Chlamydomonas reinhardtii exosome hydrogel microneedle and preparation method and application thereof

Abstract

This invention discloses a Chlamydomonas reinhardtii synergistic exosome hydrogel microneedle, its preparation method, and its application, belonging to the field of biomedical materials and microneedle delivery technology. The hydrogel microneedles prepared in this invention use methacryloxymethyl carboxymethyl chitosan and methacryloxyhyaluronic acid as the hydrogel matrix. After adding a photoinitiator to the hydrogel matrix, the microneedles are obtained through molding and ultraviolet crosslinking. The tips of the hydrogel microneedles are loaded with Chlamydomonas reinhardtii exosomes to improve transdermal delivery efficiency. The backing layer or matrix portion of the hydrogel microneedles is loaded with Chlamydomonas reinhardtii to maintain its photosynthetic activity and provide continuous local oxygen supply. This invention constructs a multifunctional therapeutic system for the synergistic effect of Chlamydomonas reinhardtii and exosomes. This system integrates in-situ photosynthetic oxygen supply, exosome anti-inflammatory repair, and local microneedle delivery onto the same platform, and can simultaneously intervene in key aspects of osteoarthritis such as hypoxia, inflammation, and insufficient tissue repair, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and microneedle delivery technology, specifically to a functionalized hydrogel microneedle loaded with Chlamydomonas reinhardtii and its exosomes using methacryloylcarboxymethyl chitosan (CMCSMA) and methacryloylhyaluronic acid (HAMA) as matrices, as well as its preparation method and specific applications. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative disease characterized by degeneration of articular cartilage, chronic inflammation, and joint dysfunction. This disease is not only accompanied by continuous degradation of the cartilage matrix, but also often manifests as multiple pathological changes, including a hypoxic microenvironment, persistent activation of inflammatory factors, and insufficient tissue repair capacity. Current treatments primarily focus on pain relief, anti-inflammation, and slowing progression, but they struggle to simultaneously address key issues such as hypoxia, inflammation, and the imbalance between tissue regeneration and these underlying conditions, thus limiting overall efficacy.

[0003] However, existing osteoarthritis treatment systems generally suffer from problems such as limited functionality, short local retention time, and insufficient regulation of complex pathological microenvironments. There is still a lack of a comprehensive treatment platform that can combine in-situ oxygen supply, anti-inflammatory regulation, repair promotion, and efficient local delivery. Summary of the Invention

[0004] The purpose of this invention is to provide a Chlamydomonas reinhardtii synergistic exosome hydrogel microneedle, and to provide a method for preparing the hydrogel microneedle and its specific applications, so as to make up for the shortcomings of the prior art in terms of local hypoxia, persistent inflammation and insufficient repair efficiency in osteoarthritis.

[0005] Microneedle delivery technology offers advantages such as minimal invasiveness, high local drug delivery efficiency, and good patient compliance, enabling it to overcome the skin barrier and improve the local utilization rate of active ingredients. Meanwhile, hydrogel materials possess excellent biocompatibility, plasticity, and loading capacity, providing a relatively mild delivery environment for extracellular vesicles, active cells, or drugs. Exosomes have natural advantages in anti-inflammatory and tissue repair properties, while Chlamydomonas reinhardtii exhibits stable photosynthetic capacity, continuously producing oxygen under light conditions, potentially improving the hypoxic state of osteoarthritis lesions.

[0006] This invention uses natural high-molecular-weight chitosan and hyaluronic acid as a base, and obtains methacrylamide-modified carboxymethyl chitosan (CMCSMA) and methacrylamide-modified hyaluronic acid (HAMA) through methacrylation modification, and constructs photocrosslinkable hydrogel microneedle carriers using them. This system utilizes the microneedle tip to achieve precise transdermal delivery of exosomes, and loads Chlamydomonas reinhardtii into the microneedle backing layer or matrix portion, thereby forming a synergistic therapeutic mode of "oxygen supply-anti-inflammatory-regeneration" locally.

[0007] Chlamydomonas reinhardtii can continuously release oxygen under light conditions to improve the local hypoxic microenvironment in osteoarthritis; exosomes can regulate inflammatory responses, promote macrophage polarization to the M2 phenotype, and enhance chondrocyte proliferation, migration, and matrix synthesis; CMCSMA / HAMA hydrogel microneedles provide stable structural support, good mechanical puncture performance, and a sustained-release platform for active ingredients, thereby achieving local microenvironment remodeling and tissue repair.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A Chlamydomonas reinhardtii synergistic exosome hydrogel microneedle is disclosed. The hydrogel microneedle is composed of a hydrogel matrix of methacryloyl carboxymethyl chitosan (CMCSMA) and methacryloyl hyaluronic acid (HAMA). After adding a photoinitiator to the hydrogel matrix, the microneedle structure is obtained by molding and ultraviolet crosslinking. The tip of the hydrogel microneedle is loaded with Chlamydomonas reinhardtii exosomes to improve transdermal delivery efficiency. The backing layer or matrix portion of the hydrogel microneedle is loaded with Chlamydomonas reinhardtii to maintain its photosynthetic activity and provide local continuous oxygen supply.

[0009] The hydrogel microneedles have the following characteristics: the microneedles have complete morphology and regular tips; the hydrogel microneedles have a porous structure with good balanced swelling performance, porosity and stability; the microneedles have sufficient mechanical strength and skin insertion capability, with the strength of a single microneedle tip >0.1 N, which can form microchannels about 200-300 μm deep in the skin; at the same time, the system has good antioxidant capacity, and the Chlamydomonas reinhardtii loaded in the hydrogel can maintain oxygen production activity for a long time under light conditions.

[0010] Furthermore, a precursor system was selected with 4%–5% HAMA and 1%–2% CMCSMA, an exosome concentration of 40 μg / mL, and 5 × 10⁻⁶ Chlamydomonas reinhardtii dosage. 7 Cells / mL.

[0011] Furthermore, the Chlamydomonas reinhardtii exosomes were obtained from the Chlamydomonas reinhardtii culture supernatant by differential centrifugation and ultracentrifugation.

[0012] A method for preparing Chlamydomonas reinhardtii synergistic exosome hydrogel microneedles includes the following steps: (1) Cultivate Chlamydomonas reinhardtii and extract its exosomes; (2) Preparation of methacrylated carboxymethyl chitosan (CMCSMA) and methacrylated hyaluronic acid (HAMA). (3) Prepare a hydrogel precursor solution by mixing CMCSMA and HAMA, and the hydrogel precursor solution also includes a photoinitiator; (4) After mixing the exosomes with the hydrogel precursor solution, introduce them into the tip of the microneedle mold; after mixing Chlamydomonas reinhardtii with the hydrogel precursor solution, fill the backing layer. (5) The hydrogel microneedles were obtained after centrifugation-assisted molding and cross-linking under 365 nm ultraviolet light and demolding.

[0013] Further, in step (1), after the Chlamydomonas reinhardtii culture reaches the plateau phase, the culture supernatant is collected, and the intact algal cells and large fragments are removed by centrifugation at 4℃ and 2000g for 10 min. The remaining fragments are then removed by centrifugation at 4℃ and 10000g for 30 min. The supernatant is then filtered through a 0.22 μm filter membrane and ultracentrifuged at 4℃ and 100000g for 70 min to collect the exosome precipitate. After resuspending with PBS and washing by ultracentrifugation again, the purified Chlamydomonas reinhardtii exosomes can be obtained.

[0014] Further, in step (2), CMCSMA is obtained by grafting carboxymethyl chitosan with methacrylic anhydride; HAMA is obtained by modifying hyaluronic acid with methacrylic anhydride; both are preferably reacted under pH 8-9 conditions, and purified products are obtained after dialysis and freeze-drying.

[0015] Further, in step (3), it is preferred to prepare a hydrogel precursor solution with 4% or 5% HAMA by mass and 1% or 2% CMCSMA by mass; and add 0.1% photoinitiator 2959 to it, and dissolve it completely before use.

[0016] Further, in steps (4) and (5), 300 μL of the precursor mixture containing exosomes is added to the microneedle mold, and centrifuged at 3000 rpm for 5 min to allow it to enter the tip of the needle. Then, the precursor mixture containing Chlamydomonas reinhardtii is filled into the backing layer. Finally, the microneedles are cross-linked under a 365 nm UV lamp for 10 min and gently demolded to obtain the composite microneedles.

[0017] The application of the hydrogel microneedles in the preparation of products for treating osteoarthritis.

[0018] The application of the hydrogel microneedles in the preparation of products that improve local hypoxia in osteoarthritis, inhibit inflammatory response, promote chondrocyte proliferation and migration, promote cartilage matrix synthesis, and delay joint degeneration.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: 1. A multifunctional therapeutic system combining Chlamydomonas reinhardtii and exosomes was constructed. This system integrates in-situ photosynthetic oxygen supply, exosome anti-inflammatory repair, and microneedle local delivery onto a single platform, enabling simultaneous intervention in key aspects of osteoarthritis such as hypoxia, inflammation, and insufficient tissue repair.

[0020] 2. A functionalized hydrogel microneedle carrier based on CMCSMA / HAMA was constructed. This carrier possesses excellent mechanical properties, gel-forming properties, and transdermal capabilities, enabling efficient local delivery of exosomes while providing a relatively stable survival environment for Chlamydomonas reinhardtii, thereby prolonging the oxygen supply time.

[0021] 3. The hydrogel microneedles exhibit good biocompatibility and pro-repair activity. In vitro experiments show that this system can promote chondrocyte proliferation and migration, enhance ALP and GAG expression, reduce ROS levels, and exert anti-inflammatory effects by regulating macrophage polarization.

[0022] 4. The hydrogel microneedles demonstrated good therapeutic potential in an in vivo model of osteoarthritis. They can reduce bone destruction and osteophyte formation, protect cartilage tissue structure, upregulate the anti-inflammatory factor IL-4 and downregulate the pro-inflammatory factor TNF-α, and promote joint repair by regulating inflammation-related pathways and the expression of cartilage metabolism-related genes.

[0023] 5. The hydrogel microneedles are made entirely of naturally derived materials and bioactive ingredients, and are green, biosafe, and have transformation potential. They can provide a minimally invasive and highly effective new treatment option for chronic degenerative diseases such as osteoarthritis. Attached Figure Description

[0024] Figure 1 Figure (I) shows the density curve (A), optical morphology (B), growth density curve (C), and zeta potential (D) of Chlamydomonas reinhardtii; Figure (II) shows the zeta potential (A), particle size (B), and transmission electron microscopy (TEM) morphology characterization (C) of the extracted Chlamydomonas reinhardtii exosomes.

[0025] Figure 2 The images show the Fourier transform infrared spectra of CMCSMA (A) and HAMA (B), and the hydrogen nuclear magnetic resonance spectra of CMCSMA (C) and HAMA (D).

[0026] Figure 3 In the middle: (I) the antioxidant capacity of hydrogels with different ratios: DPPH free radical scavenging (A) and ABTS free radical scavenging (B); (II) the rheological determination of the hydrogel matrix: equilibrium swelling ratio (A) and porosity detection results (B); (III) the gelation time curves: (A) H4C1, (B) H4C2, (C) H5C1, (D) H5C2.

[0027] Figure 4 The image shows the scanning electron microscope (SEM) morphology (A), mechanical properties (B), and H&E stained sections (C) of the composite microneedle array and its insertion ability into rat skin.

[0028] Figure 5Figure 1 shows the activity results of Chlamydomonas reinhardtii in hydrogel: continuous oxygen production capacity of Chlamydomonas reinhardtii in gel (A), oxygen production and consumption of Chlamydomonas reinhardtii in gel (B), chlorophyll content of Chlamydomonas reinhardtii in hydrogel (C), and detection of chlorophyll absorption peak in hydrogel (D).

[0029] Figure 6 (I) Morphological diagram of primary rat chondrocytes (A) and results of exosome-induced CCK-8 in primary rat chondrocytes (B); (II) Relative proliferation rate of primary rat chondrocytes (CCK-8) by microneedle extract and exosomes (A), live / dead staining results (B), Transwell three-dimensional cell migration experiment (C) (D), and fluorescence evaluation diagram of mitochondrial membrane potential protection (JC-1) (E); (III) Modeling experiment of chondrocytes damaged by H2O2 (A), protective effect of composite hydrogel microneedle extract on chondrocytes damaged by H2O2 (B), and ROS clearance in chondrocytes damaged by H2O2 by composite hydrogel microneedle extract (C); (IV) Results of anti-inflammatory repair test (A) Immunofluorescence of COL II, (B) Immunofluorescence of SOX9, (C) Macrophage polarization experiment (*p<0.05,**p<0.01, ***p<0.001).

[0030] Figure 7 In the table, (Ⅰ) represents the blood compatibility of the functionalized hydrogel microneedles, where (A) is and (B) is; (Ⅱ) represents the results of the histological safety evaluation of major organs.

[0031] Figure 8 Micro-CT images of the knee joints of rats in different treatment groups.

[0032] Figure 9 HE staining and Safranin-Fix Green staining results of the knee joints of rats in different treatment groups.

[0033] Figure 10 Immunohistochemical staining results of Col II, IL-6 and IL-10 in the knee joints of rats in different treatment groups.

[0034] Figure 11 Immunofluorescence staining results of the knee joints of rats in different treatment groups.

[0035] Figure 12 The results of serum IL-4 (A) and TNF-α levels in rats under different treatment groups (B).

[0036] Figure 13 The results of transcriptomic differential expression (A), KEGG (B), GO enrichment (C), and GSEA analysis (D) are shown. Detailed Implementation

[0037] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following detailed description is provided in conjunction with specific embodiments and accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention; any equivalent substitutions, parameter adjustments, or process optimizations made based on the concept of this invention should be included within the scope of protection of this invention.

[0038] Example 1: Culture of Chlamydomonas reinhardtii and extraction and identification of its exosomes This embodiment illustrates the methods for culturing Chlamydomonas reinhardtii, screening during the plateau phase, and extracting and characterizing its exosomes.

[0039] (1) Culture and morphological observation of Chlamydomonas reinhardtii: Chlamydomonas reinhardtii was inoculated into a suitable culture medium and cultured under light conditions. During the culture process, 10 μL of algal solution was taken and diluted 25 times, dropped onto a glass slide, and the morphology of Chlamydomonas reinhardtii was observed under an inverted microscope. Its cell size, morphological integrity and dispersion state were recorded.

[0040] (2) Detection of the growth status of Chlamydomonas reinhardtii: 5 mL of Chlamydomonas reinhardtii suspension in the plateau phase was transferred to fresh culture medium for further culture. Samples were taken at fixed time points, and the absorbance value (OD750) was measured at 750 nm using a UV-Vis spectrophotometer to plot the growth curve. At the same time, algal suspensions of different densities were prepared by centrifugation and resuspending, and OD750 was measured and density curves were plotted to determine the appropriate culture stage and subsequent exosome extraction time.

[0041] (3) Detection of surface potential of Chlamydomonas reinhardtii: Take 1 mL of Chlamydomonas reinhardtii suspension and dilute it 25 times. Place it in a particle size potential analyzer to measure the Zeta potential in order to evaluate its dispersion stability and surface charge characteristics.

[0042] (4) Extraction of Chlamydomonas reinhardtii exosomes: Chlamydomonas reinhardtii culture medium that entered the plateau phase on day 4 of culture was selected, and exosomes were extracted using differential centrifugation combined with ultracentrifugation. First, centrifugation was performed at 4 ℃ and 2000 g for 10 min to remove intact algal cells and large fragments; after collecting the supernatant, centrifugation was performed at 4 ℃ and 10000 g for 30 min to further remove residual cell debris; the obtained supernatant was filtered through a 0.22 μm sterile filter membrane and transferred to an ultracentrifuge tube, and ultracentrifuged at 4 ℃ and 100000 g for 70 min to enrich the extracellular vesicles. After discarding the supernatant, the precipitate was gently resuspended with pre-cooled PBS buffer and washed again by ultracentrifugation under the same conditions; finally, the precipitate was resuspended in 100 μL of sterile PBS to obtain the Chlamydomonas reinhardtii exosome suspension, which was stored at -20 ℃ for short-term storage and -80 ℃ for long-term storage.

[0043] (5) Morphology and particle size characterization of exosomes: 10 μL of exosome suspension was dropped onto a Parafilm sealing film, and the copper mesh of the carrier film was placed face down on the surface of the droplet for adsorption for 10-15 min. After removing the residual liquid, the vesicles were negatively stained with 2% phosphotungstic acid for 3-5 min, and then air-dried at room temperature. The morphology of the vesicles was observed under a transmission electron microscope. Another appropriate amount of exosome sample was diluted and its potential, particle size distribution and particle concentration were detected by Zeta potential analysis and nanoparticle tracking analysis (NTA).

[0044] Results analysis: such as Figure 1 As shown in (A), (B), (C), and (D) of (I), *Chlamydomonas reinhardtii* appears as a regular oval or elliptical cell under an optical microscope, with clear cell outlines, plump morphology, and good dispersion. No obvious aggregation, breakage, or mechanical damage was observed, indicating that its culture condition is good. The OD750 growth curve shows that *Chlamydomonas reinhardtii* undergoes an adaptation period and a rapid proliferation period after inoculation, gradually entering a plateau phase after day 4, with OD750 tending to stabilize. Therefore, *Chlamydomonas reinhardtii* cultured for 4 days has a high cell density and a relatively stable physiological state, making it suitable as a material source for subsequent exosome extraction and hydrogel loading.

[0045] The cell density standard curve showed a good linear relationship between Chlamydomonas reinhardtii cell density and OD750 value, providing a quantitative basis for subsequent algal cell addition. Zeta potential results showed that the surface of Chlamydomonas reinhardtii was negatively charged and the peak shape was relatively concentrated, indicating that it had a relatively stable dispersion state in the culture system. These results demonstrate that the culture system used in this invention can obtain morphologically intact, highly active, and controllable numbers of Chlamydomonas reinhardtii cells.

[0046] like Figure 1 (II) The Chlamydomonas reinhardtii exosomes obtained after differential centrifugation, filtration, and ultracentrifugation, as shown in (A), (B), and (C), exhibited typical membranous vesicle structures under transmission electron microscopy, with clear boundaries and relatively intact morphology, consistent with the morphological characteristics of extracellular vesicles. Zeta potential detection showed that the exosomes had relatively stable surface charges, and NTA particle size distribution results showed that their particle sizes were mainly within the nanoscale range and relatively concentrated. Based on the morphology, potential, and particle size results, it can be concluded that this method can stably obtain well-dispersed Chlamydomonas reinhardtii-derived exosomes with the required particle size from the supernatant of Chlamydomonas reinhardtii culture during the plateau phase, providing a reliable basis for subsequent evaluation of microneedle tip loading and anti-inflammatory repair functions.

[0047] Example 2: Preparation and physicochemical property evaluation of CMCSMA / HAMA hydrogel matrix material This embodiment illustrates the preparation of methacryloyl carboxymethyl chitosan (CMCSMA) and methacryloyl hyaluronic acid (HAMA), as well as the evaluation methods for the morphology, swelling, porosity, gelation, and antioxidant properties of CMCSMA / HAMA hydrogel matrices.

[0048] (1) Preparation of CMCSMA: 2.0 g of carboxymethyl chitosan (CMCS) powder was added to deionized water and stirred for 2-3 h to dissolve it completely. Then, methacrylic anhydride (MA) was added dropwise to the CMCS solution at a rate preferably of 0.5 mL / min. After the addition was completed, the pH of the reaction system was adjusted to 8-9 with NaOH solution at room temperature, and the reaction was continued. After the reaction was completed, the product solution was transferred to a dialysis bag and dialyzed for 3 days to remove unreacted small molecules and impurities. Finally, CMCSMA was obtained by freeze-drying.

[0049] (2) Preparation of HAMA: 2.0 g of hyaluronic acid (HA) was dissolved in 150 mL of deionized water and stirred overnight until completely dissolved; then dimethylformamide (DMF) and methacrylic anhydride (MA) were added dropwise to the solution, and the pH of the reaction system was maintained at 8-9. After reacting overnight, NaCl was added, and then the solution was transferred to a dialysis bag and dialyzed with deionized water at 4 °C for 3 days. Finally, HAMA was obtained by freeze drying.

[0050] (3) Chemical structure characterization of CMCSMA and HAMA: The sample to be tested was ground with dry KBr powder at a ratio of about 1:100 and then pressed into a tablet. The infrared absorption spectrum was collected by Fourier transform infrared spectrometer. Another dry sample was dissolved in an appropriate amount of deuterated reagent, filtered and transferred into a standard NMR tube. Its characteristic proton peaks were analyzed by nuclear magnetic resonance hydrogen spectrum.

[0051] (4) Hydrogel preparation: CMCSMA and HAMA were dissolved in different mass fractions to prepare hydrogel precursor solutions. The system with 4% HAMA and 1% CMCSMA was named H4C1; similarly, H4C2, H5C1 and H5C2 groups were prepared. 0.1% photoinitiator 2959 was added to the precursor solution and stirred thoroughly until completely dissolved. The solution was then crosslinked under 365 nm ultraviolet light irradiation to form a hydrogel.

[0052] (5) Characterization of hydrogel morphology: After freeze-drying hydrogels with different ratios, cross sections were cut and gold sputtered for conductive treatment at an accelerating voltage of 8.00 kV for 200 s. The three-dimensional pore structure inside the hydrogel was observed by scanning electron microscopy (SEM), and the pore size and network morphology were recorded.

[0053] (6) Determination of equilibrium swelling rate of hydrogel: The freeze-dried hydrogel sample was immersed in PBS solution, removed at the set time point, and the excess liquid on the surface was removed and weighed until the mass was basically stable. The equilibrium swelling rate was calculated to evaluate the water absorption capacity and hydration stability of the hydrogel.

[0054] (7) Determination of hydrogel porosity: Place a known mass of dry hydrogel sample into a known volume of anhydrous ethanol. After it has fully permeated and reached saturation, record the total volume. After removing the sample, record the remaining ethanol volume. Calculate the porosity based on the volume change.

[0055] (8) Determination of hydrogel gelation time and rheological properties: The gelation process was detected by a rheometer. A 20 mm plate was selected, the gap was set to 1 mm, the equilibrium time was 30 s, and the test temperature was 25 ℃. The fixed shear strain was set to 1%, the oscillation frequency was 0.1~10 Hz, and the changes of storage modulus G′ and loss modulus G″ with time were recorded under 365 nm ultraviolet light irradiation to evaluate the photocrosslinking gelation ability and the stability of the gel network.

[0056] (9) Determination of antioxidant properties of hydrogels: The antioxidant capacity of hydrogels was evaluated using ABTS and DPPH free radical scavenging experiments. In the ABTS experiment, 7 mmol / L ABTS stock solution was mixed with 2.45 mmol / L potassium persulfate solution at a 1:1 ratio and reacted in the dark for 12–16 h to generate a free radical working solution. This solution was then co-incubated with each group of hydrogel precursor solutions or precursor solutions containing exosomes, and the absorbance was measured at 734 nm. In the DPPH experiment, the hydrogel precursor solution was mixed with the DPPH working solution and incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm, and the free radical scavenging rate was calculated.

[0057] (10) Evaluation of the activity of Chlamydomonas reinhardtii in hydrogel: Chlamydomonas reinhardtii was mixed with hydrogel precursor solution and cross-linked, and then placed in PBS solution with nitrogen-filled to reduce dissolved oxygen concentration. It was cultured under a stable incandescent light source, and the dissolved oxygen change was recorded in real time by a portable dissolved oxygen meter. At the same time, its oxygen consumption rate was measured under dark conditions, and chlorophyll was extracted by 75% ethanol. The absorbance at 680 nm and the ultraviolet absorption spectrum were measured to evaluate the metabolic activity of Chlamydomonas reinhardtii in hydrogel and the chlorophyll retention.

[0058] Results analysis: such as Figure 2As shown in (A), (B), (C), and (D), CMCSMA exhibits a new characteristic absorption peak at approximately 1700 cm⁻¹ compared to CMCS, which is mainly associated with the methacrylamide group, indicating that methacrylic anhydride has been successfully grafted onto the carboxymethyl chitosan molecular chain. HAMA, compared to HA, shows a new characteristic absorption peak near approximately 1550 cm⁻¹, suggesting the introduction of methacrylamide-related groups into the hyaluronic acid molecular structure. Further analysis using 1H NMR spectroscopy reveals that both CMCSMA and HAMA exhibit double bond proton peaks in the 5.5–6.2 ppm region and methyl proton peaks near approximately 1.9 ppm, further confirming the successful methacrylamide modification of both natural polymers. These results demonstrate that both CMCSMA and HAMA possess photocrosslinking reaction sites and can serve as base materials for the subsequent construction of photocurable hydrogel microneedle matrices.

[0059] like Figure 3 As shown, CMCSMA / HAMA hydrogels with different ratios all exhibited good antioxidant, swelling, and gel-forming properties. ABTS and DPPH free radical scavenging experiments (I, (A) and (B)) showed that the hydrogel matrix itself has a certain free radical scavenging ability, and the scavenging effect was further enhanced after the introduction of Chlamydomonas reinhardtii exosomes, indicating that the system can provide a certain buffer for oxidative stress. As shown in (II, (A) and (B), the equilibrium swelling rate results showed that all groups of hydrogels could reach swelling equilibrium in a short time, indicating that they have a fast water absorption response and good hydration stability; the porosity test results showed that the hydrogels with different ratios all have a certain porous structure, which can provide space for exosome loading, material exchange, and oxygen diffusion.

[0060] As shown in (III), the rheological results indicate that after UV irradiation, the storage modulus G′ of each group of hydrogels gradually increased and exceeded the loss modulus G″, indicating that the system gradually transformed from a sol state to a gel network dominated by elastic characteristics. With the increase of HAMA and CMCSMA concentrations, the density of crosslinkable groups increased, the gel network formation rate accelerated, and the structural stability was enhanced. Considering the antioxidant capacity, swelling performance, pore structure, and gelation behavior, the H5C2 group has both good structural stability and functional carrying capacity (as shown in (IV) (A), (B), (C), and (D)), making it suitable as a preferred matrix for the subsequent preparation of functionalized microneedles.

[0061] Example 3: Preparation and evaluation of the physicochemical properties of functionalized hydrogel microneedles This embodiment illustrates the preparation method of functionalized hydrogel microneedles loaded with Chlamydomonas reinhardtii exosomes and Chlamydomonas reinhardtii, as well as their morphology, elemental distribution, mechanical properties, skin insertion ability, and in-situ oxygen supply performance.

[0062] (1) Preparation of blank hydrogel microneedles: Prepare H5C2 hydrogel precursor solution, add 0.1% photoinitiator 2959 and stir until fully dissolved. Add 300 μL of precursor solution to the microneedle mold, centrifuge at 3000 rpm for 5 min to allow the precursor solution to fully enter the needle tip area; then continue to add precursor solution to fill the mold backing layer, irradiate under 365 nm UV lamp for 10 min for crosslinking, and gently demold to obtain blank hydrogel microneedles.

[0063] (2) Preparation of composite functionalized microneedles: Chlamydomonas reinhardtii exosomes and hydrogel precursor solution were thoroughly mixed. 300 μL of the mixed solution was added to the microneedle mold and centrifuged at 3000 rpm for 5 min to allow the exosome-containing precursor solution to enter the needle tip. Then, excess exosome-containing solution in the backing layer was scraped off. Chlamydomonas reinhardtii and hydrogel precursor solution were mixed again and filled into the microneedle backing layer. The overflow solution was scraped off. Finally, the microneedles were irradiated under a 365 nm UV lamp for 10 min to crosslink and solidify the precursor system. The microneedles were then demolded to obtain Chlamydomonas reinhardtii photosynthetic oxygen supply synergistic exosome functionalized hydrogel microneedles.

[0064] (3) Characterization of microneedle morphology: After drying, the microneedle samples were sputtered with gold at an accelerating voltage of 8.00 kV for 200 s. The overall morphology of the microneedle array, the integrity of the needle tip and the structure of the needle body were observed by scanning electron microscopy.

[0065] (4) Microneedle element distribution detection: Based on SEM observation, energy dispersive X-ray spectroscopy (EDS) was used to perform surface scanning analysis of elements such as C, N, O, and Na in the microneedles to evaluate the uniformity of the microneedle material composition distribution.

[0066] (5) Microneedle mechanical property test: The compressive strength of the microneedles was evaluated using a universal testing machine. During the test, the microneedles were placed horizontally on the platform with the needle tip pointing vertically upward; the initial distance between the test probe and the needle tip was set to 0.5 cm, and the probe was compressed downward at a speed of 0.4 mm / min, and the displacement and force changes were recorded in real time.

[0067] (6) Evaluation of skin insertion capability: Take rat skin tissue, press the microneedle patch on the skin surface for a certain period of time and then remove it. Then fix, dehydrate, embed and section the skin, perform H&E staining, and observe the microchannel morphology and penetration depth formed by the microneedles under an optical microscope.

[0068] (7) Evaluation of oxygen supply performance of microneedles: The hydrogel microneedles loaded with Chlamydomonas reinhardtii were placed in PBS solution and the dissolved oxygen content was detected by a portable dissolved oxygen meter under incandescent light. At the same time, dark conditions were set and the oxygen consumption of algal cell respiration was recorded to evaluate the oxygen metabolism characteristics of the microneedle system under light and non-light conditions.

[0069] Results analysis: such as Figure 4 As shown in (A), (B), and (C), a regular and well-formed hydrogel microneedle array can be obtained by using an H5C2 hydrogel precursor solution for molding and UV cross-linking. Scanning electron microscopy results show that the microneedles are regular quadrangular pyramids with sharp tips and intact structures, without obvious collapse, breakage, or deformation. The height of a single microneedle is approximately 600 μm, and the substrate width is approximately 300 μm, indicating that this photocross-linking molding process has good stability and repeatability.

[0070] Mechanical property results showed that microneedles with different formulations all possessed a certain compressive strength, capable of withstanding the mechanical forces required during skin insertion, with a single needle mechanical strength > 0.1 N. As the concentration of the hydrogel precursor increased, the compressive strength of the microneedles gradually increased, with the H5C2 group exhibiting superior mechanical properties, meeting the requirements of penetrating the stratum corneum while maintaining needle tip integrity. H&E staining of rat skin showed that after pressure, the microneedles formed obvious microchannels in the skin tissue, with a penetration depth of approximately 200–300 μm, and the surrounding tissue structure remained largely intact, without extensive tearing or severe damage. These results indicate that the hydrogel microneedles prepared in this invention possess excellent minimally invasive puncture capabilities and can serve as an effective carrier for the local delivery of exosomes and active components of Chlamydomonas reinhardtii.

[0071] like Figure 5 As shown in (A), (B), (C), and (D), the hydrogel system loaded with *Chlamydomonas reinhardtii* exhibits stable oxygen production capacity under light conditions. Under light, the dissolved oxygen concentration in PBS increases rapidly and remains relatively stable for a considerable period; under dark conditions, the system shows a certain oxygen consumption trend, indicating that the encapsulated *Chlamydomonas reinhardtii* still possesses active photosynthetic and respiratory metabolic capabilities. Chlorophyll content and absorption spectrum results show that *Chlamydomonas reinhardtii* within the hydrogel maintains high chlorophyll activity after cultivation, and the characteristic absorption peak does not show significant shift, indicating that the CMCSMA / HAMA hydrogel network can provide a mild and stable survival environment for *Chlamydomonas reinhardtii*. These results demonstrate that this functionalized microneedle system not only possesses stable physical puncture and delivery capabilities but can also achieve localized continuous oxygen supply through the photosynthesis of *Chlamydomonas reinhardtii*.

[0072] Example 4: Cellular experiments and in vitro anti-inflammatory and repair evaluation of functionalized hydrogel microneedles This embodiment illustrates the in vitro biological effects of functionalized hydrogel microparticles on primary rat chondrocytes and macrophages, including cell compatibility, screening for optimal exosome concentration, cell migration, cartilage matrix formation, antioxidant protection, maintenance of cartilage phenotype, and macrophage polarization.

[0073] (1) Extraction of primary chondrocytes: Male SD rats aged 2-3 weeks were euthanized by cervical dislocation, and the hair on both hind limbs was removed. The rats were then disinfected in 75% ethanol for 5 min. The knee joint was exposed in a clean bench, and hyaline cartilage was scraped from the articular surface. After washing with PBS 2-3 times, the cartilage was minced. 5-10 mL of EDTA-trypsin was added, and the rats were incubated at 37 ℃ and 5% CO2 for 30 min. After discarding the trypsin, the rats were washed with PBS, and 5 mL of 0.2% type II collagenase solution was added. The rats were digested at 37 ℃ for 4-5 h. The digestion solution was filtered through a 70 μm filter, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in complete culture medium and seeded in T25 culture flasks. After 48 h, the medium was changed to remove non-adherent cells. The medium was changed every 3 days thereafter. Chondrocytes of passage 5 or less were used for the experiment.

[0074] (2) Effect of exosomes on chondrocyte proliferation: Chondrocytes in the logarithmic growth phase were diluted to 2×10⁻⁶ in DMEM / F12 complete medium. 4 Cells / mL were seeded at 200 μL per well in 96-well plates. After adhesion, the experimental groups were added to complete culture medium containing 20 μg / mL, 40 μg / mL, and 80 μg / mL Chlamydomonas reinhardtii exosomes, while the control group was added to ordinary complete culture medium. The cells were cultured for 24, 48, and 72 h, respectively. At each time point, the cells were incubated with CCK-8 solution for 2 h, and the absorbance was measured at 450 nm to calculate the relative cell proliferation rate.

[0075] (3) Chondrocyte compatibility evaluation: Primary chondrocytes were adjusted to 2×10 4 Cells / mL were seeded in 96-well plates. After adhesion, the experimental groups were treated with the four different hydrogel extracts mentioned above, or with a hydrogel extract containing 40 μg / mL exosomes, while the control group was treated with fresh complete culture medium. Relative cell proliferation was assessed using the CCK-8 assay after 1, 3, and 5 days of culture. Simultaneously, Calcein-AM / PI live / dead staining was used to observe cell viability and cell death under a fluorescence microscope.

[0076] (4) Chondrocyte migration assay: The effects of hydrogels and exosomes on chondrocyte migration were evaluated using Transwell chambers. Chondrocytes were subjected to serum-free starvation for 24 h before the experiment, and then the cell density was adjusted to 3 × 10⁶ cells / h using serum-free DMEM / F12 medium. 5cells / mL. Add 100 μL of cell suspension to the upper chamber and basal culture medium, H5C2 hydrogel extract, or hydrogel extract containing 40 μg / mL exosomes to the lower chamber. After 24 h of culture, fix with methanol for 20 min, stain with 0.1% crystal violet for 20 min, wipe away unmigrated cells from the upper chamber, observe under a microscope and photograph. If necessary, wash off the crystal violet with 33% acetic acid, measure absorbance at 570 nm and calculate relative migration rate.

[0077] (5) Evaluation of ALP activity and glycosaminoglycan formation: Chondrocytes from generations 2 to 5 were subjected to a 2×10⁻⁶ saturation assay. 5 Cells / wells were seeded in 6-well plates pretreated with 0.1% gelatin. After adhesion, H5C2 hydrogel extract or exosome extract containing 40 μg / mL was added for intervention. The medium was changed every 2 days. ALP staining and ALP activity quantification were performed on days 7 and 14. For glycosaminoglycan detection, chondrocytes were seeded at 5 × 10⁻⁶ wells. 4 Cells / mL were seeded in 24-well plates, fixed with 4% paraformaldehyde after 3 days of intervention, and then stained with Alsin Blue and Toluidine Blue to evaluate the production of acidic mucopolysaccharides and GAGs in the extracellular matrix of chondrocytes.

[0078] (6) Antioxidant capacity and mitochondrial protection experiment: chondrocytes were subjected to 2×10 4 Cells / mL were seeded in 96-well plates. After adhesion, different concentrations of H2O2 were added to induce oxidative damage, and the appropriate damage concentration was screened using the MTT assay. Subsequently, a positive control group, an H2O2 damage group, an H5C2 hydrogel extract group, and a group containing 40 μg / mL exosome hydrogel extract were set up. After treatment for 2 h, cell viability was detected by the MTT assay. At the same time, the cells were incubated with the DCFH-DA fluorescent probe for 30 min to evaluate the intracellular ROS level. JC-1 staining was performed for 20–30 min to evaluate changes in mitochondrial membrane potential.

[0079] (7) Immunofluorescence of chondrocyte-associated proteins: Chondrocytes were induced with 10 ng / mL IL-1β for 24 h to simulate the inflammatory microenvironment of osteoarthritis; subsequently, they were treated with complete culture medium, H5C2 hydrogel extract, and hydrogel extract containing 40 μg / mL exosomes, respectively. After treatment, the cells were fixed with 4% paraformaldehyde for 20 min, permeated with 0.5% Triton X-100 for 10 min, blocked with 1% BSA for 1 h, and incubated overnight at 4 ℃ with SOX9 or COL II primary antibody, respectively; the next day, the cells were incubated with fluorescent secondary antibody in the dark for 1 h, and after DAPI counterstaining, fluorescence microscopy and relative expression intensity analysis were performed.

[0080] (8) Macrophage polarization experiment: RAW 264.7 macrophages were polarized at a concentration of 2 × 10⁻⁶ cells / mL. 4 Cells / mL were seeded in 96-well plates, and after adhesion, 100 ng / mL LPS was added to induce M1 polarization for 24 h. Subsequently, the medium was replaced with complete medium, H5C2 hydrogel extract, or hydrogel extract containing 40 μg / mL exosomes for further intervention. After treatment, the cells were fixed and blocked, and incubated overnight at 4 °C with iNOS primary antibody (M1 marker) or CD163 primary antibody (M2 marker), respectively. Then, fluorescent secondary antibody and DAPI were added for counterstaining, and the expression of each marker was observed under a fluorescence microscope.

[0081] (9) Detection of cartilage-related gene expression: chondrocytes were seeded into gelatin-pretreated 6-well plates and cultured for 14 days. RNA was extracted and cDNA was obtained by reverse transcription. The expression of cartilage-related genes such as COL I, COL II and Acan was detected by qPCR. GAPDH was used as an internal reference and the relative expression level was calculated by the 2^-ΔΔCt method.

[0082] Results analysis: such as Figure 6 (I) As shown, Chlamydomonas reinhardtii exosomes did not exhibit significant toxicity to primary rat chondrocytes within the range of 20–80 μg / mL, and the relative cell proliferation rate of each group remained at a high level. Among them, the 40 μg / mL group showed a more significant proliferative effect, indicating that this concentration is more suitable for subsequent composite microneedle construction. CCK-8 and live / dead staining results showed that the hydrogel microneedle extracts of different ratios had good compatibility with chondrocytes, with green live cells being dominant and no large number of red dead cells observed. Compared with the hydrogel group alone, the H5C2+Exo group had a greater number of cells, suggesting that Chlamydomonas reinhardtii exosomes can further enhance the promoting effect of the hydrogel microneedle system on chondrocyte growth.

[0083] like Figure 6 As shown in (II), the Transwell migration assay revealed a certain number of migrating cells in each treatment group. The H5C2+Exo group showed the highest number of chondrocytes migrating beneath the membrane, with significantly higher crystal violet staining area and relative migration rate compared to the hydrogel-only group. This result indicates that exosome-composite hydrogel microneedles can enhance the three-dimensional migration and recruitment capacity of chondrocytes, which is beneficial for cell replenishment in the damaged area and subsequent tissue repair. JC-1 staining results showed that after H2O2 modeling, the red fluorescence of chondrocytes decreased while the green fluorescence increased, indicating a decrease in mitochondrial membrane potential. After H5C2+Exo intervention, the red / green fluorescence ratio significantly recovered, indicating that it can maintain mitochondrial membrane potential stability and alleviate mitochondrial damage and early apoptosis caused by oxidative stress.

[0084] like Figure 6As shown in (III), H2O2 treatment resulted in a dose-dependent decrease in chondrocyte viability, which can be used to construct a stable oxidative damage model. After treatment with H5C2 hydrogel extract and the H5C2+Exo complex system, the viability of damaged chondrocytes recovered to varying degrees, with the H5C2+Exo group showing the most significant protective effect. DCFH-DA fluorescence staining showed that the H2O2 model group exhibited significantly enhanced green fluorescence, indicating a large accumulation of ROS; while the H5C2+Exo group showed significantly weakened green fluorescence, indicating that this system can effectively reduce excessive intracellular ROS, thereby exerting an antioxidant protective effect.

[0085] like Figure 6 (IV) As shown, IL-1β induction significantly decreased the expression of SOX9 and COL II in chondrocytes, suggesting that the inflammatory microenvironment disrupted chondrocyte phenotypic stability and matrix synthesis function. After intervention with the H5C2+Exo complex system, both the nuclear positive signal of SOX9 and the fluorescence intensity of COL II were significantly enhanced, indicating that this system can maintain the chondrocyte phenotype under inflammatory stimulation and promote the expression of cartilage matrix-related proteins. Macrophage polarization experiments showed that LPS induction significantly enhanced the iNOS positive signal, suggesting the formation of the M1 inflammatory phenotype; H5C2+Exo treatment decreased iNOS expression while significantly enhancing the CD163 positive signal, indicating that exosome-composite hydrogel microneedles can promote the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory and repairing M2 type. In summary, the hydrogel microneedle system of this invention exhibits good cell compatibility, migration-promoting, antioxidant, mitochondrial protective, and anti-inflammatory repair effects in vitro.

[0086] Example 5: Application and Mechanism Analysis of Functionalized Hydrogel Microneedles in a Rat Model of Osteoarthritis This embodiment illustrates the in vivo treatment method, safety evaluation, and mechanism analysis of functionalized hydrogel microneedles in a rat osteoarthritis model.

[0087] (1) Blood compatibility test: Fresh whole blood from rabbits was collected, anticoagulated with sodium citrate, and mixed with an equal volume of physiological saline. The mixture was centrifuged at 4 ℃ and 1500 r / min for 10 min, the supernatant was discarded, and the washing was repeated 4-6 times until the supernatant was colorless and transparent, and red blood cell precipitate was obtained. The red blood cells were diluted with physiological saline to a 2% (v / v) suspension. The irradiated sterilized hydrogel sample was extracted in physiological saline to prepare test solutions of different concentrations. An extraction solution group with 40 μg / mL exosomes, a negative control group (physiological saline), and a positive control group (deionized water) were set up. Each group was mixed with an equal volume of red blood cell suspension, incubated at 37 ℃ for 1 h, centrifuged, and the absorbance of the supernatant was measured at 545 nm to calculate the hemolysis rate.

[0088] (2) Establishment of a rat osteoarthritis model: Sodium monoiodoacetate (MIA) powder was dissolved in sterile physiological saline and stirred in the dark to prepare a 40 μg / mL solution. Male Wistar rats, approximately 250 g in size and 7–8 weeks old, were selected and acclimatized for 7 days, followed by a 12-hour fast with free access to water. Anesthesia was performed using 5% isoflurane, and after confirmation, 2%–3% isoflurane was maintained. The rats were fixed in a supine position, and the right knee joint was selected. After shaving and disinfection, the knee joint was flexed to approximately 90°. 50 μL of MIA solution was drawn into a syringe and injected into the joint cavity through the infrapatellar ligament. After confirming the needle was inside the joint cavity, the solution was slowly injected, held for 10–15 seconds, and then withdrawn. The knee joint was passively flexed and extended several times to promote drug distribution. The osteoarthritis model was obtained approximately 14 days after model establishment.

[0089] (3) Grouping and treatment of rats: Rats that successfully established the model were randomly divided into Control group (normal growth group), Model group (modeling saline treatment), MN group (hydrogel microneedle group), MN+Exo group (hydrogel microneedle + exosome), and MN+Exo+Chl group (hydrogel microneedle + exosome + Chlamydomonas reinhardtii). During treatment, rats were anesthetized with isoflurane, the hair on the knee joint was shaved and cleaned and disinfected, and after the skin was dry, the corresponding microneedle patch was pressed onto the knee joint for 5 minutes and fixed with transparent medical tape. The microneedles were changed every 2-3 days during the treatment period. Rats were sacrificed in the second and fourth weeks of treatment, and the knee joint tissue was fixed in 4% paraformaldehyde solution for later use.

[0090] (4) Micro-CT detection: Knee joint samples were taken 2 weeks and 4 weeks after treatment. After fixation with 4% paraformaldehyde for 24 hours, Micro-CT scans were performed to reconstruct three-dimensional images of joint bone tissue and to perform bone morphometric analysis to evaluate joint bone destruction, osteophyte formation and joint structural integrity.

[0091] (5) H&E and Safranin-Fix-Green staining: Intact knee joint tissue was taken, fixed for 48 h, rinsed with running water, and decalcified in 10% EDTA decalcification solution. Then, it was dehydrated by gradient ethanol, cleared with xylene, embedded in paraffin, and sectioned at 5 μm. H&E staining was used to observe the integrity of cartilage surface, cell arrangement, synovial inflammation, and subchondral bone structure. In Safranin-Fix-Green staining, the rehydrated sections were first stained with iron hematoxylin, then stained with 0.02% Fast Green for 1–3 min, differentiated with 1% acetic acid for 10–15 s, stained with 1% Safranin O for 2–5 min, dehydrated and cleared, and then mounted to observe the retention of chondroitin proteoglycans and matrix repair.

[0092] (6) Immunofluorescence of joint tissue: Knee cartilage tissue treated for 2 weeks was stained with COL II, SOX9, MMP13 and HIF-1α immunofluorescence. After dewaxing and rehydration, the sections were heated in pH 6.0 citrate buffer for antigen retrieval, washed with PBS, treated with 3% hydrogen peroxide, blocked with 5% BSA for 30 min, and incubated overnight at 4 ℃ with the corresponding primary antibody. The next day, the sections were incubated at room temperature for 60 min with fluorescent secondary antibody, counterstained with DAPI, mounted and observed under a fluorescence microscope.

[0093] (7) Immunohistochemistry of joint tissue: Knee joint tissues treated for 2 weeks and 4 weeks were subjected to COL II, IL-6 and IL-10 immunohistochemical staining. After dewaxing, rehydration, antigen retrieval and blocking, the corresponding primary antibody was added and incubated overnight at 4 °C; the next day, the secondary antibody was added and incubated, DAB staining was performed, hematoxylin counterstaining was performed, dehydrated and cleared and mounted, and the distribution and intensity of positive staining were observed under a microscope.

[0094] (8) ELISA detection: Whole blood was collected via the abdominal aorta 2 weeks after treatment. After standing at room temperature for 2 h, the blood was centrifuged at 3000 rpm for 15 min at 4 ℃ and serum was collected. The levels of IL-4 and TNF-α were detected according to the ELISA kit instructions: After adding samples to the standard wells, blank wells and sample wells, the detection antibody was added and incubated at 37 ℃ for 1 h. After washing, streptavidin working solution was added, and the mixture was incubated again and washed. The chromogenic substrate was added and reacted in the dark for 30 min. Finally, the stop solution was added and the OD value was read at 450 nm.

[0095] (9) Transcriptomic analysis: Joint tissues from the Sham group and MN+Exo+Chl group 2 weeks after treatment were subjected to transcriptomic sequencing. Combined with differentially expressed gene analysis, KEGG pathway enrichment, GO functional annotation and GSEA analysis, the molecular mechanism of the composite microneedle system regulating the inflammatory microenvironment and cartilage repair in osteoarthritis was explored.

[0096] (10) In vivo safety evaluation: 4 weeks after the end of treatment, the heart, liver, spleen, lungs and kidneys of rats in each group were collected, fixed with 4% paraformaldehyde for 24 h, and after routine dehydration, clearing, embedding, 5 μm sectioning and H&E staining, the presence of pathological changes such as inflammatory cell infiltration, tissue necrosis, vacuolar degeneration or structural disorder was observed under an optical microscope.

[0097] Results analysis: such as Figure 7As shown in the blood compatibility experiment, no obvious gross hemolysis was observed in the hydrogel extract at different concentrations and in the composite system loaded with 40 μg / mL Chlamydomonas reinhardtii exosomes. The hemolysis rate in each experimental group was lower than the safety evaluation threshold for biomedical materials, and no significant adverse changes occurred due to increased material concentration or the addition of exosomes, indicating that the hydrogel microneedles and their exosome composite system have good blood compatibility. H&E staining results of major organs showed that the heart, spleen, liver, lung, and kidney tissues of rats in each group remained structurally intact after treatment, with no obvious inflammatory cell infiltration, tissue necrosis, vacuolar degeneration, or structural disorder, suggesting that this functionalized microneedle system has good in vivo biocompatibility during this experimental period.

[0098] like Figure 8 As shown in the results, Micro-CT scans revealed that the Control group had intact joint structures and smooth articular surfaces with no obvious bone erosion or osteophyte formation. The Sham group showed irregular articular surfaces with significant bone destruction and osteophyte formation, indicating a successful establishment of the osteoarthritis model. The MN group still showed significant joint damage, suggesting that the microneedle matrix alone has limited protective effect on bone structure. The MN+Exo group showed reduced bone destruction and relatively improved articular surfaces, indicating that exosomes have certain anti-inflammatory and repair-promoting effects. The MN+Exo+Chl group had the most intact joint structure, with minimal bone erosion and osteophyte formation, approaching normal levels. These results indicate that exosomes and Chlamydomonas reinhardtii photosynthetic oxygen supply have a synergistic protective effect, reducing OA-related bone structure destruction and delaying joint degeneration.

[0099] like Figure 9 As shown, H&E staining and Safranin-Fixed Green staining further verified the effects of different treatment groups on cartilage tissue structure and matrix preservation. The Sham group showed rough cartilage surface, reduced chondrocyte count, thinner cartilage layer, and significant proteoglycan loss. While the MN group could alleviate damage to some extent, the cartilage surface remained discontinuous, resulting in limited repair. The MN+Exo group showed a smoother cartilage surface and deeper matrix staining. The MN+Exo+Chl group had a thicker cartilage layer, more regular cell arrangement, significantly improved surface continuity, and more pronounced and evenly distributed Safranin O staining, indicating effective preservation and regeneration of proteoglycans and cartilage matrix. These results demonstrate that microneedles combined with exosomes and Chlamydomonas reinhardtii oxygen supply can significantly promote cartilage structure repair and matrix reconstruction, with effects superior to microneedles alone or microneedles loaded with exosomes.

[0100] like Figure 10As shown, immunohistochemical results of joint tissue revealed that COL II positive expression was significantly weakened in the Sham group, indicating continuous loss of cartilage-specific matrix in osteoarthritis; the MN group showed only mild recovery; the COL II positive area expanded in the MN+Exo group, indicating that exosomes can promote cartilage matrix synthesis; the COL II staining was strongest and more evenly distributed in the MN+Exo+Chl group, indicating that the combined treatment can effectively promote type II collagen deposition in cartilage. Regarding inflammatory factors, IL-6 positive staining was strong in the Sham group, indicating continuous activation of local joint inflammation; IL-6 expression decreased in the MN+Exo group, and IL-6 signaling was further weakened in the MN+Exo+Chl group. Conversely, the anti-inflammatory factor IL-10 was underexpressed in the model state, but significantly enhanced after combined intervention with exosomes and Chlamydomonas reinhardtii. These results demonstrate that the system of this invention can simultaneously inhibit pro-inflammatory responses and activate anti-inflammatory repair responses, thereby improving the local immune microenvironment of OA.

[0101] like Figure 11 As shown, immunofluorescence staining further revealed the treatment mechanism from the perspectives of cartilage synthesis, degradation, and hypoxia response. The Control group showed abundant expression of COL II and SOX9, indicating intact cartilage structure and normal matrix synthesis function. The Sham group showed significantly decreased COL II and SOX9 signals, while MMP13 and HIF-1α expression was enhanced, suggesting loss of chondrocyte phenotype, aggravated matrix degradation, and a significant hypoxia response under OA conditions. The MN+Exo group enhanced COL II and SOX9 expression and decreased MMP13 expression, indicating that exosomes promote cartilage matrix synthesis and inhibit matrix degradation. The MN+Exo+Chl group showed the strongest COL II and SOX9 signals and the lowest MMP13 and HIF-1α expression, indicating that photosynthetic oxygen supply from *Chlamydomonas reinhardtii* can further alleviate local hypoxia, reduce HIF-1α-related responses, and jointly inhibit matrix degradation with exosomes.

[0102] like Figure 12 As shown, ELISA results revealed that the Sham group exhibited decreased IL-4 levels and increased TNF-α levels, indicating a significant pro-inflammatory state after modeling. The MN+Exo group showed increased IL-4 levels and decreased TNF-α levels, suggesting that exosomes possess a good anti-inflammatory regulatory effect. The MN+Exo+Chl group showed further increases in IL-4 and further decreases in TNF-α, exhibiting the best anti-inflammatory effect. These results corroborate the immunohistochemical findings of decreased IL-6 and increased IL-10, demonstrating that the synergistic effect of exosomes and Chlamydomonas reinhardtii photosynthetic oxygen supply can induce the formation of an anti-inflammatory microenvironment.

[0103] like Figure 13As shown, transcriptomic analysis revealed a large number of differentially expressed genes in the MN+Exo+Chl group compared to the Sham group, suggesting that the combined treatment can induce extensive regulation of transcriptional levels in joint tissues. KEGG enrichment results showed that the differentially expressed genes were mainly enriched in pathways related to immune regulation, inflammatory response, and cell metabolism, such as cytokine-receptor interactions, phagosomes, lysosomes, PI3K, and MAPK. GO analysis further showed that the differentially expressed genes were closely related to processes such as immune response, inflammation regulation, cell migration, vesicle transport, and receptor binding. GSEA results also indicated significant changes in gene sets related to cytokine-mediated signaling, chemokine activity, antigen processing and presentation, and protein homeostasis. Combined with immunofluorescence and immunohistochemical results, it is believed that the MN+Exo+Chl system may gradually shift the pathological state of OA tissue from one dominated by inflammation, hypoxia, and matrix degradation to one dominated by anti-inflammation, oxygen supply, and cartilage matrix repair by regulating pathways related to inflammatory response, hypoxia response, oxidative stress, and cartilage matrix metabolism.

[0104] The above embodiments demonstrate that the Chlamydomonas reinhardtii photosynthetic oxygen supply synergistic exosome functionalized hydrogel microneedles disclosed in this invention can improve the local hypoxia and inflammatory microenvironment of osteoarthritis, promote chondrocyte proliferation and migration and cartilage matrix synthesis through the synergistic effects of microneedle local delivery, exosome anti-inflammatory repair and Chlamydomonas reinhardtii in situ oxygen supply, and exhibit good safety and repair effects in vivo.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Any modifications, equivalent substitutions, parameter adjustments, and process optimizations made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Chlamydomonas reinhardtii-synergistic exosome hydrogel microneedle, characterized in that, The hydrogel microneedles are composed of a hydrogel matrix of methacryloyl carboxymethyl chitosan (CMCSMA) and methacryloyl hyaluronic acid (HAMA). After adding a photoinitiator to the hydrogel matrix, the microneedles are obtained through molding and UV crosslinking. The tips of the hydrogel microneedles are loaded with Chlamydomonas reinhardtii exosomes, and the backing layer or matrix portion of the hydrogel microneedles is loaded with Chlamydomonas reinhardtii.

2. The Chlamydomonas reinhardtii synergistic exosome hydrogel microneedles as described in claim 1, characterized in that, The hydrogel microneedles have a complete morphology and regular tips. The internal structure of the hydrogel microneedles is porous and has good balanced swelling properties, porosity, stability and antioxidant properties. The strength of the tips of the hydrogel microneedles is >0.1 N, forming microchannels with a depth of about 200 to 300 μm in the skin.

3. The Chlamydomonas reinhardtii synergistic exosome hydrogel microneedles as described in claim 1, characterized in that, The hydrogel microneedles contained 4 wt%–5 wt% methacrylamide hyaluronic acid (HAMA) and 1 wt%–2 wt% methacrylamide carboxymethyl chitosan (CMCSMA); the concentration of Chlamydomonas reinhardtii exosomes added was 40 μg / mL, and the amount of Chlamydomonas reinhardtii was 5 × 10⁻⁶. 7 Cells / mL.

4. The method for preparing the Chlamydomonas reinhardtii synergistic exosome hydrogel microneedles according to claim 1, characterized in that, Includes the following steps: (1) Cultivate Chlamydomonas reinhardtii and extract its exosomes; (2) Preparation of methacrylated carboxymethyl chitosan (CMCSMA) and methacrylated hyaluronic acid (HAMA); (3) Prepare a hydrogel precursor solution by mixing CMCSMA and HAMA, and the hydrogel precursor solution also includes a photoinitiator; (4) After mixing the exosomes with the hydrogel precursor solution, introduce them into the tip of the microneedle mold; after mixing Chlamydomonas reinhardtii with the hydrogel precursor solution, fill the backing layer. (5) The hydrogel microneedles are obtained after centrifugation-assisted molding and ultraviolet cross-linking, and then demolded.

5. The preparation method according to claim 4, characterized in that, In step (1), after Chlamydomonas reinhardtii is cultured to the plateau phase, the culture supernatant is collected, intact algal cells and large fragments are removed by centrifugation, residual fragments are removed by centrifugation, the supernatant is filtered using a filter membrane, and exosome precipitate is collected by ultracentrifugation. After resuspending in PBS and washing by ultracentrifugation again, purified Chlamydomonas reinhardtii exosomes can be obtained.

6. The preparation method according to claim 4, characterized in that, In step (2), CMCSMA was obtained by grafting carboxymethyl chitosan with methacrylic anhydride; HAMA was obtained by modifying hyaluronic acid with methacrylic anhydride; both were reacted under pH 8-9 conditions and purified by dialysis and freeze-drying.

7. The preparation method according to claim 4, characterized in that, In step (3), a hydrogel precursor solution is prepared with 4% or 5% HAMA and 1% or 2% CMCSMA by mass; and 0.1% photoinitiator is added to it and fully dissolved before use.

8. The preparation method according to claim 4, characterized in that, In steps (4) and (5), the hydrogel precursor solution containing exosomes is added to the microneedle mold, centrifuged to allow it to enter the needle tip, and then the precursor mixture containing Chlamydomonas reinhardtii is filled into the backing layer. Finally, the microneedles are cross-linked under UV light and gently demolded to obtain composite microneedles.

9. The use of the hydrogel microneedles of claim 1 in the preparation of products for treating osteoarthritis.

10. The use of the hydrogel microneedles of claim 1 in the preparation of products that improve local hypoxia in osteoarthritis, inhibit inflammatory response, promote chondrocyte proliferation and migration, promote cartilage matrix synthesis and delay joint degeneration.

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