Preparation of engineered mitochondria based on microfluidic chip and its joint-targeted therapy

Abstract

This invention provides a method for preparing engineered mitochondria based on a microfluidic chip and its application in joint-targeted therapy, relating to the field of biomedical technology. The engineered mitochondrial system based on the microfluidic chip includes a microfluidic chip, mitochondria, and dendritic polylysine. The mitochondria bind to the dendritic polylysine within the microfluidic chip through electrostatic interactions. This application successfully prepares engineered mitochondria based on a microfluidic chip and discloses its application in targeted therapy for arthritis. The dendritic polylysine has a positively charged surface, thus enabling it to target inflammatory sites and inhibit the release of inflammatory factors. Mitochondria may influence the microenvironment of rheumatoid arthritis by affecting the polarization state of macrophages, thereby exerting a therapeutic effect. Simultaneously, mitochondria can regulate the expression of inflammatory factors, reducing the expression of pro-inflammatory factors such as IL-6 and IL-1β, thereby alleviating the inflammatory response.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the preparation of engineered mitochondria based on microfluidic chips and their joint-targeted therapy. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized primarily by erosive arthritis. The peak age of onset is 45-60 years, but it can occur at any age. Although the etiology and pathogenesis of RA are not fully understood, its basic pathological changes are clearly identified as synovitis and pannus formation, gradually leading to the destruction of articular cartilage and bone, ultimately resulting in joint deformity and loss of function. RA is a highly disabling disease and a significant cause of disability in my country, with the disability rate increasing as the disease progresses. Despite the availability of various treatments, a portion of patients still have poor prognoses, making its treatment a persistent challenge in the medical field.

[0003] Traditional drug treatments have limited efficacy and numerous side effects. In recent years, nanomedicines have shown great potential in disease treatment due to their advantages such as targeting and controlled release. However, how to efficiently prepare nanomedicines with specific functions and achieve targeted delivery to specific sites remains a key focus and challenge in current research. Therefore, we urgently need to develop new therapeutic approaches. Summary of the Invention

[0004] The purpose of this invention is to address the lack of targeted treatment options for rheumatoid arthritis in the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An engineered mitochondrial system based on a microfluidic chip includes a microfluidic chip, mitochondria, and dendritic polylysine, wherein the mitochondria bind to the dendritic polylysine within the microfluidic chip through electrostatic interactions.

[0007] Preferably, the microfluidic chip is formed by irreversibly sealing two layers, both of which are made of light- and air-permeable PDMS polymer. The lower layer is a concave, curved S-shaped channel, and the upper layer is a raised fishbone structure, which is embedded in the S-shaped channel.

[0008] Preferably, the microfluidic chip mainly consists of an inlet pool, a mixing chamber, and an outlet pool. The mixing chamber is embedded with a fishbone structure, which allows the liquid to form a chaotic flow.

[0009] Preferably, it is prepared by the following method:

[0010] S1: Fabrication of microfluidic chips;

[0011] S2: Obtaining a dendritic polylysine solution;

[0012] S3: Mitochondrial preparation;

[0013] S4: Preparation of engineered mitochondria: The mitochondria in S3 and the dendritic polylysine solution obtained in S2 are slowly injected into the microfluidic chip through a microfluidic pump. After passing through the fishbone structure, the liquid produces chaotic flow. The dendritic polylysine is anchored to the mitochondria through electrostatic interaction. The resulting product is the engineered mitochondria.

[0014] Preferably, the specific steps of S1 are as follows:

[0015] First, the shape of the microfluidic chip is designed, a sketch of the chip is constructed using drawing software, and a mask is fabricated.

[0016] The silicon wafer was cleaned with a plasma cleaner for 60 seconds, then photoresist was evenly applied to the silicon dioxide wafer, baked at 90°C for 30 minutes, and the chip pattern was printed after the silicon wafer was cooled to room temperature.

[0017] Bake at 90°C for 30 minutes, cool, and wash away residual photoresist with ethyl lactate. Then bake at 110°C for 20 minutes.

[0018] Add PDMS solution to the mold, remove air bubbles using a vacuum pump, and let it solidify overnight;

[0019] The solidified PDMS block was removed, and the upper and lower layers were sterilized by overnight ultraviolet irradiation, followed by plasma treatment for 30-45 seconds for sealing.

[0020] The PDMS15s were subjected to plasma cleaning and oxidation treatment to form O-Si-O covalent bonds on the PDMS surface. Then, the upper and lower PDMS layers were sealed together to obtain a microfluidic chip.

[0021] Preferably, the concentration of the dendritic polylysine solution in S2 is 0.5 mg / mL.

[0022] Preferably, the specific steps of S3 are as follows: take the heart of a 6-8 week old bablc mouse, place it on ice, cut the heart tissue into small pieces, add trypsin and digest on ice for 20 minutes, centrifuge to remove the trypsin, add mitochondrial separation reagent, homogenize 20 times, centrifuge at 1000g for 5 minutes, discard the precipitate, keep the supernatant, centrifuge the supernatant at 3500g for 10 minutes to obtain a white precipitate, add PBS to wash and centrifuge to obtain the white precipitate, which is the extracted mitochondria.

[0023] This application also provides the application of an engineered mitochondrial system based on a microfluidic chip in the preparation of a drug for treating rheumatoid arthritis, wherein the engineered mitochondrial system is the engineered mitochondrial system described above.

[0024] Preferably, the engineered mitochondrial system targets inflammatory sites through the positive charge on the surface of dendritic polylysine, while inhibiting the release of inflammatory factors, and affects the polarization state of macrophages through mitochondria, thereby influencing the microenvironment of rheumatoid arthritis and thus playing a therapeutic role.

[0025] Preferably, the engineered mitochondrial system reduces the expression of pro-inflammatory factors by regulating the expression of inflammatory factors through mitochondria, thereby alleviating the inflammatory response.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] This application provides a method for preparing engineered mitochondria based on microfluidic chips and its joint-targeted therapy. The engineered mitochondria mentioned in this application have good anti-inflammatory effects in vitro and can target inflammatory sites after being modified with dendritic polylysine, thus showing broad application prospects in the treatment of rheumatoid arthritis. Attached Figure Description

[0028] Figure 1 This is a mask design diagram of a microfluidic chip according to one embodiment of the present invention. Figure 1 a is the mask design diagram of the lower S-shaped channel. Figure 1 b is the mask design diagram of the upper fishbone structure.

[0029] Figure 2 This invention relates to the construction of a microfluidic chip and the detection of particle size potential of engineered mitochondria in one embodiment of the present invention. Figure 2 a is a bright-field photograph of a microfluidic chip. Figure 2 b represents the particle size analysis of the engineered mitochondria. Figure 2 c represents the potential detection of the engineered mitochondria.

[0030] Figure 3 This invention describes the in vitro anti-inflammatory ability of engineered mitochondria detected using q-PCR technology in one embodiment of the invention. Figure 3 a) is the expression level of IL-6 in macrophages after LPS stimulation and the addition of engineered mitochondria. Figure 3 b is the expression level of TNF-α in macrophages after LPS stimulation and the addition of engineered mitochondria. Figure 3 c represents the expression level of CD86 in macrophages after LPS stimulation with engineered mitochondria.

[0031] Figure 4 This invention relates to an embodiment of the inflammatory targeting capability of engineered mitochondria detected by in vivo imaging in small animals after engineered mitochondrial transplantation. Figure 4a is an in vivo image of engineered mitochondrial transplanted mice at different time points. Figure 4 b is a fluorescence imaging image of the major organs removed after the mouse was sacrificed.

[0032] Figure 5 This invention relates to a method for detecting joint damage in mice after engineered mitochondrial transplantation using MicroCT. Figure 5 Image a is a representative MicroCT image of the joints of mice in different treatment groups. Figure 5 b represents the statistical results of bone mineral density (BMD) in different groups of mice. Figure 5 c represents the statistical results of bone volume fraction (BV / TV) in different groups of mice. Figure 5 d represents the statistical result of the number of trabeculae (Tb.N) in different groups of mice.

[0033] Figure 6 This invention relates to an embodiment of the study where, after engineered mitochondrial transplantation, ELISA was used to detect the expression levels of serum inflammatory factors in mice from different groups. Figure 6 a represents the statistical results of serum IL-6 expression levels in mice from different treatment groups. Figure 6 b represents the statistical results of serum IL-1β expression levels in mice from different treatment groups. Figure 6 c represents the statistical results of serum TNF-α expression levels in mice from different treatment groups. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] An engineered mitochondrial system based on a microfluidic chip includes a microfluidic chip, mitochondria, and dendritic polylysine, wherein the mitochondria bind to the dendritic polylysine within the microfluidic chip through electrostatic interactions.

[0036] This application also provides a method for preparing the above-mentioned engineered mitochondrial system, comprising the following steps:

[0037] S1: Fabrication of microfluidic chips:

[0038] First, the shape of the microfluidic chip is designed. In one embodiment, the microfluidic chip is formed by irreversibly sealing two layers, both of which are made of light- and air-permeable PDMS polymer. The lower layer is a concave, curved S-shaped channel, and the upper layer is a raised fishbone structure, which is embedded in the S-shaped channel.

[0039] Please see Figure 1 a and b, the microfluidic chip mentioned above mainly consists of an inlet pool, a mixing chamber, and an outlet pool. The mixing chamber is embedded with a fishbone structure, which allows the liquid to form a chaotic flow.

[0040] Based on the shape of the designed microfluidic chip, a sketch of the chip is constructed using drawing software, such as AutoCAD, and a mask is made.

[0041] The silicon wafer was cleaned with a plasma cleaner for 60 seconds, then photoresist was evenly applied to the silicon dioxide wafer, baked at 90°C for 30 minutes, and the chip pattern was printed after the silicon wafer cooled to room temperature.

[0042] Bake the wafer at 90°C for 30 minutes, then cool it and wash away the residual photoresist with ethyl lactate. Bake the film at 110°C for 20 minutes.

[0043] Add PDMS solution to the mold, remove air bubbles using a vacuum pump, and let it solidify overnight.

[0044] The solidified PDMS block was removed, and the upper and lower layers were sterilized by overnight ultraviolet irradiation, followed by plasma treatment for 30-45 seconds for sealing.

[0045] The PDMS15s were subjected to plasma cleaning and oxidation treatment to form O-Si-O covalent bonds on the PDMS surface. Then, the upper and lower PDMS layers were sealed together to obtain a microfluidic chip.

[0046] S2: Obtaining the dendritic polylysine solution:

[0047] Weigh 0.5 mg of dendritic polylysine and dissolve it in 1 mL of PBS to obtain a dendritic polylysine solution with a concentration of 0.5 mg / mL.

[0048] S3: Preparation of mitochondria:

[0049] Bablc mice aged 6-8 weeks were euthanized by cervical dislocation. The hearts of the 6-8 week old Bablc mice were removed and placed on ice. The heart tissue was minced and digested with trypsin on ice for 20 minutes. The trypsin was removed by centrifugation. Mitochondrial separation reagent was added, and the mixture was homogenized 20 times. The mixture was centrifuged at 1000g for 5 minutes, and the precipitate was discarded. The supernatant was centrifuged at 3500g for 10 minutes to obtain a white precipitate. The precipitate was washed with PBS and centrifuged again. The white precipitate obtained was the extracted mitochondria.

[0050] S4: Preparation of engineered mitochondria

[0051] The mitochondria in S3 and the dendritic polylysine solution obtained in S2 were slowly injected into the microfluidic chip via a microfluidic pump. After passing through the fishbone structure, the liquid produced chaotic flow, and the dendritic polylysine was anchored to the mitochondria through electrostatic interaction. The resulting product is the engineered mitochondria.

[0052] Based on the engineered mitochondria described above, this application provides the application of engineered mitochondria in the preparation of drugs for treating rheumatoid arthritis. These mitochondria target inflammatory sites through the positive charge on the surface of dendritic polylysine, while simultaneously inhibiting the release of inflammatory factors. Furthermore, by influencing the polarization state of macrophages through mitochondria, they affect the microenvironment of rheumatoid arthritis, thereby exerting a therapeutic effect. Simultaneously, mitochondria regulate the expression of inflammatory factors, reducing the expression of pro-inflammatory factors such as IL-6 and IL-1β, thereby alleviating the inflammatory response.

[0053] The above content will be explained in conjunction with specific verification experiments:

[0054] Example 1: Measurement of particle size and potential of engineered mitochondria:

[0055] First, a sketch of the chip was constructed using AutoCAD, and a mask was created. A PDMS microfluidic chip was successfully fabricated, as shown in the following figure. Figure 2 As shown in a.

[0056] Different ratios of dendritic lysine solution and mitochondria were prepared, with concentration ratios of mitochondrial solution and dendritic lysine solution of 1:1, 1:5, and 1:10, respectively. These were then thoroughly mixed in a microfluidic chip, and the particle size and potential of the engineered mitochondria were measured. Figure 2 As shown in b, the particle size of the engineered mitochondria obtained with different configuration ratios is approximately 500 nm. Figure 2 As shown in Figure c, as the proportion of dendritic polylysine solution gradually increases, the potential of engineered mitochondria turns positive and gradually increases.

[0057] Example 2: Verification of the anti-inflammatory ability of engineered mitochondria in vitro:

[0058] Macrophages were seeded in 6-well plates, and LPS (1 μg / mL) was added for 6 h to induce macrophage inflammation. Engineered mitochondria were then added for co-culture, and the expression levels of macrophage inflammatory factors were detected using qPCR. Figure 3 As shown in figure a, after LPS stimulation, the expression level of IL-6 in macrophages significantly increased, while engineered mitochondria significantly reduced its expression, showing a concentration gradient trend. Figure 3 As shown in b, LPS stimulation significantly increased TNF-α expression in macrophages, while engineered mitochondria significantly reduced its expression, exhibiting a concentration gradient trend. Figure 3 As shown in c, after LPS stimulation, the expression level of CD86 in macrophages increased significantly, while the expression of CD86 in engineered mitochondria decreased significantly, showing a concentration gradient trend.

[0059] Example 3: Verifying that engineered mitochondria can target joint sites

[0060] Six-week-old male DBA / 1 mice were used to induce a collagen-induced arthritis (CIA) mouse model. Free DID and DID-labeled engineered mitochondria were injected into the CIA mice, respectively. In vivo imaging was used to observe the dye distribution in the mice. The results are as follows: Figure 4 As shown in figure a, the fluorescence intensity at the joints of mice injected with DID-labeled engineered mitochondria was significantly higher than that in the free dye group, suggesting that engineered mitochondria can accumulate more quickly and in greater quantities at the joints. Figure 4 b shows that most of the DID dye was enriched in the liver and lungs of mice. Compared with mice injected with free DID dye, mice injected with DID-labeled engineered mitochondria showed significantly higher fluorescence intensity in the joints, suggesting that engineered mitochondria can target the joints of mice.

[0061] Example 4: Verifying that engineered mitochondria can alleviate joint damage and systemic inflammation in CIA mice:

[0062] Six-week-old male DBA / 1 mice were selected to induce a CIA (cancer-associated arthritis) mouse model using collagen. After successful modeling, mice were randomly divided into a healthy control group, a CIA group, a CIA+G3K treatment group, a CIA+Mito treatment group, and a CIA+Mito@G3K treatment group. Administering the medication via tail vein for 4 weeks, twice weekly. After 4 weeks, serum and joint samples were collected. MicroCT was used to observe bone destruction in the joints. Figure 5 As shown in figure a, CIA mice suffered severe joint damage, while the damage was significantly reduced after engineered mitochondrial transplantation. Figure 5 Images b, c, and d show that bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone number (Tb.N) were significantly lower in CIA mice compared to healthy mice, and this trend was significantly reversed after engineered mitochondrial transplantation. Figure 6 The study showed that the serum IL-6 expression level was increased in the CIA group mice, and the expression level decreased in all groups of mice after treatment. The engineered mitochondrial group mice showed the best treatment effect. Figure 6 b shows that the serum IL-1β expression level of mice in the CIA group is increased, and the expression level of mice in all groups decreases after treatment. The engineered mitochondrial group of mice showed the best treatment effect. Figure 6 c showed that the serum TNF-α expression level was increased in the CIA group mice, and the expression level decreased in all groups of mice after treatment. The engineered mitochondrial group mice showed the best treatment effect.

[0063] This application provides a method for preparing engineered mitochondria based on microfluidic chips and its joint-targeted therapy. The engineered mitochondria mentioned in this application have good anti-inflammatory effects in vitro and can target inflammatory sites after being modified with dendritic polylysine, thus showing broad application prospects in the treatment of rheumatoid arthritis.

Claims

1. An engineered mitochondrial system based on a microfluidic chip, characterized in that: The microfluidic chip includes a microfluidic chip, mitochondria, and dendritic polylysine at a concentration of 0.5 mg / mL. The microfluidic chip is composed of two layers of PDMS polymer, with the lower layer being a concave, curved S-shaped channel and the upper layer being a convex fishbone structure embedded in the S-shaped channel. When the mitochondria and the dendritic polylysine solution are injected into the microfluidic chip, the liquid undergoes chaotic flow after passing through the fishbone structure. The mitochondria bind to the dendritic polylysine within the microfluidic chip through electrostatic interactions.

2. The engineered mitochondrial system based on a microfluidic chip according to claim 1, characterized in that: The microfluidic chip mainly consists of an inlet pool, a mixing chamber, and an outlet pool. The mixing chamber is embedded with a fishbone structure, which allows the liquid to form a chaotic flow.

3. The engineered mitochondrial system based on a microfluidic chip according to claim 1, characterized in that: It is prepared by the following method: S1: Fabrication of microfluidic chips; S2: Obtaining a dendritic polylysine solution; S3: Mitochondrial preparation; S4: Preparation of engineered mitochondria: The mitochondria in S3 and the dendritic polylysine solution obtained in S2 are slowly injected into the microfluidic chip through a microfluidic pump. After passing through the fishbone structure, the liquid produces chaotic flow. The dendritic polylysine is anchored to the mitochondria through electrostatic interaction. The resulting product is the engineered mitochondria.

4. The engineered mitochondrial system based on a microfluidic chip according to claim 3, characterized in that: The specific steps of S1 are as follows: First, the shape of the microfluidic chip is designed, a sketch of the chip is constructed using drawing software, and a mask is fabricated. The silicon wafer was cleaned with a plasma cleaner for 60 seconds, then photoresist was evenly coated onto the silicon dioxide wafer, baked at 90°C for 30 minutes, and the chip pattern was printed after the silicon wafer cooled to room temperature. Bake the wafer at 90℃ for 30 min, cool it, wash away the residual photoresist with ethyl lactate, and bake the film at 110℃ for 20 min. Add PDMS solution to the mold, remove air bubbles using a vacuum pump, and let it solidify overnight; The solidified PDMS block was removed, and the upper and lower layers were sterilized by overnight ultraviolet irradiation, followed by plasma treatment for 30-45 seconds for sealing. The PDMS was subjected to plasma cleaning and oxidation treatment for 15 seconds to form O-Si-O covalent bonds on the PDMS surface. Then, the upper and lower PDMS layers were sealed together to obtain a microfluidic chip.

5. The engineered mitochondrial system based on a microfluidic chip according to claim 3, characterized in that: The specific steps of S3 are as follows: Take the heart of a 6-8 week old bablc mouse, place it on ice, cut the heart tissue into small pieces, add trypsin and digest on ice for 20 min, centrifuge to remove trypsin, add mitochondrial separation reagent, homogenize 20 times, centrifuge at 1000 g for 5 min, discard the precipitate, keep the supernatant, centrifuge the supernatant at 3500 g for 10 min to obtain a white precipitate, add PBS to wash and centrifuge to obtain the white precipitate, which is the extracted mitochondria.

6. The application of an engineered mitochondrial system based on a microfluidic chip in the preparation of drugs for treating rheumatoid arthritis, characterized in that: The engineered mitochondrial system is the engineered mitochondrial system described in any one of claims 1-5.

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