Use of coacervates in the preparation of carriers for mitochondrial transplantation

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of condensates in the preparation of vectors for mitochondrial transplantation. This invention utilizes FmocWYRGRL short peptide phase separation to form condensates, which can rapidly and gently encapsulate mitochondria, extending their in vitro activity maintenance time to 24 hours and improving their cellular uptake efficiency, while also exhibiting good biocompatibility and lumen retention capacity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of condensates in the preparation of vectors for mitochondrial transplantation. Background Technology

[0002] Mitochondria, as the "energy factories" of eukaryotic cells, play a crucial role in generating adenosine triphosphate (ATP) through oxidative phosphorylation. They also participate in vital processes such as calcium homeostasis, regulation of reactive oxygen species (ROS), and programmed cell death. Mitochondrial dysfunction is closely associated with a variety of major diseases, including neurodegenerative diseases (such as Alzheimer's and Parkinson's), metabolic diseases, cardiovascular diseases, reproductive dysfunction, osteoarthritis, and the aging process. Traditional treatment strategies (such as antioxidants, metabolic cofactor supplementation, or gene therapy) primarily focus on alleviating symptoms or slowing the progression of the disease, failing to fundamentally repair or replace the damaged mitochondrial network, and especially unable to reverse widespread cellular damage that has already occurred.

[0003] To overcome this limitation, mitochondrial transplantation technology has been proposed as an innovative therapeutic concept. Its core lies in the direct delivery of healthy, fully functional exogenous mitochondria into target cells or tissues, aiming to replace or supplement defective mitochondria and restore cellular energy metabolism and physiological function. This technology was initially validated in in vitro cell models. In recent years, research has expanded to in vivo animal models and early clinical trials, showing significant potential, particularly in the field of acute ischemic injury (such as myocardial infarction and stroke). Transplanted healthy mitochondria can be taken up by damaged cells, rapidly increasing local ATP levels, reducing oxidative stress and cell death, and improving tissue function recovery.

[0004] Despite the immense therapeutic potential of mitochondrial transplantation, a core bottleneck in its clinical application lies in the extreme scarcity of efficient and targeted mitochondrial delivery systems. Effectively delivering exogenous healthy mitochondria to the target lesion area and ensuring their efficient uptake by diseased cells is a critical hurdle that current technology struggles to overcome. Furthermore, extracted mitochondria are highly susceptible to inactivation, typically maintaining activity for only 2-6 hours. Free mitochondria are easily recognized and cleared by the immune system or subjected to enzymatic degradation in systemic circulation, resulting in significant losses during delivery. Simultaneously, the lack of effective targeting mechanisms makes it difficult for mitochondria to precisely accumulate in specific damaged tissues or cells; non-specific distribution not only reduces efficacy but may also lead to off-target risks. Most critically, as relatively large organelles, mitochondria are extremely inefficient at passively crossing the cell membrane barrier; the mechanisms of active uptake by recipient cells (such as endocytosis) are unclear and inefficient, severely limiting the number of functional mitochondria that successfully enter the cytoplasm and reach their target sites. Therefore, developing delivery technologies that can maintain in vitro mitochondrial activity, achieve precise targeting, and efficiently mediate mitochondrial transmembrane transport is the primary challenge that urgently needs to be addressed to advance mitochondrial transplantation from concept to clinical application.

[0005] Existing targeted delivery vectors for mitochondria mainly include liposomes, cationic polymers, and cell-penetrating peptides. Although they have certain delivery capabilities, these vectors still have the following problems in mitochondrial transplantation: they cannot prolong the activity of isolated mitochondria in vitro, and the vectors are mainly taken up by cells through non-specific endocytosis. Mitochondria, as micron-sized organelles, have extremely low transmembrane efficiency. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of condensates in the preparation of vectors for mitochondrial transplantation.

[0007] The application of condensates in the preparation of vectors for mitochondrial transplantation, wherein the condensates are obtained by phase separation of a short peptide, the short peptide being FmocWYRGRL, with the following structural formula: .

[0008] The present invention utilizes the condensate formed by the phase separation of FmocWYRGRL short peptides to rapidly and gently encapsulate mitochondria, extending their in vitro activity to 24 hours and improving their cellular uptake efficiency, while also exhibiting good biocompatibility and cavity retention capacity.

[0009] Preferably, the preparation step of the aggregate is as follows: mixing the short peptide solution with a phase separation crowding agent solution to obtain the aggregate; The concentration of the short peptide solution is 50 μM to 1000 μM, the concentration of the phase separation crowding agent solution is 1 mM to 1000 mM, and the volume ratio of the short peptide solution to the phase separation crowding agent solution is 1:90 to 110.

[0010] Preferably, the phase separation crowding agent is sodium chloride, ATP, ADP, tannic acid, sodium heparin, or chondroitin sulfate.

[0011] Preferably, the mitochondrial solution is mixed with the phase separation crowding agent solution, and then the short peptide solution is added and mixed thoroughly.

[0012] Preferably, the concentration of the mitochondrial solution is 1 μg / mL to 50 μg / mL, the concentration of the short peptide solution is 1 mM to 100 mM, the concentration of the phase separation crowding agent solution is 1 mM to 1000 mM, and the volume ratio is 1 to 20:1 to 10:1000.

[0013] Preferably, the short peptide is obtained by solid-phase peptide synthesis from amino acids protected by 2-chlorotriphenylmethyl chloro resin and 9-fluorenylmethoxycarbonyl groups.

[0014] Preferably, the vector is used for live mitochondrial transplantation in eukaryotic cells.

[0015] An encapsulated mitochondria for delivery, obtained by encapsulating mitochondria in the aforementioned condensate.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The short peptide in this invention has a peptide sequence rich in arginine and tyrosine, WYRGRL, and is capped by an Fmoc group. The FmocWYRGRL peptide enables the condensate delivery system to achieve joint cavity retention. Simultaneously, the Fmoc group enhances the peptide's self-assembly stability.

[0017] In this invention, condensates can be easily and conveniently prepared using the FmocWYRGRL peptide. In the presence of a congestant, they can be prepared within seconds and stabilize into uniform droplets with a size of approximately 1-2 μm within about one hour. These condensates exhibit good biocompatibility and can encapsulate various types of small molecule fluorescent probes, nucleic acids, proteins, nanoparticles, etc., with fast encapsulation speed and high efficiency. This invention also modifies the structure of peptides rich in arginine and tyrosine to form peptides with phase-separation properties and applies them to mitochondrial transplantation.

[0018] In this invention, condensates can rapidly encapsulate mitochondria, and the formation of turbid condensates can be observed with the naked eye. The environment within the condensates is mild, which is conducive to the stability of mitochondria, and no cumbersome preparation process is required. The method of mitochondrial transplantation using condensates is convenient to operate, has a very high uptake efficiency, and has great application potential.

[0019] This invention utilizes biocompatible condensates to significantly prolong the activity and functional stability of isolated mitochondria. These condensates can mimic the intracellular environment, providing necessary physical support and biochemical protection for isolated mitochondria, effectively slowing down their energy decay and structural degradation, thereby greatly improving the survival rate and functional integrity of mitochondria during in vitro preservation. Attached Figure Description

[0020] Figure 1 Mass spectrum of the FmocWYRGRL peptide that forms aggregates according to the present invention.

[0021] Figure 2 The high-performance liquid chromatogram of the FmocWYRGRL peptide forming aggregates of the present invention is shown, wherein the nested table shows the mobile phase components and chromatographic conditions.

[0022] Figure 3 Optical diagram of the condensate of the present invention.

[0023] Figure 4 : Particle size distribution diagram of the aggregates of the present invention.

[0024] Figure 5 : The effect of peptide and sodium chloride concentrations on aggregate formation in this invention.

[0025] Figure 6 : The effect of the peptides and other crowding agents of the present invention on aggregate formation.

[0026] Figure 7 The cytotoxicity of the FmocWYRGRL peptide that forms aggregates according to the present invention.

[0027] Figure 8 SDS gel electrophoresis image of mitochondria encapsulated in condensates according to the present invention.

[0028] Figure 9 The ATP concentration of mitochondria encapsulated in the condensate of the present invention after 24 hours.

[0029] Figure 10 The condensate of this invention provides fluorescent images of mitochondria to chondrocytes.

[0030] Figure 11 The present invention provides a flow cytometry-based quantitative method for delivering mitochondria to chondrocytes via condensate delivery.

[0031] Figure 12 Preparation process of FmocWYRGRL. Detailed Implementation

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0033] Example 1 Preparation of a peptide with the sequence FmocWYRGRL: This example utilizes 2-chlorotriphenylmethyl chloro resin and Fmoc-protected amino acids to design and synthesize via solid-phase peptide synthesis (SPPS). The structural formula is as follows: .

[0034] Preparation process as follows Figure 12 As shown.

[0035] First, the resin was expanded by weighing 2 mmol of resin into a solid-phase synthesis tube, adding 10 mL of dichloromethane, and bubbling for 20 min. Then, it was washed three times with N,N-dimethylformamide.

[0036] Weigh 5 mmol of Fmoc-leucine into a glass bottle, add 10 mL of N,N-dimethylformamide and 5 mmol of N,N-diisopropylethylamine, and sonicate until completely dissolved. Add the solution to the resin, purge with nitrogen for 30 min, and then wash three times with N,N-dimethylformamide. Add 10 mL of blocking solution N,N-diisopropylethylamine / methanol / dichloromethane (volume ratio 5 / 15 / 80), purge with nitrogen for 10 min, dry the solution, add another 10 mL of blocking solution, purge with nitrogen for 10 min, and then wash three times with N,N-dimethylformamide. Add 10 mL of a 5% piperazine + 2% DBU mixture, purge with nitrogen for 10 min, and then wash five times with N,N-dimethylformamide.

[0037] Weigh Fmoc-Pbf-arginine (6.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (6 mmol) into a glass bottle, add 10 mL of N,N-dimethylformamide, then add N,N-diisopropylethylamine (12 mmol), and sonicate until completely dissolved. Add the solution to the resin and react under nitrogen for 30 min. Wash three times with N,N-dimethylformamide. Add 10 mL of a mixture of 5% piperazine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), react under nitrogen for 10 min, and then wash five times with N,N-dimethylformamide.

[0038] Weigh Fmoc-glycine (6.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (6 mmol) into a glass bottle, add 10 mL of N,N-dimethylformamide, then add N,N-diisopropylethylamine (12 mmol), and sonicate until completely dissolved. Add the solution to the resin and react under nitrogen for 30 min. Wash three times with N,N-dimethylformamide. Add 10 mL of a mixture of 5% piperazine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), react under nitrogen for 10 min, and then wash five times with N,N-dimethylformamide.

[0039] Weigh Fmoc-Pbf-arginine (6.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (6 mmol) into a glass bottle, add 10 mL of N,N-dimethylformamide, then add N,N-diisopropylethylamine (12 mmol), and sonicate until completely dissolved. Add the solution to the resin and react under nitrogen for 30 min. Wash three times with N,N-dimethylformamide. Add 10 mL of a mixture of 5% piperazine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), react under nitrogen for 10 min, and then wash five times with N,N-dimethylformamide.

[0040] Weigh Fmoc-tBu-tyrosine (6.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (6 mmol) into a glass bottle, add 10 mL of N,N-dimethylformamide, then add N,N-diisopropylethylamine (12 mmol), and sonicate until completely dissolved. Add the solution to the resin and react under nitrogen for 30 min. Wash three times with N,N-dimethylformamide. Add 10 mL of a mixture of 5% piperazine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), react under nitrogen for 10 min, and then wash five times with N,N-dimethylformamide.

[0041] Weigh Fmoc-Boc-tryptophan (6.0 mmol) and O-benzotriazole-tetramethylurea hexafluorophosphate (6 mmol) into a glass bottle, add 10 mL of N,N-dimethylformamide, and then add N,N-diisopropylethylamine (12 mmol). Sonicate until completely dissolved. Add the solution to the resin and react under nitrogen atmosphere for 30 min.

[0042] The tube was washed five times each with N,N-dimethylformamide, dichloromethane, methanol, and n-hexane. 20 mL of a mixture of 95% trifluoroacetic acid and 5% dichloromethane was carefully added along the inner wall of the tube, and the reaction was allowed to proceed for 2 hours. The product was collected and the trifluoroacetic acid was removed using a rotary evaporator; the product FmocWYRGRL precipitated with diethyl ether. Figure 1 As shown, the structure of FmocWYRGRL was confirmed to be correct by mass spectrometry. Figure 2 As shown, the purity of FmocWYRGRL was 97.25% as confirmed by high performance liquid chromatography.

[0043] Example 2: Preparation of FmocWYRGRL condensates and their application in mitochondrial delivery (1) Characteristics of short peptide phase separation to form aggregates Weigh 10.73 mg of the short peptide FmocWYRGRL and dissolve it in 0.1 mL of distilled water to obtain a 100 mM stock solution. Prepare a 500 mM sodium chloride solution for later use.

[0044] Add 1 μL of the short peptide FmocWYRGRL to 100 μL of 500 mM sodium chloride, mix well and let stand for 0.5 h. After standing, observe under a confocal microscope.

[0045] After mixing, a noticeable change in the turbidity of the solution can be observed immediately, such as... Figure 3 As shown, the formation of small droplets can be observed under a confocal microscope, proving that the peptides underwent liquid-liquid phase separation and formed aggregates. Particle size analysis revealed that the size of these aggregates is approximately 2-3 μm, such as... Figure 4As shown.

[0046] (2) Screening of conditions for short peptide phase separation to form aggregates 10.73 mg of the short peptide FmocWYRGRL was weighed and dissolved in 0.1 mL of distilled water to obtain a 100 mM stock solution. Sodium chloride solutions with concentrations of 1000 mM, 500 mM, 200 mM, 100 mM, and 50 mM were prepared for later use. The concentration of aggregates formed by phase separation of the short peptide was screened by observing changes in turbidity at different concentration ratios.

[0047] like Figure 5 As shown, the formation of aggregates was observed under conditions of sodium chloride concentrations above 500 mM and peptide concentrations above 50 μM.

[0048] The ability of other crowding agents to form aggregates was further investigated. Short peptide FmocWYRGRL aqueous solutions with concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM and 1 mM were prepared. 100 μL of each solution was added to ATP (100 mM), ADP (100 mM), chondroitin sulfate (100 mM), tannic acid (100 mM) and heparin sodium (100 mM), mixed well and allowed to stand for 0.5 h. After standing, the change in absorbance was recorded using an ELISA reader.

[0049] like Figure 6 As shown, the turbidity of the mixed solution changed significantly, indicating that the short peptide FmocWYRGRL can also form aggregates in the presence of these crowding agents.

[0050] (3) Biosafety of short peptides The biosafety of FmocWYRGRL was assessed using chondrocyte C28 / I2. Chondrocytes were seeded in 96-well plates and cultured for 24 h. Then, different concentrations of FmocWYRGRL were added and incubated for a total of 48 h. Cell viability was then assessed using a CCK8 assay kit.

[0051] like Figure 7 As shown, at a concentration of 250 μM, cell viability remained above 90%, indicating that FmocWYRGRL has good biosafety.

[0052] (4) Encapsulation and delivery of mitochondria by short peptide condensates The mitochondrial transplantation effect of FmocWYRGRL condensates was evaluated using chondrocyte C28 / I2. Chondrocytes were seeded in 12-well plates and cultured for 24 h. 10.73 mg of the short peptide FmocWYRGRL was weighed and dissolved in 0.1 mL of distilled water to obtain a 100 mM stock solution. 10 μL of mitochondria extracted from 10,000 bone mesenchymal stem cells (concentration of 5 μg / mL within half an hour of extraction) was added to 1 mL of 154 mM sodium chloride solution, mixed thoroughly, and 5 μL of FmocWYRGRL was added. The mixture was allowed to stand for 30 min, and condensate formation was observed under a microscope. Unencapsulated mitochondria were separated by centrifugation at 800 g, and the precipitate was used to confirm the encapsulation of mitochondria in the condensates by SDS gel electrophoresis. Theoretically, mitochondria usually require centrifugation of more than 3500 g to precipitate; the FmocWYRGRL peptide-mitochondrial complex precipitated by centrifugation at 800 g.

[0053] Figure 8 The SDS-PAGE analysis of the precipitate clearly detected characteristic mitochondrial protein bands, which were essentially consistent with the mitochondrial protein bands in the control group, confirming that mitochondria were encapsulated within the condensates. Figure 8 ).

[0054] It is worth noting that mitochondria typically survive only about 6 hours in vitro. However, by assessing the ATP production capacity of mitochondria, it was found that condensates can maintain mitochondrial activity up to 24 hours. The results are as follows... Figure 9 .

[0055] Donor mitochondria were stained with a mitochondrial red probe and centrifuged to remove unbound probes. Recipient chondrocytes were stained with a mitochondrial green probe and washed three times with PBS to remove unbound probes. The condensate containing stained mitochondria was added to the recipient chondrocytes and incubated for 4 h. The expression of red fluorescence was observed under a confocal microscope.

[0056] like Figure 10 As shown, after co-incubation for 4 h, red-labeled mitochondria were clearly observed to enter the cells and did not colocalize with the green fluorescence in the recipient cells.

[0057] Quantitative detection of intracellular red fluorescence intensity using flow cytometry ( Figure 11 The condensate-loaded mitochondria significantly improved mitochondrial uptake levels, increasing them by approximately 50% compared to the mitochondrial-only group.

[0058] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0059] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0060] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a coacervate for the preparation of a carrier for mitochondrial transplantation, characterized in that, The aggregate was obtained by phase separation of a short peptide, the short peptide being FmocWYRGRL, with the following structural formula: 。 2. Use according to claim 1, characterized in that, The preparation steps of the aggregate are as follows: mixing the short peptide solution with the phase separation crowding agent solution to obtain the aggregate; The concentration of the short peptide solution is 50 μM to 1000 μM, the concentration of the phase separation crowding agent solution is 500 mM to 1000 mM, and the volume ratio of the short peptide solution to the phase separation crowding agent solution is 1:90 to 110.

3. Use according to claim 2, characterized in that, The phase separation crowding agent is sodium chloride, ATP, ADP, tannic acid, sodium heparin, or chondroitin sulfate.

4. Use according to claim 2, characterized in that, When the condensate is used as a carrier to encapsulate mitochondria, the mitochondrial solution is mixed with a phase separation crowding agent solution, and then the short peptide solution is added and mixed thoroughly.

5. The application according to claim 4, characterized in that, The concentration of the mitochondrial solution is 1 μg / mL to 50 μg / mL, the concentration of the short peptide solution is 1 mM to 100 mM, and the concentration of the phase separation crowding agent solution is 1 mM to 1000 mM, with a volume ratio of 1 to 20:1 to 10:1000.

6. Use according to claim 1, characterized in that, The short peptide is obtained by solid-phase peptide synthesis from amino acids protected by 2-chlorotriphenylmethyl chloro resin and 9-fluorenylmethoxycarbonyl groups.

7. The use according to claim 1, characterized in that, The vector is used for live mitochondrial transplantation in eukaryotic cells.

8. An encapsulated mitochondrion for delivery, wherein, The mitochondria are obtained by encapsulating them in the condensate described in claim 2.

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