Preparation of engineered mitochondria based on micro-fluidic chip and joint targeted therapy of engineered mitochondria

Abstract

The invention provides preparation of engineered mitochondria based on a micro-fluidic chip and joint targeted therapy of the engineered mitochondria, and relates to the technical field of biomedicine.The engineered mitochondria system based on the micro-fluidic chip comprises the micro-fluidic chip, mitochondria and dendritic polylysine, the mitochondria is combined with the dendritic polylysine in the micro-fluidic chip through electrostatic interaction. The invention successfully prepares the engineered mitochondria based on the micro-fluidic chip, and further discloses application of the engineered mitochondria in arthritis targeted therapy. The surface of the dendritic polylysine is positively charged, so that the dendritic polylysine can target an inflammatory site and inhibit release of inflammatory factors, mitochondria possibly affects the polarization state of macrophages so as to affect the microenvironment of rheumatoid arthritis and further play a treatment role, and meanwhile, the mitochondria can regulate expression of the inflammatory factors, so that the rheumatoid arthritis treatment effect is improved. The expression of proinflammatory factors such as IL-6, IL-1beta and the like is reduced, so that the inflammatory response is relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the preparation of engineered mitochondria based on a microfluidic chip and joint-targeted treatment thereof. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease with erosive arthritis as its main clinical manifestation. The peak age of onset is 45 to 60 years old, but it can occur at any age. Although the etiology and pathogenesis of RA have not yet been fully elucidated, its basic pathological changes are synovitis and pannus formation, which gradually cause destruction of articular cartilage and bone, ultimately leading to joint deformity and loss of function. RA is a highly disabling disease and an important cause of disability in the Chinese population. As the disease progresses, the disability rate of RA patients continues to rise. Although many treatment options are available, some patients still have a poor prognosis, so its treatment has always been a difficult problem in the medical community.

[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 targeted properties and controlled release. However, efficient preparation of nanomedicines with specific functions and targeted delivery to specific sites remain key research priorities and challenges. Therefore, the development of new treatment modalities is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem of lack of targeted treatment plan for rheumatoid arthritis in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An engineered mitochondrial system based on a microfluidic chip comprises a microfluidic chip, mitochondria and dendritic polylysine, wherein the mitochondria are combined with the dendritic polylysine in the microfluidic chip through electrostatic interaction.

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

[0008] Preferably, the microfluidic chip is mainly composed of an inlet pool, a mixing chamber and an outlet pool, and a fishbone structure is embedded in the mixing chamber, so that the liquid can form a chaotic flow.

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

[0010] S1: Preparation of microfluidic chip;

[0011] S2: Obtaining 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, and 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 made.

[0016] Use a plasma cleaner to clean the silicon wafer for 60 seconds, then evenly coat the photoresist on the silicon dioxide wafer, bake the wafer at 90° for 30 minutes, and then print the chip pattern after the silicon wafer cools to room temperature;

[0017] Bake the film at 90° for 30 minutes, then use ethyl lactate to remove the remaining photoresist after cooling, and bake the film at 110° for 20 minutes;

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

[0019] The solidified PDMS block was dug out, and the upper and lower layers were sterilized by UV irradiation overnight, and then sealed by plasma treatment for 30-45 seconds;

[0020] The PDMS was oxidized using a plasma cleaner for 15 seconds to form O-Si-O covalent bonds on the PDMS surface. The upper and lower PDMS layers were then sealed 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: removing the heart of a 6-8 W bablc mouse, placing it on ice, chopping the heart tissue, adding trypsin to digest on ice for 20 minutes, centrifuging to remove the trypsin, adding mitochondrial isolation reagent, homogenizing 20 times, centrifuging at 1000g for 5 minutes, discarding the precipitate, retaining the supernatant, and centrifuging the supernatant at 3500g for 10 minutes to obtain a white precipitate, adding PBS to wash and then centrifuging to obtain a white precipitate, which is the extracted mitochondria.

[0023] The present application also provides an 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 affecting the polarization state of macrophages through mitochondria, thereby affecting the microenvironment of rheumatoid arthritis and thus playing a therapeutic role.

[0025] Preferably, the engineered mitochondrial system regulates the expression of inflammatory factors through mitochondria, reduces the expression of pro-inflammatory factors, and thus alleviates the inflammatory response.

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

[0027] The present application provides a method for preparing engineered mitochondria based on a microfluidic chip and their joint-targeted treatment. The engineered mitochondria mentioned in the present application have good anti-inflammatory effects in vitro and can target inflammatory sites after being modified with dendritic polylysine. Therefore, they have broad application prospects in the treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0030] Figure 3 In one embodiment of the present invention, the anti-inflammatory ability of the engineered mitochondria constructed in vitro was detected by q-PCR technology. Figure 3 a is the expression level of IL-6 in macrophages detected by adding engineered mitochondria after LPS stimulation of macrophages. Figure 3 b is the expression level of TNF-α in macrophages after LPS stimulation by adding engineered mitochondria. Figure 3 c is the expression level of macrophage CD86 after LPS stimulation of macrophages and the addition of engineered mitochondria.

[0031] Figure 4 In one embodiment of the present invention, the inflammation targeting ability of engineered mitochondria is detected by in vivo imaging of small animals after engineered mitochondria transplantation. Figure 4a is the in vivo imaging of mice after engineered mitochondria transplantation at different time periods. Figure 4 b is the fluorescence imaging of the main organs after the mice were killed.

[0032] Figure 5 In one embodiment of the present invention, MicroCT was used to detect joint damage in mice after engineered mitochondria transplantation. Figure 5 a Representative MicroCT images of the joints of mice in different treatment groups. Figure 5 b is the statistical results of bone mineral density (BMD) of mice in different groups. Figure 5 c is the statistical results of bone volume fraction (BV / TV) of mice in different groups. Figure 5 d is the statistical results of the trabecular number (Tb.N) of mice in different groups.

[0033] Figure 6 In one embodiment of the present invention, the expression levels of serum inflammatory factors in each group of mice were detected by ELISA after the engineered mitochondria were transplanted. Figure 6 a is the statistical results of serum IL-6 expression levels in mice in different treatment groups. Figure 6 b is the statistical results of serum IL-1β expression levels in mice in different treatment groups. Figure 6 c is the statistical results of serum TNF-α expression levels in mice in different treatment groups. DETAILED DESCRIPTION

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

[0035] An engineered mitochondrial system based on a microfluidic chip comprises a microfluidic chip, mitochondria and dendritic polylysine, wherein the mitochondria are combined with the dendritic polylysine in the microfluidic chip through electrostatic interaction.

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

[0037] S1: Preparation of microfluidic chip:

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

[0039] See also Figure 1 a, b, The above-mentioned microfluidic chip mainly consists of an inlet pool, a mixing chamber and an outlet pool. The mixing chamber is inlaid with a fishbone structure, and the liquid can form chaotic flow.

[0040] According to the shape of the designed microfluidic chip, a sketch of the chip is constructed using drawing software, and a mask is made. The drawing software may use Auto CAD.

[0041] Use a plasma cleaner to clean the silicon wafer for 60 seconds, then evenly apply photoresist on the silicon dioxide wafer, bake the wafer at 90° for 30 minutes, and then engrave the chip pattern after the silicon wafer cools to room temperature.

[0042] Bake the film at 90° for 30 minutes, and after cooling, use ethyl lactate to wash away the residual photoresist, and fry the film at 110° for 20 minutes.

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

[0044] The solidified PDMS block was dug out, and the upper and lower layers were sterilized by UV irradiation overnight, and then sealed by plasma treatment for 30-45s.

[0045] The PDMS was oxidized using a plasma cleaner for 15 seconds to form O-Si-O covalent bonds on the PDMS surface. The upper and lower PDMS layers were then sealed to obtain a microfluidic chip.

[0046] S2: Obtaining dendritic polylysine solution:

[0047] 0.5 mg of dendrimer polylysine was weighed and dissolved in 1 mL of PBS to obtain a dendrimer polylysine solution with a concentration of 0.5 mg / mL.

[0048] S3: Preparation of mitochondria:

[0049] 6-8 week old bablc mice were killed by cervical dislocation, and the hearts of 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, and mitochondrial isolation reagent was added. The mixture was homogenized 20 times and centrifuged at 1000 g for 5 minutes. The precipitate was discarded and the supernatant was retained. The supernatant was centrifuged at 3500 g for 10 minutes to obtain a white precipitate. The white precipitate was washed with PBS and then centrifuged to obtain the extracted mitochondria.

[0050] S4: Preparation of engineered mitochondria

[0051] 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, and the dendritic polylysine is anchored to the mitochondria through electrostatic action. The resulting product is the engineered mitochondria.

[0052] Based on the engineered mitochondria described above, the present application provides the use of engineered mitochondria in the preparation of drugs for the treatment of rheumatoid arthritis, which targets the inflammatory site through the positive charge on the surface of dendritic polylysine, while inhibiting the release of inflammatory factors, and affecting the polarization state of macrophages through mitochondria, thereby affecting the microenvironment of rheumatoid arthritis and thus playing a therapeutic role. At the same time, mitochondria regulate the expression of inflammatory factors and reduce the expression of pro-inflammatory factors such as IL-6, IL-1β, etc., thereby alleviating the inflammatory response.

[0053] The following is an elaboration of the above content in combination with specific verification experiments:

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

[0055] First, Auto CAD was used to create a sketch of the chip and a mask. A PDMS microfluidic chip was successfully produced. Figure 2 As shown in a.

[0056] Different ratios of dendritic lysine solution and mitochondria were prepared, so that the concentration ratio of mitochondrial solution to dendritic lysine solution was 1:1, 1:5, and 1:10, respectively. The mixture was evenly 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 about 500 nm. Figure 2 As shown in (c), as the proportion of the dendritic polylysine solution gradually increased, the potential of the engineered mitochondria turned positive and gradually increased.

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

[0058] Macrophages were seeded in 6-well plates and stimulated with LPS (1 μg / mL) 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 q-PCR technology. Figure 3 As shown in a, after LPS stimulation, the expression level of IL-6 in macrophages increased significantly, while the engineered mitochondria significantly reduced its expression, and the trend was a concentration gradient. Figure 3 As shown in b, after LPS stimulation, the expression level of TNF-α in macrophages increased significantly, while the engineered mitochondria significantly reduced its expression, and the trend was a concentration gradient. Figure 3 As shown in c, after LPS stimulation, the expression level of CD86 in macrophages increased significantly, while engineered mitochondria significantly reduced its expression in a concentration gradient trend.

[0059] Example 3: Verification that engineered mitochondria can target joints

[0060] 6-week-old male DBA / 1 mice were used to induce a CIA mouse model of arthritis using collagen. Free DID and DID-labeled engineered mitochondria were injected into the CIA mice, and the distribution of the dye in the mice was observed by in vivo imaging. The results are as follows: Figure 4 As shown in a, the fluorescence intensity of the joints of mice injected with DID-labeled engineered mitochondria was significantly higher than that of the free dye group, suggesting that engineered mitochondria can aggregate faster and more in the joints. Figure 4 b shows that most of the DID dye is enriched in the liver and lungs of mice. Compared with the group of mice injected with free DID dye, the fluorescence intensity of the joints of mice injected with DID-labeled engineered mitochondria is significantly higher, suggesting that engineered mitochondria can target the joints of mice.

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

[0062] Six-week-old male DBA / 1 mice were selected and a collagen-induced CIA mouse model was established. After model establishment, the mice were divided into a healthy control group, a CIA group, a CIA+G3K group, a CIA+Mito group, and a CIA+Mito@G3K group. Treatment was continued via the tail vein twice weekly for 4 weeks. Serum and joints were collected after 4 weeks. MicroCT was used to observe bone destruction in the joints. Figure 5 As shown in a, CIA mice suffered severe joint damage, while the damage was significantly alleviated after engineered mitochondria transplantation. Figure 5 b, c, and d show that the bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular number (Tb.N) of CIA mice were significantly lower than those of healthy mice, and this trend was significantly reversed after engineered mitochondrial transplantation. Figure 6 a shows that the expression level of serum IL-6 in mice in the CIA group increased, and the expression levels of mice in each group decreased after treatment. The mice in the engineered mitochondria group showed the best therapeutic effect. Figure 6 b shows that the expression level of serum IL-1β in mice in the CIA group was increased, and the expression levels in mice in all groups decreased after treatment. The mice in the engineered mitochondria group showed the best therapeutic effect. Figure 6 c shows that the expression level of serum TNF-α increased in mice in the CIA group, and the expression levels of mice in all groups decreased after treatment. The mice in the engineered mitochondria group showed the best therapeutic effect.

[0063] This application provides a method for preparing engineered mitochondria based on a microfluidic chip and their joint-targeted treatment. 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. Therefore, they have broad application prospects in the treatment of rheumatoid arthritis.

Claims

1. An engineered mitochondrial system based on a microfluidic chip, characterized by: The invention comprises a microfluidic chip, mitochondria and dendritic polylysine, wherein the mitochondria are combined with the dendritic polylysine in the microfluidic chip through electrostatic interaction.

2. The microfluidic chip-based engineered mitochondria according to claim 1, characterized in that: The microfluidic chip is made of two irreversibly sealed layers, both of which are made of light-permeable and breathable PDMS polymer. The lower layer is a concave, curved S-shaped channel, and the upper layer is a convex fishbone structure, which is embedded in the S-shaped channel.

3. The microfluidic chip-based engineered mitochondria according to claim 2, characterized in that: The microfluidic chip is mainly composed of an inlet pool, a mixing chamber and an outlet pool. The mixing chamber is inlaid with a fishbone structure, and the liquid can form a chaotic flow.

4. The microfluidic chip-based engineered mitochondria according to claim 1, characterized in that: It is prepared by the following method: S1: Preparation of microfluidic chip; S2: Obtaining 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, and the dendritic polylysine is anchored to the mitochondria through electrostatic interaction. The resulting product is the engineered mitochondria.

5. The microfluidic chip-based engineered mitochondria according to claim 4, 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 made. Use a plasma cleaner to clean the silicon wafer for 60 seconds, then evenly coat the photoresist on the silicon dioxide wafer, bake the wafer at 90° for 30 minutes, and then print the chip pattern after the silicon wafer cools to room temperature; Bake the film at 90° for 30 minutes, then cool it down and use ethyl lactate to remove the remaining photoresist, and bake the film at 110° for 20 minutes. Add PDMS solution to the mold, use a vacuum pump to remove bubbles, and let it solidify overnight; The solidified PDMS block was dug out, and the upper and lower layers were sterilized by UV irradiation overnight, and then sealed by plasma treatment for 30-45 seconds; The PDMS was oxidized using a plasma cleaner for 15 seconds to form O-Si-O covalent bonds on the PDMS surface. The upper and lower PDMS layers were then sealed to obtain a microfluidic chip.

6. The microfluidic chip-based engineered mitochondria according to claim 4, characterized in that: The concentration of the dendritic polylysine solution in S2 is 0.5 mg / mL.

7. The microfluidic chip-based engineered mitochondria according to claim 4, characterized in that: The specific steps of S3 are as follows: remove the heart of a 6-8 week old bablc mouse, place it on ice, mince the heart tissue, add trypsin to digest on ice for 20 minutes, centrifuge to remove the trypsin, add mitochondrial isolation reagent, homogenize 20 times, centrifuge at 1000g for 5 minutes, discard the precipitate, retain the supernatant, centrifuge the supernatant at 3500g for 10 minutes to obtain a white precipitate, add PBS to wash, and then centrifuge to obtain a white precipitate, which is the extracted mitochondria.

8. Use of an engineered mitochondrial system based on a microfluidic chip in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The engineered mitochondrial system is the engineered mitochondrial system according to any one of claims 1 to 7.

9. The microfluidic chip-based engineered mitochondria according to claim 8, characterized in that: The engineered mitochondrial system targets inflammatory sites through the positive charge on the surface of dendritic polylysine, and affects the polarization state of macrophages through mitochondria, inhibiting the release of inflammatory factors, thereby affecting the microenvironment of rheumatoid arthritis and further playing a therapeutic role.

10. The microfluidic chip-based engineered mitochondria according to claim 8, characterized in that: The engineered mitochondrial system regulates the expression of inflammatory factors through mitochondria, reduces the expression of pro-inflammatory factors, and thus alleviates the inflammatory response.

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