Fluorinated polysarcosine mitochondrial targeting micelle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a fluorinated polysarcosine mitochondrial targeting micelle as well as a preparation method and application thereof. A fluorine-containing alkyl chain is used as a hydrophobic block, polysarcosine is used as a hydrophilic block, the micelle is self-assembled, and the general formula of the micelle is shown in the formula (1), wherein m and n are positive integers. The preferable structure CF1-PPar24 of the fluorinated polysarcosine mitochondrial targeting micelle is that an alkyl chain is replaced by a fluorocarbon chain to optimize the micelle, so that the blood environment stability and the in-vivo circulation time of the micelle are improved, and the tumor tissue accumulation effect of a drug is enhanced; the neutral mitochondrial targeting carrier is constructed by adopting fluorocarbon chain-polysarcosine, so that the toxicity and off-target effect are reduced while the targeting property is retained, the application defect of PEG (Polyethylene Glycol) materials is avoided, and the purpose of improving the drug delivery performance is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fluorinated polysarcosine mitochondrial targeted micelle, its preparation method, and its application. Background Technology

[0002] Polysarcosine (PSar) is a polypeptide composed of N-methylglycine residues linked by peptide bonds. Its repeating unit is –[–NHCH2COCH3–]–, and its main chain is identical to that of natural polypeptides. It has only one methyl group on its side chain and possesses a protein-like backbone. Known properties include excellent water solubility (>500 mg / mL). -1 It exhibits extremely weak interaction with plasma proteins and the reticuloendothelial phagocytic system (RES); its main chain peptide bonds can be slowly recognized by intracellular proteases and eventually degraded into sarcosine (a natural metabolite in the human body), without cumulative toxicity; its chain segments are highly flexible and have high steric hindrance, enabling it to form a 3–5 nm thick hydrophilic protective layer, giving micelles "stealth" properties. Therefore, PSA has been widely regarded as a next-generation alternative hydrophilic shell for polyethylene glycol (PEG).

[0003] The self-assembly principle of fluorinated amphiphilic polymers. Fluorinated alkyl groups (–(CF2) n CF3, n≥2) has extremely low surface energy (~12mN·m -1 Fluorine-carbon chains exhibit an oleophobic effect incompatible with alkyl hydrocarbons. Existing research indicates that when the DP of the fluorocarbon chain is ≥2, a dense fluorine core can be formed through fluorine-fluorine interactions, reducing the micelle CMC by 1–2 orders of magnitude. The water permeability of the fluorine-containing core is 30–50% lower than that of the hydrocarbon phase, significantly inhibiting drug diffusion and leakage in the bloodstream. The core formed by the fluorocarbon chain has an affinity for the lipid raft region of the cell membrane, increasing the penetration depth of tumor tissue by 2–3 times. Currently, fluorination strategies have been used in polyester systems such as polylactic acid (PLA), polycaprolactone (PCL), and polycarbonate, but have not yet been combined with polysarcosine.

[0004] Mitochondrial targeting ligands and their mechanisms of action. Mitochondrial membrane potential (ΔΨm, –180 mV) drives the enrichment of lipophilic cations. Known ligands include triphenylphosphine (TPP). + TPP: logP ~4.5, with an enrichment fold increasing 10-fold for each additional carbon chain; Mitochondrial penetrating peptide (MPP): sequence KLALKLALKALKAALKLA, carrying 4–5 positive charges, can enter mitochondria via a dual mechanism of electrostatics and membrane potential; after conjugation of the above ligands with drugs or carriers, the mitochondrial / cytoplasmic concentration ratio can reach 50–200. However, using TPP alone... + MPP may have problems such as high positive charge toxicity, strong plasma protein adsorption, and uncontrolled distribution in vivo.

[0005] In the prior art, patent CN120484249A discloses a fluorine-modified polysarcosinate lipid, its preparation method, and its applications. This invention, through chemical modification of polysarcosinate lipids with different fluorine-containing groups, yields fluorine-modified polysarcosinate lipids that can replace PEGylated lipids in the preparation of lipid nanoparticles (LNPs) for loading nucleic acids and other drugs, thereby improving drug delivery performance. This patent mainly features a 3-block structure, with an N,N-dialkyl chain as the hydrophobic core, polysarcosinate as the hydrophilic block, and -CH2CF3 as the liposome surface functional ligand for performance improvement; however, it does not exhibit good self-assembly performance or mitochondrial targeting. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to design and provide a technical solution for fluorinated polysarcosine mitochondrial-targeting micelles, their preparation method, and applications. This solution can reduce the critical micelle concentration (CMC), solving the problems of premature dissociation and drug leakage caused by blood dilution after intravenous injection; increase the penetration of polysarcosine micelles into tumor sites; endow micelles with better mitochondrial targeting ability without introducing high positive charges, optimizing their subcellular organelle targeting distribution efficiency; and overcome the immunogenicity risks, non-degradability, and potential toxicity associated with polyethylene glycol (PEG) as a hydrophilic block in nanomicelles.

[0007] This invention is specifically achieved through the following technical solutions: The first aspect of this invention provides fluorinated polysarcosine mitochondrial-targeting micelles, which are self-assembled into micelles using fluorinated alkyl chains as hydrophobic blocks and polysarcosine as hydrophilic blocks, and their general formula is shown in formula (1): Equation (1) Where m and n are positive integers.

[0008] Furthermore, the fluorinated polysarcosine mitochondrial targeted micelles have m=1 and n=24.

[0009] Furthermore, the critical micelle concentration of the preferred structure of the targeted micelles is less than 0.1 mg·L⁻¹. -1 .

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned fluorinated polysarcosine mitochondrial targeted micelles, wherein the fluorinated polysarcosine mitochondrial targeted micelles are obtained by reacting N-ethylpolysarcosine-25 with perfluorobutyric acid under the catalysis of 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), and anhydrous dichloromethane (DCM).

[0011] Furthermore, the above preparation method specifically includes the following steps: S.1 Dissolve N-ethylpolysarcosine-25 in DCM; S.2 Dissolve perfluorobutyric acid in DCM, add DCC, and after a pre-reaction of 3-5 hours, add a catalytic amount of DMAP; S.3 The N-ethyl polysarcosine-25 solution prepared in S.1 is slowly added to the polymer solution prepared in S.2, and the mixture is stirred in an ice bath at 0°C to pre-equilibrate. After a period of time, the mixture is moved to room temperature and stirred for 20-30 hours, during which dicyclohexylurea is generated. After the S.4 reaction is completed, the product is filtered to remove dicyclohexylurea and residual small molecule impurities, and the crude fluorinated polysarcosine product CF1-PSar is obtained. 24 Transfer to a dialysis bag and dialyze in a pure water environment for 2-4 days; S.5 Finally, the purified product was lyophilized to obtain the dried fluorinated polysarcosine product CF1-PSar. 24 .

[0012] Furthermore, step S.4 removes substances including filtration, transfer, and dialysis.

[0013] The third aspect of this invention provides the application of the above-mentioned fluorinated polysarcosine mitochondrial-targeting micelles in the preparation of biological agents.

[0014] A fourth aspect of the present invention provides a biological agent containing the above-described fluorinated polysarcosine mitochondrial-targeting micelles.

[0015] The fifth aspect of this invention provides the application of the above-described fluorinated polysarcosine mitochondrial targeting micelles or preparation method in targeted drug delivery.

[0016] The present invention has the following beneficial effects: By replacing alkyl segments with fluorocarbon segments, and utilizing the dual hydrophobic-oleophobic effect and strong fluorine-fluorine interaction, the CMC of micelles is reduced by two orders of magnitude, ensuring that they remain intact even under high blood dilution after intravenous injection and preventing premature drug leakage.

[0017] By utilizing the affinity between the core and lipid valve of fluorine-fluorine micelles, as well as their surface charge neutrality, plasma protein adsorption and reticuloendothelial phagocytosis are reduced, prolonging the in vivo circulation time. At the same time, the penetration depth of tumor tissue is enhanced, and the effective accumulation of lesions is increased.

[0018] A non-charged mitochondrial targeting carrier is used, which is composed of a fluorinated amphiphilic compound consisting of a fluorocarbon chain (F7)-polysarcosine (25). This makes the overall charge of the carrier close to neutral, which significantly reduces peripheral toxicity and off-target effects while retaining mitochondrial targeting.

[0019] Using polystyrene as a hydrophilic block can avoid the potential for long-term or repeated use of PEG to induce the production of anti-PEG antibodies in the human body, as well as the non-degradability of PEG and the chronic toxicity caused by organ accumulation (such as in the liver and spleen) due to long-term accumulation. Attached Figure Description

[0020] Figure 1 It is the general formula for fluorinated polysarcosine amphiphilic polymers; CF m —PSar n : Among them CF m For hydrophobic blocks, PSar n It is a hydrophilic segment; Figure 2 The effect of perfluoroalkyl chain length on CMC value; Figure 3 The effect of polysarcosine chain length on CMC value; Figure 4 For CF1-PSar 24 Electron micrograph of self-assembled micelles; Figure 5 For 3D tumor sphere permeability assessment, scale bar 50 μm; Figure 6 For CF1-PSar 24 Cellular uptake efficiency of self-assembled micelles in different cells; Figure 7 For CF1-PSar 24 Targeted analysis of self-assembled micelle subcellular organelles; Figure 8 For CF1-PSar 24 Toxicity analysis of self-assembled micelles in different cells; Figure 9 For CF1-PSar 24 Schematic diagram of the self-assembled micelle synthesis process. Detailed Implementation

[0021] The technical features and advantages of the invention will be described in more detail below with reference to the accompanying drawings.

[0022] Example 1: CF1-PSar 24 Synthesis methods of self-assembled micelles Under dry, anhydrous conditions (glassware dried and cooled, with nitrogen / argon protection throughout if necessary), perfluorobutyric acid (428 mg, 2 mmol) was dissolved in 30 mL of anhydrous dichloromethane (DCM), and dicyclohexylcarbodiimide (DCC, 412 mg, 2 mmol) was added and stirred for 4 hours. Then, a catalytic amount of 4-dimethylaminopyridine (DMAP, 36.6 mg, 0.3 mmol) was added, and the mixture was transferred to an ice bath. Then, terminal amino-terminated N-ethylpolysarcosine-25 (3.64 g, 2 mmol) dissolved in DCM (100 mL) was slowly added dropwise to the activated perfluorobutyric acid solution using a constant pressure funnel. At this point, white dicyclohexylurea (DCU) can usually be observed to gradually form and precipitate. After the addition was complete, the ice bath was removed, and the mixture was transferred to room temperature to continue the reaction for 24 hours to ensure complete reaction. After the reaction was complete, the reaction solution was filtered at room temperature (using a sintered glass funnel or filter membrane) to remove the DCU precipitate. The filter cake was then washed 1–2 times with a small amount of anhydrous dichloromethane to recover the adsorbed product. The filtrates were combined and transferred to a 1.5 kDa dialysis bag, and dialyzed in deionized water for 72 hours, with the water changed periodically to remove residual small molecule impurities. Finally, the purified product was lyophilized to obtain the dried fluorinated polysarcosine product CF1-PSar. 24 (3.68 g, yield 91.27%), general formula as follows Figure 1 As shown, the synthesis path is... Figure 9 The preferred structure is m=1, n=24.

[0023] Example 2: CF1-PSar 24 Determination of Critical Micelle Concentration (CMC) of Self-Assembled Micelles The CMC of micelles was determined using pyrene as a hydrophobic fluorescent probe. The specific steps were as follows: 8 × 10⁸ pyrene was added to the test tube. - 5 M's pyrene chloroform solution was then added to polymer solutions prepared in chloroform and serially diluted 10-fold to achieve a final polymer concentration ranging from 100 to 10. -8 mg·mL⁻¹. The test tubes were vacuum-dried overnight, hydrated with PBS, and incubated with shaking at room temperature for 24 h. Fluorescence intensity was measured using a Tecan Infinite M1000 Pro microplate reader, with excitation wavelengths of 338 nm and 334 nm, and emission wavelength of 390 nm. The ratio of fluorescence intensity (I) was plotted on the x-axis as the logarithm of polymer concentration. 338 / I 334 Plotting the curve on the ordinate, the CMC value was determined by the intersection of the two tangent lines of the fitted curve. The results show that the micelle CMC value decreases with increasing perfluoroalkyl chain length. Figure 2 ), increases with increasing polysarcosine chain length ( Figure 3 ); among them CF1-PSar24 The CMC of the self-assembled micelles was 0.08 mg·L⁻¹. -1 This indicates that it can maintain structural stability even under highly diluted conditions, a characteristic that helps it maintain its integrity during in vivo circulation.

[0024] Example 3: CF 1- PSar 24 Measurement of self-assembled micelle size The morphology and particle size of the micelles were observed using transmission electron microscopy (TEM). A drop of the micelle dispersion was placed on a 400-mesh carbon film copper grid (Ted Pella Corporation), allowed to air dry, and then observed and imaged using a JEOL JEM-2010 transmission electron microscope (Nippon Electron Corporation). The results showed that CF1-PSar 24 The self-assembled micelles are regularly spherical with a particle size distribution ranging from 80 to 100 nm. Figure 4 This size range is advantageous for its enrichment in tumor tissue by enhancing penetration and retention effects.

[0025] Example 4: CF1-PSar 24 Self-assembled micelles for assessing the permeability of 3D tumor spheres First, a HeLa cell spheroid model was established: HeLa cells were pre-coated in 96-well plates with 1.5% (w / v) agarose medium solution. After curing at room temperature, cells were seeded at a density of 5 × 10³ cells per well. After centrifugation at 1500 rcf for 15 min, the cells were cultured at 37℃ and 5% CO2 to form dense spheroids. Spheroids cultured for 4-5 days were then compared with PEG-PE micelles labeled with rhodamine B-phosphatidylethanolamine (Rh-PE) (control group) and CF1-PSar, respectively. 24 Self-assembled micelles (experimental group) were incubated for 4 h. After incubation, the spheres were transferred to new well plates and gently washed with PBS. Z-axis tomographic images were acquired at 20 μm intervals using confocal microscopy for analysis. Compared with traditional PEG-PE micelles, CF1-PSar 24 Self-assembled micelles exhibit superior deep penetration ability of tumor spheres. Figure 5 This demonstrates its potential advantage in penetrating the barrier of solid tumors.

[0026] Example 5: CF1-PSar 24 Evaluation of the uptake capacity of self-assembled micelles in different cell lines CF1-PSar was labeled with Rh-PE (1%, w / w) as a fluorescent probe. 24 Micelles. Before the experiment, mouse macrophages (RAW264.7), mouse fibroblasts (NIH3T3), and human cervical adenocarcinoma epithelial cells (HeLa) were sampled at 1×10⁻⁶ cells per well.5 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured for 24 h. Subsequently, labeled micelles were diluted with serum-free medium and co-incubated with the cells for 1 h. Cells were washed three times with PBS buffer to remove uninternalized micelles / dye. Cell uptake of the micelles was observed using a Nikon Ti Eclipse fluorescence microscope system. CF1-PSar 24 Self-assembled micelles can be effectively taken up by various cell lines such as RAW264.7, NIH3T3, and HeLa. Figure 6 This indicates that it has a broad spectrum of cell internalization capabilities.

[0027] Example 6: CF1-PSar 24 Subcellular organelle targeting analysis of self-assembled micelles in different cell lines HeLa cells were distributed at a density of 1 × 10⁻⁶ cells per well. 5 After being seeded at a density of [number] cells and cultured for 24 h, cells were co-incubated with Rh-PE-labeled micelles in serum-free medium at 37°C. Subsequently, specific organelles were stained using commercial dyes according to the supplier's recommended protocol: endosomes / lysosomes (LYSO-ID® Green), mitochondria (Rh123 or MitoView™ Green), endoplasmic reticulum (ER-Tracker™ Green), and Golgi apparatus (NBD C6-ceramide). To investigate the mitochondrial targeting mechanism, FITC-labeled polymer components were calibrated to the same fluorescence intensity and co-incubated with HeLa cells for 1 h. Mitochondria were counterstained with MitoView™ 633, and nuclei were stained with Hoechst 33258 for 15 min. Imaging and co-localization were performed using a Nikon Ti Eclipse confocal microscope system. Co-localization analysis showed that CF1-PSar [missing information - likely a specific cell type or component]. 24 Self-assembled micelles and mitochondrial markers are highly co-labeled ( Figure 7 This clearly indicates that it is primarily located in mitochondria and possesses unique organelle targeting capabilities.

[0028] Example 7: CF1-PSar 24 Toxicity analysis of self-assembled micelles in different cell lines 24 h before cell treatment, RAW264.7, NIH3T3, and HeLa cells were seeded in 96-well plates at a density of 2 × 10³ cells per well. Different concentrations of CF1-PSar were then added. 24Self-assembled micelles were added to complete growth medium and co-incubated with cells at 37°C for 48 h. Cell viability was assessed using CellTiter-Blue® cell viability assay kit (Promega): a mixture of 90 μL complete growth medium and 10 μL reagent was added to each well, and after incubation at 37°C for 2 h, fluorescence intensity was measured using a microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm, and cell viability was calculated. The results showed that at 1×10⁻⁶ cells / wells, cell viability was significantly improved. -5 Up to 1 mg / mL -1 Within the concentration range, CF1-PSar 24 Self-assembled micelles did not significantly affect the survival rate of the three test cell types. Figure 8 This demonstrates that the nanocarrier exhibits good biocompatibility and safety within this concentration range.

Claims

1. A fluorinated polysarcosine mitochondrial-targeting micelle, characterized in that, Using fluorinated alkyl chains as hydrophobic blocks and polysarcosine as hydrophilic blocks, micelles are self-assembled, and their general formula is shown in formula (1): Equation (1) Where m and n are positive integers.

2. The fluorinated polysarcosine mitochondrial-targeting micelles as described in claim 1, characterized in that, m=1, n=24.

3. The fluorinated polysarcosine mitochondrial-targeting micelles as described in claim 1, characterized in that, The critical micelle concentration of the targeted micelles is less than 0.1 mg·L⁻¹. -1 .

4. The method for preparing fluorinated polysarcosine mitochondrial-targeting micelles as described in claim 1, characterized in that, The fluorinated polysarcosine mitochondrial-targeting micelles were obtained by reacting N-ethylpolysarcosine-25 with perfluorobutyric acid under the catalysis of DMAP, DCC, and DCM.

5. The preparation method according to claim 4, characterized in that, Specifically, the following steps are included: S.1 Dissolve N-ethylpolysarcosine-25 in DCM; S.2 Dissolve perfluorobutyric acid in DCM, add DCC, and after a pre-reaction of 3-5 hours, add a catalytic amount of DMAP; S.3 The N-ethyl polysarcosine-25 solution prepared in S.1 is slowly added to the polymer solution prepared in S.2, and the mixture is stirred in an ice bath at 0°C to pre-equilibrate. After a period of time, the mixture is moved to room temperature and stirred for 20-30 hours, during which dicyclohexylurea is generated. After the S.4 reaction is completed, the product is filtered to remove dicyclohexylurea and residual small molecule impurities, and the crude fluorinated polysarcosine product CF1-PSar is obtained. 24 Transfer to a dialysis bag and dialyze in a pure water environment for 2-4 days; S.5 Finally, the purified product was lyophilized to obtain the dried fluorinated polysarcosine product CF1-PSar. 24 .

6. The preparation method according to claim 5, characterized in that, Step S.4 involves the removal of substances including filtration, transfer, and dialysis.

7. The use of fluorinated polysarcosine mitochondrial targeting micelles as described in any one of claims 1-2 in the preparation of biological agents.

8. A biological agent comprising fluorinated polysarcosine mitochondrial-targeting micelles as described in any one of claims 1-2.

9. The application of the fluorinated polysarcosine mitochondrial targeting micelles as described in any one of claims 1-2 or the preparation method as described in any one of claims 4-6 in targeted drug delivery.

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