NO concentration was significantly reduced, and the BMSCs-TAT-AZM-NPs@Gel group had a stronger NO inhibition effect, which was also significantly different from the other treatment groups ( * P<0.05, ** P < 0.01, **** P<0.0001).

[0151] 4.5 Determination of oxidative stress levels in serum and spinal cord tissue

[0152] After treatment, MDA content was determined using the TBA colorimetric method, MPO activity was determined using ELISA, and SOD activity in serum and spinal cord tissue was determined using the WST-1 method. All experiments were performed strictly according to the kit instructions. The specific steps were as follows:

[0153] (1) SOD enzyme activity determination

[0154] Serum sample pretreatment: Pretreat the serum according to the above serum treatment method.

[0155] Tissue sample pretreatment: After accurately weighing the spinal cord tissue, add 9 volumes of physiological saline and homogenize in an ice bath. Centrifuge at 700×g for 10 minutes and collect the supernatant for subsequent measurement. Prepare the appropriate amount of substrate application solution and enzyme working solution according to the kit instructions. Calculate the SOD activity of serum and tissue samples according to the formula:

[0156] ①SOD inhibition ratio(%)=((OD control -OD control blank )-(OD sample -OD sample blank )) / (OD control -OD controlblank )×100%

[0157] ②SOD activity in serum (U / mL)=SOD inhibition ratio / 50%×reactant dilution factor×sample dilution tactor

[0158] ③SOD actiVity in tissues(U / mg protein)=SOD inhibition ratio / 50%×reactant dilution factor / total concentration of sample protein(mg protein / mL)

[0159] (2) MDA content determination

[0160] Serum sample pretreatment: Pre-treat according to the serum treatment method described above. Tissue sample pretreatment: Accurately weigh the spinal cord tissue and add 9 times the volume of physiological saline to homogenize the tissue. Process according to the kit instructions. Calculate the MDA content in serum and tissue samples according to the formula:

[0161] ①MDA concentration in serum (nmol / mL)=(OD sample -OD control ) / (OD standard -OD blank )×standard concentration(10nmol / mL)×sample dilutionfactor

[0162] ②MDA concentration in tissues(nmol / mg protein)=(OD sample -OD control ) / (OD standard -OD blank )×standard concentration(10nmol / mL) / total concentration of sample protein(mg protein / mL)(Note: * nmol / mgprot is nanomoles / milligram protein)

[0163] (3) MPO enzyme activity assay

[0164] Serum sample pretreatment: Add the same volume of Reagent 2 to the sample serum and mix thoroughly. Take 0.9 mL of the above mixture and add 0.1 mL of Reagent 3, mix thoroughly and place in a water bath.

[0165] Tissue sample pretreatment: Accurately weigh the spinal cord tissue, add Reagent 2 at a ratio of 1:19, and homogenize. Take 0.9 mL and add 0.1 mL of Reagent 3, and incubate in a 37°C water bath for 15 minutes. Prepare the colorimetric reagent and serum sample according to the kit instructions. Calculate the MPO activity in the serum according to the formula:

[0166] MPO activity in serum(U / L)=(OD sample -OD cintrol ) / (11.3×V sample )

[0167] Prepare appropriate amounts of colorimetric reagent and tissue sample according to the kit instructions. Calculate MPO activity in spinal cord tissue according to the formula:

[0168] MPO actiVity in tissues(U / g)=(OD sample -OD control ) / (11.3×M tissue )

[0169] This study used MPO, MDA, and SOD as indicators to examine the changes in the content of oxidative factors in the lesion after treatment with each preparation group. Figure 14 As shown in Figure 2, after spinal cord injury, the levels of MPO and MDA in serum and spinal cord tissue increased significantly ( # P<0.05, ## P < 0.01, #### P<0.0001), the activity of SOD was significantly reduced ( ## P < 0.01, #### P < 0.0001), indicating that after the rats were modeled, a more obvious peroxidation reaction occurred at the injury site, leading to an increase in oxidative factors, thereby causing more serious secondary damage. Compared with the Saline group, the MPO and MDA concentrations in serum and spinal cord tissue of SCI rats were significantly reduced after treatment with AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel. Correspondingly, the SOD enzyme activity in serum and spinal cord tissue was significantly increased. Compared with the Saline group, the MPO, MDA content and SOD enzyme activity of each treatment group were significantly different ( # P<0.05, ## P < 0.01, ### P < 0.001, ####P<0.0001), indicating that each treatment group had a certain therapeutic effect. The BMSCs-TAT-AZM-NPs@Gel group showed the best oxidative stress effect, and there was a significant difference compared with the other treatment groups ( * P<0.05, ** P < 0.01, *** P < 0.001, **** P<0.0001).

[0170] 5. Spinal cord histopathological observation

[0171] 5.1 Tissue sampling

[0172] Rats were anesthetized by intraperitoneal injection of 80–100 mg / kg sodium phenobarbital. Approximately 4 mL of cardiac blood was collected and centrifuged at 700 × g for 5 min. The supernatant serum was collected and stored for later use. After the rats were sacrificed, the spinal cords were dissected and washed multiple times with saline. The spinal cords were then fixed with 4% paraformaldehyde and stored at room temperature until further use.

[0173] 5.2 H&E staining

[0174] After treatment with each preparation group, spinal cord tissue was removed from each group and stained with hematoxylin-eosin. The spinal cord tissue of each group of experimental rats was pathologically analyzed based on the staining results to examine the improvement of neuronal necrosis, microglial proliferation and neurophagocytic phenomenon.

[0175] After H&E staining, the pathological changes of the spinal cord tissue of rats in each group 35 days after surgery were shown in Figure 15 As shown in A, under optical microscope observation, the staining results of some experimental groups showed neuronal necrosis (area indicated by black arrows), microglial proliferation (area indicated by blue arrows), neurophagocytosis (area indicated by yellow arrows), and other pathological changes. However, by observing the pathological changes of the Healthy group, the above pathological phenomena were not found. The experimental results show that these pathological phenomena are related to the modeling and treatment methods.

[0176] Compared with the Healthy group, no obvious pathological changes were found in the spinal cord tissue of the rats in the Sham group, while more pathological phenomena were found in the spinal cord tissue of the rats in the Saline group, indicating that the lesions were related to the modeling of this experiment. Compared with the Saline group, the neurophagocytic phenomenon in the AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs and BMSCs-TAT-AZM-NPs@Gel groups was alleviated. At the same time, the pathological features such as microglial proliferation and neuronal necrosis were also improved to varying degrees, indicating that the AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, BMSCs-TA The T-AZM-NPs@Gel groups all had different degrees of therapeutic and improvement effects on spinal cord injury. Among them, the degree of spinal cord tissue pathological changes in the BMSCs-TAT-AZM-NPs@Gel group was relatively mild, and the degree of spinal cord tissue pathological changes in the AZM, BMSCs, TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs groups were slightly alleviated, indicating that the therapeutic effects of the AZM, BMSCs, TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs groups on SCI were not as significant as those of the BMSCs-TAT-AZM-NPs@Gel group, which also shows that the BMSCs-TAT-AZM-NPs@Gel group can accelerate the repair of damaged tissue.

[0177] The above pathological observation results were quantitatively analyzed. Figure 15 As shown in C, the improvement of the BMSCs-TAT-AZM-NPs@Gel group was significantly better than that of the other groups ( * P<0.1, *** P < 0.001, **** P<0.0001).

[0178] 5.3 Nissl staining

[0179] Thirty-five days after surgery, spinal cord tissue was removed from each group and Nissl staining was performed. Changes in nerve cells in the tissue were observed under an optical microscope. Nissl staining is a staining method that specifically monitors nerve cell survival. Analysis of Nissl staining provides a visual indicator of nerve cell survival.

[0180] After drug treatment, spinal cord tissue was removed from each group and made into wax slides for tissue TUNEL staining. By analyzing the TUNEL staining results, the apoptosis of nerve cells can be directly observed.

[0181] The results of Nissl staining of the spinal cord tissues of rats in each group 35 days after surgery are shown in the figure. Figure 15As shown in B, after modeling, the number of Nissl bodies (indicated by red arrows) at the injury site showed a significant downward trend, indicating that the modeling led to neuronal loss. Figure 15 The statistical results showed that compared with the Healthy group, the number of Nissl bodies in the Sham group did not change significantly. Compared with the treatment group, the number of Nissl bodies in the spinal cord tissue of rats in the Saline group was significantly reduced, and there was a significant difference. ( #### P < 0.0001), indicating that the loss of neurons in the Saline group was the most serious. Compared with the Saline group, the number of Nissl bodies in the spinal cord tissue of rats in the AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups gradually increased ( ### P < 0.001, #### P<0.0001). The experimental results showed that AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups all had therapeutic effects on SCI; among them, the therapeutic effect of BMSCs-TAT-AZM-NPs@Gel group was the most obvious. There was also a significant difference compared with other treatment groups ( * P<0.05, *** P < 0.001, **** P<0.0001).

[0182] 5.4 TUNEL staining

[0183] 35 days after surgery, the spinal cord tissues of the rats were taken out. The TUNEL staining results of the spinal cord tissues of the rats in each group are shown in Figure 16 As shown in A, after spinal cord injury, the number of apoptotic cells in the Saline group was the highest compared to the other groups. After treatment with different drug groups, the apoptosis of nerve cells was significantly improved. Figure 16 B Statistical results show that compared with the Healthy group, the apoptosis rate of the Sham group was slightly higher, but there was no significant difference, while the apoptosis percentage of the spinal cord tissue cells in the Saline group was significantly increased, with a significant difference ( #### P<0.0001). Compared with the Saline group, the apoptosis of spinal cord nerve cells in the AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups was significantly improved ( ### P < 0.001, ####P<0.0001); the percentage of apoptotic cells in the BMSCs-TAT-AZM-NPs@Gel group was significantly lower than that in the AZM, BMSCs, TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs groups ( * P<0.05, ** P<0.01), indicating that BMSCs-TAT-AZM-NPs@Gel can inhibit cell apoptosis induced by spinal cord injury.

[0184] 6 Immunohistochemistry

[0185] Thirty-five days after surgery, rats were anesthetized, and 2-cm-long spinal cord segments were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned coronally. 30-μm-thick tissue sections were prepared for immunohistochemistry. Sections were incubated in 5% serum-0.3% Triton X-100 for 20 minutes and then incubated overnight at 4°C with primary antibodies. Primary antibodies used were neurofilament 200 kDa (NF200; 1:500, Proteintech) to identify axons, myelin basic protein (MBP; 1:200, Proteintech) to identify myelin, or glial fibrillary acidic protein (GFAP; 1:500, Biss) to identify glial cells. Sections were then incubated with appropriate secondary antibodies for 2 hours in the dark. After three washes with PBS, tissue sections were stained with DAB developer and nuclei were counterstained with hematoxylin for 3 minutes. The sections were then dehydrated and mounted on slides with neutral gelatin.

[0186] Stained sections were imaged at 400x magnification using a fluorescence microscope (Life Technologies, USA) and a digital trinocular micro-camera system (BA400digital, Mcaudi). Morphological analysis, counting of neurons and apoptotic cells, was performed using ImageJ software. Antibody staining levels were quantified using the Halo data analysis system.

[0187] Use anti-MF 200 Antibodies were used to stain the injury site to assess axonal survival and regeneration, anti-MBP antibody was used to assess the number of myelinated axons, and anti-GFAP antibody was used to track glial scar formation. Figure 17 As shown in Figure 2, after 5 weeks of treatment, a large amount of axonal loss was observed in the saline group and the Blank Gel group, which was manifested by the NF loss around the injury site. 200 ( Figure 17 A) and MBP( Figure 17 B) value decreased, GFAP( Figure 17 C) values ​​increased. This indicates severe axonal damage and the formation of dense glial scars. AZM, BMSCs, TAT-AZM-NPs, BMSCs-TAT-AZM-NPs, and BMSCs-TAT-AZM-NPs@Gel groups showed NF 200and lower expression of MBP and higher expression of GFAP. In particular, BMSCs-TAT-AZM-NPs@Gel were associated with more myelinated axons, NF 200 The results were consistent with the idea that AZM and BMSCs promote tissue repair through their anti-apoptotic, antioxidant, and anti-inflammatory properties. Furthermore, BMSCs-TAT-AZM-NPs@Gel was associated with more intact and spared myelin sheaths and longer axonal extensions.

[0188] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

Claims

1. A novel hydrogel-based BMSCs-loaded azithromycin preparation, characterized in that: The novel preparation is composed of albumin-azithromycin-nanoparticles sequentially encapsulated by rat bone marrow mesenchymal stem cells and chitosan hydrogel; wherein the mass ratio of the albumin-azithromycin-nanoparticles, rat bone marrow mesenchymal stem cells, and chitosan hydrogel is 1:1-2:1-2; the albumin-azithromycin-nanoparticles are nanoparticles formed by encapsulating azithromycin in albumin, and the mass ratio of the azithromycin to the albumin is 1:4-5; The preparation method of the albumin-azithromycin-nanoparticles comprises: The main drug azithromycin, excipients and chloroform are ultrasonically dissolved to obtain an oil phase; the pH value of a human serum albumin solution with a mass fraction of 1% to 5% is adjusted to 3.5 to 4.5 and then used as an aqueous phase; the oil phase and the aqueous phase are mixed and sheared to obtain milky white colostrum; the colostrum is subjected to rotary evaporation to remove chloroform to obtain albumin-azithromycin-nanoparticles, namely TAT-AZM-NPs, wherein the excipients are cholesterol TAT-PEG2000-Chol and lecithin.

2. A method for preparing the novel hydrogel-based BMSCs-loaded azithromycin preparation according to claim 1, characterized in that: The method comprises: The main drug azithromycin, excipients, and chloroform are ultrasonically dissolved to obtain an oil phase; a 1% to 5% human serum albumin solution is adjusted to a pH of 3.5 to 4.5 to serve as an aqueous phase; the oil phase and the aqueous phase are mixed and sheared to obtain milky white colostrum; the colostrum is subjected to rotary evaporation to remove chloroform to obtain albumin-azithromycin-nanoparticles, namely TAT-AZM-NPs; wherein the excipients are cholesterol TAT-PEG2000-Chol and lecithin; The albumin-azithromycin-nanoparticles were mixed with rat bone marrow mesenchymal stem cells and incubated together in an incubator. The mixture was then digested, centrifuged, the supernatant was discarded, and fresh culture medium was added for resuspending. Chitosan hydrogel was then added, mixed, and cultured to obtain a new hydrogel-based BMSCs-loaded azithromycin preparation, namely BMSCs-TAT-AZM-NPs@Gel.

3. The preparation method according to claim 2, characterized in that The mass ratio of the azithromycin to the excipient is 1:3-7; wherein the mass ratio of the cholesterol to the lecithin is 1:1; and the mass ratio of the azithromycin to the human serum albumin is 1:4-5.

4. The preparation method according to claim 2, characterized in that The mass ratio of the albumin-azithromycin-nanoparticles to the rat bone marrow mesenchymal stem cells and chitosan hydrogel is 1:1-2:1-2; the rat bone marrow mesenchymal stem cells adopt rat bone marrow mesenchymal stem cell suspension, and each milliliter of the cell suspension contains 10 5 pcs~2ⅹ10 5 cells.

5. The preparation method according to claim 2, characterized in that The preparation method of the chitosan hydrogel comprises: The chitosan powder is dissolved in hydrochloric acid to obtain a chitosan solution; sodium β-glycerophosphate is dissolved in deionized water to obtain a sodium β-glycerophosphate solution; the sodium β-glycerophosphate solution is added to the chitosan solution under stirring in an ice-water bath, and the mixture is then placed in a constant temperature water bath at 37±2° C. to obtain a chitosan hydrogel.

6. The preparation method according to claim 5, characterized in that The mass ratio of the chitosan powder to the sodium β-glycerophosphate is 1:

8.

7. The preparation method according to claim 2, characterized in that The method for preparing rat bone marrow mesenchymal stem cells comprises: isolating and obtaining rat tibias and femurs, then gently cutting off the epiphyseal ends with sterile scissors to expose the bone marrow cavity of the epiphyseal ends, then rinsing with fresh culture medium for multiple times, and passing the rinsing liquid through a 200-mesh sieve, collecting, and centrifuging to obtain a cell suspension; diluting the cell suspension with culture medium and then inoculating it, and digesting and passage the cells after they grow to 80% to 90% of the culture flask to obtain rat bone marrow mesenchymal stem cells.

8. Use of the novel hydrogel-based BMSCs-loaded azithromycin preparation according to claim 1 in the preparation of a drug for treating spinal cord injury.

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