High-stability engineered exosome based on rigid hydrophobic anchoring and micelle disassembly and purification as well as preparation method and application of high-stability engineered exosome

Abstract

The invention relates to the technical field of biomedicine nanotechnology, in particular to a high-stability engineered exosome based on rigid hydrophobic anchoring and micelle disassembly and purification as well as a preparation method and application of the high-stability engineered exosome. According to the invention, a ternary surface structure of rigid hydrophobic anchoring-hydrophilic polymer spacer arm-targeting ligand (L-S-T) is constructed, cholesterol hemisuccinate or multi-branched lipid is selected as an anchoring group, and the problem that conventional phospholipid modification is easy to fall off in vivo and in an albumin-containing preparation is obviously solved; a technology of'micellar insertion after temperature control 'combined with'CMC critical concentration adjustment and purification' is adopted, lossless insertion of functional molecules is realized by utilizing thermodynamic driving force, and residual micelles are induced to be disintegrated into monomers by adjusting the concentration of a system to be lower than the concentration of critical micelles, so that efficient removal of free impurities is realized through conventional ultrafiltration. The engineered exosome has the advantages of long circulation, high targeting and excellent storage stability, and has important clinical application and commercial transformation prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanotechnology, specifically to a highly stable engineered exosome based on rigid hydrophobic anchoring and micellar disassembly and purification, its preparation method, and its application. Background Technology

[0002] Exosomes, as an endogenous nanocarrier, have attracted much attention in the field of drug delivery. However, existing technologies (such as US20210276318A1 or WO2022 / 026234A1), although involving lipid insertion modification, still face core challenges: 1. Instability: Traditional DSPE modifications are easily removed by serum albumin through a lipid exchange mechanism (e.g., Figure 4 (As shown on the left).

[0003] The fundamental reason is that the hydrophobic portion of traditional bi-tailed phospholipids is highly flexible, resulting in insufficient binding energy with the exosome membrane and a lack of physical spatial barrier against competitive binding to albumin.

[0004] 2. Purification difficulties: Existing techniques often neglect the removal of residual micelles (15-25 nm) after the reaction.

[0005] Because their particle size is similar to that of exosomes, they are difficult to separate by conventional filtration, resulting in residual micelles competitively binding to lesion targets, potentially affecting targeting efficiency and treatment efficacy.

[0006] 3. Process adaptability: High-temperature intercalation can easily lead to protein inactivation, while low-temperature intercalation is inefficient.

[0007] Therefore, there is an urgent need in this field for an innovative engineering strategy that can enable modified exosomes to remain highly stable in the bloodstream while ensuring their efficient targeting of lesion sites. Summary of the Invention

[0008] Based on this, one embodiment of this application provides a highly stable engineered exosome based on rigid hydrophobic anchoring and micelle disassembly and purification, as well as its preparation method and application.

[0009] This invention provides an overall solution of "high stability anchoring + CMC-induced purification".

[0010] 1. High-stability structural design (double-locking structure) This invention abandons the traditional double-chain phospholipid (DSPE) and innovatively introduces rigid hydrophobic anchoring groups (such as cholesterol derivatives) or multivalent hydrophobic groups (such as... Figure 2 A in Figure 2The core of the concept (as shown in B of

[0011] Principle: Cholesterol structure has rigid steroidal ring, which can be specifically inserted into the "lipid raft" microdomain of the exosome membrane (as shown in Figure 1

[0012] Other rigid planar hydrophobic structures (such as pentacyclic triterpenes, specific polycyclic aromatic hydrocarbons) can also resist the extraction of albumin through similar strong hydrophobic interactions and steric hindrance.

[0013] Surprising effect of multi-tailed lipids: experiments have found that when the number of hydrophobic tail chains is ≥ 3 (such as tri-palmitate based on glycerol skeleton), their hydrophobic binding energy increases exponentially, and even in the non-lipid raft region, they can resist the extraction of albumin, showing better stability than traditional double-chain lipids.

[0014] The mechanism is that the multi-tail structure greatly reduces the probability of dissociation from the membrane through the entropy effect.

[0015] Effect: This structure is significantly better than traditional DSPE modification (as shown in A of Figure 4 and B of Figure 5 ) in resisting shedding in plasma environment and albumin-containing preparations.

[0016] Based on the same mechanism of enhancing hydrophobic binding energy and resisting extraction, the concept of the present application can also be extended to other rigid hydrophobic anchor groups that meet the above structure-function relationship.

[0017] Therefore, the cholesterol derivatives, multi-tailed lipids and other rigid hydrophobic structures all achieve ≥ 80% resistance to albumin extraction through the common mechanism of "enhanced hydrophobic binding energy / rigid insertion-high binding entropy", which are parallel means to solve the same technical problem.

[0018] The present application also provides an amphiphilic functional modification molecule having the general formula L-S-T, wherein L, S, and T are defined as before.

[0019] The present application also provides the use of the amphiphilic functional modification molecule for modifying the surface of the exosome membrane to obtain engineered exosomes with anti-albumin extraction properties.

[0020] 2. Micelle disassembly purification process (CMC shift purification) In view of the difficulty of removing free micelles, the present application proposes a universal "critical micelle concentration (CMC) adjustment-based" purification strategy (as shown in Figure 3

[0021] ​​The essence of this method lies in utilizing the physicochemical properties of amphiphilic molecules to dilute the concentration of non-inserted molecules in the system to a level below their CMC value, thereby transforming the difficult-to-separate "nanomicelles" into "monomer molecules" that are easy to filter out, thus turning the "nanoseparation problem" into a "molecular filtration problem".

[0022] The core of this method lies in utilizing the CMC properties, and its universality does not depend on specific modified molecular structures.

[0023] It should be understood that Figure 3 The process flow shown is a holistic solution based on thermodynamic principles (CMC).

[0024] Step III (CMC-regulated disassembly) is a key prerequisite for Step IV (size exclusion separation) to efficiently remove free modified molecules.

[0025] Omitting step III or changing its order (e.g., separating before diluting) will not solve the problem of residual micelle removal described in the background art.

[0026] 3. DMSO-free lyophilized formulations This formulation is based on a specific ratio of trehalose and HSA. The use of anti-extraction anchoring groups makes it possible to use HSA at high concentrations in the formulation.

[0027] After freeze-drying, a solid formulation with a specific microstructure is formed (e.g., Figure 6 (As shown).

[0028] The lyophilized formulation is characterized in that the engineered exosome particles are uniformly embedded and physically fixed in an amorphous matrix with a continuous porous network structure formed by a lyophilization protectant (such as trehalose) and a membrane stabilizer (such as human serum albumin).

[0029] The porosity of this porous matrix is ​​typically between 60% and 90%, with an average pore size ranging from 0.1 μm to 10 μm. This structure facilitates rapid reconstitution and minimizes exosome aggregation and membrane damage during the drying process, which is one of the important reasons why the lyophilized formulations of this invention can maintain high stability after reconstitution.

[0030] Technical positioning and related technical description of the present invention: This invention addresses the core challenge of intravenous delivery of nanoscale carriers such as exosomes—the high specific surface area leading to the competitive stripping of modified molecules by serum proteins (chemical shedding problem)—and provides a solution centered on "rigid anchoring and anti-extraction".

[0031] This technical path forms a clear technical distinction and complementarity with the technology developed by the applicant for the delivery of large-sized living cells (such as mesenchymal stem cells), for example, by polyethylene glycol modification to solve the problem of physical lung capillary interception caused by cell size.

[0032] The former (the present application) focuses on chemical stability, and uses rigid hydrophobic anchoring and CMC purification process to ensure the modification integrity of nanoparticles in complex biological environment; the latter focuses on physical rheology optimization and sequential control of functions, and solves the problems of mechanical jamming at the cell scale and spatiotemporal control during targeting.

[0033] Both of them solve the most specific key bottlenecks in clinical application of biological delivery carriers of different properties and different scales, and together constitute a platform technology system covering multi-scale delivery requirements. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0035] Figure 1 The figure is a schematic diagram of the engineered exosome structure of the present application, which shows the anchoring mode of the L-S-T ternary structure in the membrane lipid bilayer (taking the anchoring of cholesterol derivative in lipid raft as an example); Figure 2 The figure is a schematic diagram of the molecular structure of two embodiments, wherein Figure 2 A in the formula is cholesterol derivative anchoring, Figure 2 B in the formula is multi-tailed lipid anchoring; Figure 3 The figure is a flow chart of CMC adjustment purification process, which shows the complete process from micelle formation to final purification; Figure 4 The figure is a schematic diagram of stability comparison between the present application and prior art; Figure 5 The figure is a plasma stability experiment data graph, wherein Figure 5 A in the formula is a quantitative column chart, Figure 5 B in the formula is a histogram for detecting exosome surface targeting ligand retention rate by flow cytometry; Figure 6 The figure is a schematic diagram of the microstructure cross section of the freeze-dried preparation. DETAILED DESCRIPTION

[0036] The application will be described in further detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are only used to explain the application and not intended to limit the scope of the application. The purpose of providing these embodiments and examples is to make the disclosure of the application more thoroughly and comprehensively understood. It should also be understood that the application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the application, and the equivalent forms obtained by such changes or modifications also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application. It should be understood that the application can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings: The selection range of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0039] In this application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or more than two.

[0040] The "combination thereof", "any combination thereof", "any combination thereof" and the like used herein include all suitable combinations of any two or more listed items.

[0041] In the present application, “suitable” in “suitable combination”, “suitable manner”, “any suitable manner” and the like means that the technical solutions of the present application can be implemented, the technical problems of the present application can be solved, and the intended technical effects of the present application can be achieved.

[0042] In the present application, “further”, “still further”, “in particular” and the like are used for description purposes and represent differences in content, but should not be understood as limiting the protection scope of the present application.

[0043] In the present application, “optionally”, “optional” and “optional” mean that it can or can not be present, i.e., it means that it is selected from either of the two parallel schemes “has” or “has not”. If there are multiple “optionally” in a technical solution, and there is no contradictory or mutually restrictive relationship, each “optionally” is independent.

[0044] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution of the listed features.

[0045] In the present application, with respect to a numerical interval (i.e., a numerical range), if not specifically stated, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes both numerical end points (i.e., the minimum value and the maximum value) of the numerical range and each numerical value between the two numerical end points. If not specifically stated, when a numerical interval only points to integers within the numerical interval, including both end point integers of the numerical range and each integer between the two end point integers, in the present application, each integer is directly listed, for example, t is an integer selected from 1-10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0046] In the present application, the temperature parameter, if not specifically limited, allows for constant temperature treatment and also allows for fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0047] In the present application, both (w / w) and wt% represent weight percentage, (v / v) represents volume percentage, and (w / v) represents mass volume percentage.

[0048] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is incorporated by reference individually. Unless and to the extent conflicting with the purposes and / or technical solutions of the present application, the documents mentioned in the present application are incorporated by reference in the present application in the whole content and in the whole purpose. When the present application refers to the documents mentioned, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents mentioned, the examples, preferred modes of the relevant technical features are also incorporated by reference in the present application as far as possible. It should be understood that when the contents of the references conflict with the description in the present application, the present application is prior or is amended according to the description in the present application adaptively.

[0049] Definitions and explanations: In the present application, the "cholesterol-PEG derivative" refers to an amphiphilic molecule precursor formed by covalently connecting one end of a polyethylene glycol (PEG) chain to the hydroxyl group of cholesterol or its derivative (such as cholesterol hemisuccinate) through a covalent bond (such as an ester bond, an ether bond).

[0050] In the present application, the "rigid steroidal structure" refers to a sterol or its derivative with a cyclopentane polycyclic hydrophenanthrene core skeleton, which is not prone to conformational flipping, such as but not limited to cholesterol, cholesterol hemisuccinate (CHEMS), ergosterol, etc.

[0051] In the present application, the "multi-tail hydrophobic structure" refers to a hydrophobic structure formed by covalently connecting ≥3 C14-C22 straight-chain or branched-chain saturated fatty acid chains to the same skeleton. The saturated fatty acid chains can be straight-chain (such as palmitic acid, stearic acid) or branched-chain (such as phytanic acid); and the multi-tail structure can be connected through a glycerol, polylysine, or dendrimer skeleton, etc.

[0052] It should be noted that the present application realizes high stability of membrane anchoring through "multi-tail enhanced entropy effect". The core of this mechanism lies in the synergistic interaction of multiple (≥3) fatty acid chains with the membrane lipid bilayer, which greatly reduces the probability of dissociation from the membrane. For branched-chain fatty acids, although there are slight differences in local conformation compared with straight-chain, they still retain the hydrophobic nature of long alkyl chains and can provide multiple hydrophobic contact points through their branched structures. Therefore, based on the above-mentioned mechanism disclosed in the present application, it can be reasonably expected that the multi-tail hydrophobic structure constructed by branched-chain fatty acids can also achieve the technical effects of enhancing membrane binding stability and resisting albumin extraction. Including branched-chain fatty acids in the protection scope conforms to the core inventive concept of the present application.

[0053] The saturated fatty acid chains can be straight-chain or branched-chain; and the multi-tail structure can be connected through a glycerol, polylysine, or dendrimer skeleton, etc.

[0054] In the present invention, the "hydrophobic group with rigid planar or polycyclic aromatic structure" refers to a rigid or semi-rigid planar hydrophobic structure that can stably embed into the interior of the lipid bilayer of the exosome membrane through hydrophobic interaction.

[0055] The "critical micelle concentration (CMC)" in the present invention refers to the lowest concentration threshold at which amphiphilic molecules self-assemble from monomer state to form thermodynamically stable micellar aggregates under specific solvent environment (such as buffer system) and temperature conditions.

[0056] The numerical value depends on the chemical structure of the amphiphilic molecule and factors such as ionic strength, pH value, and temperature of the solution.

[0057] The CMC value can be determined by conventional physicochemical methods in the art, including but not limited to surface tension method, conductivity method, light scattering method, or fluorescence probe method (such as the pyrene fluorescence probe method described in Example 1).

[0058] In the context of the present invention, if there are slight differences in the values obtained by different determination methods, the value determined by the fluorescence probe method in the same buffer and temperature conditions as the reaction system is preferred.

[0059] The functional modification molecule "retention rate ≥ 80%" in the claims of the present invention, unless otherwise specified, refers to the average value obtained from three independent repeated experiments (n = 3) under standard verification experimental conditions (such as Example 3).

[0060] The "other hydrophobic groups with rigid planar or polycyclic aromatic structure" in the present invention (as defined in Claim 3) are an extension based on the same inventive concept in addition to the specific implemented cholesterol derivatives and multi-tailed lipids. The selection principle and feasibility are based on the following two points: 1. Structure-function relationship principle: The core finding of the present invention is that the rigid planar structure of the anchoring group and the resulting high hydrophobic binding energy / binding entropy are the key to resisting serum albumin extraction.

[0061] Both the steroid ring of cholesterol and the dense alkyl chain of multi-tailed lipids embody this principle.

[0062] Therefore, any hydrophobic structure that meets the following conditions can be reasonably expected to have similar functions: (a) has a rigid or semi-rigid planar / polycyclic skeleton that limits its conformational freedom in the membrane; (b) has sufficient hydrophobic surface area to produce strong hydrophobic interaction with the membrane lipid bilayer; (c) its overall size and shape are suitable for embedding in the lipid bilayer without causing serious disturbance to the membrane structure.

[0063] 2. Representative compounds exemplify the general principle: Based on the above principles, one skilled in the art can reasonably select a variety of known rigid hydrophobic structures based on the principles of structure-function relationship disclosed in the present application to make equivalent substitutions. For example: Pentacyclic triterpenoid derivatives: such as ursolic acid, oleanolic acid.

[0064] Its rigid pentacyclic triterpene skeleton is highly similar to the rigid and hydrophobic cholesteroid ring, and is a potential excellent anchoring group.

[0065] Its carboxyl group can be connected to a PEG spacer arm through reactions such as esterification to form a functional molecule.

[0066] Polycyclic aromatic hydrocarbon derivatives: such as pyrene, perylene, which are only examples to illustrate the principle of 'rigid conjugated planar structure' and are not a limitation of the present application.

[0067] It has a large planar conjugated system and strong hydrophobicity, and it is known that such structures can strongly insert and stabilize lipid membranes.

[0068] L-S-T molecules can be constructed by introducing a connecting arm (such as an alkyl chain connecting PEG) on their molecules.

[0069] Rigid macrocyclic compounds: such as some cuphane derivatives or cyclodextrin hydrophobic modifiers with rigid cavities and hydrophobic outer walls, which also meet the design principles of the present application.

[0070] One skilled in the art understands that as long as the core mechanism of 'rigid / planar hydrophobic module providing anti-extraction stability' disclosed in the present application is followed, routine chemical modifications of the above-mentioned example compounds or other similar structures to connect spacers (S) and ligands (T) and apply them to exosome modification through the MTPI and CMC purification processes described in the present application can obtain engineered exosomes with similar high stability and targeting function.

[0071] The 'MTPI' (Micelle-to-Protein Insertion) process described in the present application refers to a method of co-incubating amphiphilic functional molecules in the form of micelles with exosomes, and using thermodynamic driving force to insert them into the membrane.

[0072] This is within the scope of routine experiments under the teachings of the present application, and can be achieved without undue experimentation.

[0073] One skilled in the art understands that 'keeping the concentration of free molecules below CMC' is the core thermodynamic principle of the purification process of the present application.

[0074] The specific means to achieve this purpose are not limited.

[0075] Although the embodiment is mainly achieved by adding buffer dilution, other means that can break the micelle-monomer balance are also included in the scope of the present application.

[0076] For example, constant volume dialysis (Diafiltration) is performed using tangential flow filtration (TFF), fresh buffer is supplemented while monomers are constantly removed, and the system is always maintained in a low concentration environment; or the ionic strength or pH of the buffer is changed in specific cases to change the CMC properties of the molecules.

[0077] Any technical solution that utilizes the principle of 'disassembly below CMC' to convert nanomicelles into small molecule monomers to achieve separation from exosomes falls within the scope of the present application. The embodiments of the present application will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the guidelines given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or according to the known experimental methods in the art.

[0078] In the following specific examples, the measurement parameters of the raw materials, such as the amount, may have slight deviations within the weighing accuracy range if not specifically stated. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operation accuracy.

[0079] It should be understood that in various embodiments of the present application, the size of the sequence number of the above processes does not mean the order of execution, and the execution order of the processes should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0080] Example 1: Synthesis and CMC determination of high stability functional molecule (Chol-PEG-TFP) Raw materials: Cholesterol hemisuccinate (CHEMS), NH2-PEG-COOH (MW=5000 Da), targeting peptide (SEQ ID NO: 1).

[0081] Synthesis step: CHEMS is activated with NHS and EDC in anhydrous DMSO for 30 minutes to obtain an activated ester intermediate.

[0082] It is added dropwise to the PBS solution of NH2-PEG-COOH and reacted at room temperature for 4 hours.

[0083] After purification by ultrafiltration, the product is condensed with the C-terminal carboxyl group of the targeting peptide (TFP) under the catalysis of EDC / NHS, and after the reaction is completed, the product is purified again by ultrafiltration and freeze-dried to obtain the final product Chol-PEG-TFP.

[0084] Variants: The present example is also applicable to functional variants of SEQ ID NO: 1, as long as the variant has >80% sequence homology to SEQ ID NO: 1 and retains the ability to bind VCAM-1. The functional variants include, but are not limited to, addition of Lys or Met at the carboxy terminus of the core sequence (Ile-Glu-Leu-Leu-Gln-Ala-Arg) shown in SEQ ID NO: 1, and conservative substitutions (e.g., Glu→Asp, Leu→Ile / Val, Ala→Gly) at the 2nd, 3rd, 4th, 6th amino acid within the core sequence, and the variant has >80% sequence homology to the full-length sequence of SEQ ID NO: 1.

[0085] Specifically, the functional variants include, but are not limited to, SEQ ID NO: 2 (amino acid sequence: Ile-Glu-Leu-Leu-Gln-Ala-Arg-Lys) and SEQ ID NO: 3 (amino acid sequence: Ile-Glu-Leu-Leu-Gln-Ala-Arg-Lys-Met).

[0086] The sequence information has been submitted as a separate file.

[0087] SEQ ID NO: 1: Amino acid sequence: Ile Glu Leu Leu Gln Ala Arg.

[0088] SEQ ID NO: 2: Amino acid sequence: Ile Glu Leu Leu Gln Ala Arg Lys.

[0089] SEQ ID NO: 3: Amino acid sequence: Ile Glu Leu Leu Gln Ala Arg Lys Met.

[0090] CMC determination: The critical micelle concentration (CMC) of the product Chol-PEG-TFP was determined using pyrene fluorescence probe method.

[0091] Briefly, a series of Chol-PEG-TFP PBS solutions with concentration gradient (1 μM - 100 μM) were prepared, and a small amount of pyrene in acetone solution was added (final concentration 1 μM). After evaporation of acetone, the fluorescence intensity ratio (I1 / I3) of the first vibration peak (I1, about 373 nm) and the third vibration peak (I3, about 384 nm) of pyrene in each solution was determined at an excitation wavelength of 339 nm.

[0092] The CMC value was determined by plotting the molecular concentration as the abscissa and the I1 / I3 value as the ordinate, and the concentration corresponding to the inflection point of the curve.

[0093] The assay result is that the CMC of Chol-PEG-TFP is about 7.5 μM in PBS (pH 7.4).

[0094] The following table shows the change of CMC value under different conditions, as shown in Table 1: Table 1

[0095] Example 2: MTPI insertion membrane and CMC regulation purification process (as shown in Figure 3 ) 1. Micelle preparation: Chol-PEG-TFP is dissolved in PBS and ultrasonically dispersed to prepare a 200 μM stock solution (> CMC).

[0096] 2. Temperature-controlled membrane insertion: 1 mL of exosome suspension (1×10 11 particles / mL) is mixed with 200 μL of the above micelle stock solution, and incubated at 37°C (ensure ≤ 42°C) for 45 min.

[0097] 3. CMC regulation: 20 mL of cold PBS (4°C) is added to the reaction system to expand the total volume by about 20 times, at which time the concentration of free Chol-PEG-TFP molecules is reduced to about 5 μM (< CMC), inducing the disassembly of residual micelles into monomers.

[0098] 4. Separation: a tangential flow filtration device (TFF, 300 kDa MWCO membrane package) is used to concentrate and exchange the system after dilution.

[0099] The free monomer modification molecules are filtered out (Permeate), and the modified exosomes are retained (Retentate).

[0100] 5. Results: The exosome recovery rate in the final product is 85% ± 5% (counted by NTA), and the removal rate of free modification molecules is > 98% (calculated by measuring the molecular concentration in the filtrate by HPLC).

[0101] Comparative Example (traditional method): As a control, the same mixed reaction system without CMC regulation dilution is directly purified by traditional ultrafiltration centrifugation (100 kDa molecular weight cutoff).

[0102] The results show that the removal rate of free micelles (not inserted) is only 60%, which is significantly lower than that of the present method (> 98%), proving the necessity of the "CMC regulation disassembly" step for efficient purification.

[0103] Example 3: Plasma stability verification and synergistic effect demonstration Control 1: DSPE-PEG-TFP (traditional dual-tail phospholipid).

[0104] Control 2: Chol-TFP (rigid anchor only, no hydrophilic polymer spacer S).

[0105] Experimental Group 1: Chol-PEG-TFP (cholesterol anchor, complete L-S-T triad).

[0106] Experimental Group 2: Tripalmitin-PEG-TFP (triple-tail lipid anchor, complete L-S-T triad).

[0107] Methods: The modified exosomes of each group (membrane labeled with DiI fluorescent dye, TFP ligand labeled with FITC) were incubated in a buffer containing 2% (w / v) human serum albumin (HSA) at 37°C for 4 hours.

[0108] Sampling, detecting the mean fluorescence intensity (MFI) of exosome surface FITC by flow cytometry, and comparing with the MFI of 0 hour sample, calculating the retention rate of targeting ligand.

[0109] Results: After 4 hours of incubation, the retention rate of Control 1 (DSPE-PEG-TFP) was 58% ± 5%; the retention rate of Control 2 (Chol-TFP) was 68% ± 4%; the retention rate of Experimental Group 1 (Chol-PEG-TFP) was 91% ± 3%; the retention rate of Experimental Group 2 (Tripalmitin-PEG-TFP) was 88% ± 3% (n = 3, mean ± SD).

[0110] The data are shown in A of FIG. 1. Figure 5

[0111] Conclusion: The experimental data show that the complete L-S-T triad (Experimental Group 1) is necessary and sufficient to achieve a high retention rate of ≥80%.

[0112] The molecule with only rigid anchor (L) but lacking hydrophilic shielding layer (S) (Control 2) has better stability than traditional dual-tail phospholipid, but significantly lower than the complete L-S-T triad.

[0113] Example 4: Freeze-dried preparation Freeze-dried preparation 11 Formulation of the original solution before freeze-drying: engineered exosomes (prepared in Example 2) 1 × 10 11 particles / mL, trehalose 8% (w / v), human serum albumin (HSA) 2% (w / v), dissolved in PBS (pH 7.4).

[0114] Freeze-drying procedure: the original solution was divided into a Schlenk flask and placed in a freeze dryer.

[0115] Cool down to -40°C at 1°C / min, pre-freeze for 2 hours.

[0116] Turn on vacuum, main drying: warm up to -20°C and hold for 20 hours.

[0117] Subsequent secondary drying: warm up to 25°C and hold for 10 hours, until the product residual moisture is < 3%.

[0118] Results: white, loose structured cake was obtained.

[0119] Scanning electron microscope (SEM) observation showed that it has a typical "embedded porous matrix" microstructure (as shown in Figure 6 Exosome particles are embedded in a continuous porous network formed by trehalose and HSA.

[0120] Image analysis showed that the porosity of the porous matrix is about 75%, and the average pore size is about 2 μm.

[0121] After reconstitution, the average particle size change rate of exosomes is 10% (determined by DLS), and the retention rate of targeting functional ligand is 92% (flow detection).

[0122] The ratio of CD63 positive particle number after reconstitution to that before freezing is 96% ± 3% (NTA determination), which confirms that 2% HSA does not mask the surface markers of exosomes.

[0123] Those skilled in the art can understand that due to slight changes in specific process parameters, the microstructure of the actual product (such as pore size distribution) may vary within a certain range, but as long as it has the core structural characteristics of the "embedded porous matrix", and after reconstitution, it can achieve the technical effects of "exosome average particle size change rate ≤ 15%, and targeting functional ligand retention rate ≥ 85%", it falls within the protection scope of the present application.

[0124] Example 5: In vivo targeting efficacy Construct a VCAM-1 high expression mouse inflammation model (TNF-α induced) and a 4T1 breast cancer lung metastasis model.

[0125] DiR-labeled, model drug-loaded (such as Cy5.5-labeled siRNA) unmodified exosomes, DSPE-PEG-TFP modified exosomes and Chol-PEG-TFP modified exosomes (the present application) were injected into model mice through the tail vein.

[0126] Use a small animal live imaging system (IVIS) to image at 2 hours, 6 hours and 24 hours after injection.

[0127] The results show that the enrichment amount (measured by fluorescence intensity) of Chol-PEG-TFP modified exosomes at the inflammation site and lung micro-metastasis (high expression of VCAM-1) is 8.5 times higher than that of the unmodified group and 4.2 times higher than that of the DSPE-PEG-TFP group at the 24-hour time point, proving the broad-spectrum applicability and excellent in-vivo targeting of the delivery system for VCAM-1 related pathological characteristics.

[0128] The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the patent protection scope of the present application.

[0129] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application.

[0130] In addition, it should be understood that, after reading the above teaching content of the present application, those skilled in the art can make various modifications or changes to the present application, and the equivalent forms obtained are also within the protection scope of the present application.

[0131] It should also be understood that, on the basis of the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, and these all belong to the protection scope of the appended claims of the present application.

[0132] Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description can be used to explain the content of the claims.

Claims

1. An engineered exosome, characterized in that, The engineered exosome comprises an exosome body and an amphiphilic functional modification molecule anchored on the surface of the exosome membrane lipid bilayer; the amphiphilic functional modification molecule has a general structure L-S-T, wherein: L is a hydrophobic anchoring group selected from any one of (i) a cholesterol derivative having a rigid steroidal structure, or (ii) a multi-tail hydrophobic structure formed by C14-C22 straight-chain or branched-chain saturated fatty acid chains covalently linked to the same backbone and the number of which is greater than or equal to 3; the hydrophobic anchoring group is stably embedded in the inside of the exosome membrane lipid bilayer or the lipid raft microstructure domain through hydrophobic interaction; S is a hydrophilic polymer spacer arm that forms a hydration shield layer on the surface of the exosome membrane; T is a targeting functional ligand connected to the end of S; and the engineered exosome has anti-albumin extraction properties, and the retention rate of the functional modification molecule is greater than or equal to 80% after incubation in a buffer containing 2% (w / v) human serum albumin at 37°C for 4 hours.

2. The engineered exosome of claim 1, wherein, The hydrophobic anchoring group L is selected from: (a) a cholesterol hemisuccinate, a cholesterol formate, or a cholesterol-PEG derivative formed by covalently connecting one end of a polyethylene glycol to the hydroxyl group of the cholesterol derivative; (b) a multi-tail hydrophobic structure grafted with C14-C22 saturated fatty acid chains based on a glycerol, polylysine or dendrimer backbone.

3. An engineered exosome, characterized in that, The engineered exosome comprises an exosome body and an amphiphilic functional modification molecule anchored on the surface of the exosome membrane lipid bilayer; the amphiphilic functional modification molecule has a general structure L-S-T, wherein: L is a hydrophobic anchoring group selected from a hydrophobic group having a rigid planar or polycyclic aromatic hydrocarbon structure and capable of being stably embedded in the inside of the exosome membrane lipid bilayer through hydrophobic interaction; S is a hydrophilic polymer spacer arm that forms a hydration shield layer on the surface of the exosome membrane; T is a targeting functional ligand connected to the end of S; and the engineered exosome has anti-albumin extraction properties, and the retention rate of the functional modification molecule is greater than or equal to 80% after incubation in a buffer containing 2% (w / v) human serum albumin at 37°C for 4 hours.

4. The engineered exosome of claim 3, wherein, The hydrophobic anchoring group is selected from a pentacyclic triterpenoid derivative, a hydrophobic aromatic compound derivative having a rigid conjugated planar structure, or a rigid macrocyclic compound.

5. The engineered exosome of claim 4, wherein, The pentacyclic triterpenoid derivative is a hydrophobic modified derivative of ursolic acid or oleanolic acid.

6. The engineered exosome of claim 4, wherein, The hydrophobic aromatic compound derivative having a rigid conjugated planar structure is a polycyclic aromatic hydrocarbon derivative.

7. The engineered exosome of claim 4, wherein, The rigid macrocyclic compound is a calixarene derivative or a hydrophobically modified cyclodextrin.

8. The engineered exosome of any one of claims 1 to 7, wherein, The hydrophilic polymer spacer arm S is a polyethylene glycol with a molecular weight of 2 kDa to 10 kDa; The targeting functional ligand T is selected from a polypeptide, an antibody fragment or a nucleic acid aptamer capable of specifically binding to VCAM-1, ICAM-1, E-selectin, Integrins, HER2 or EGFR.

9. The engineered exosome of any one of claims 1 to 7, wherein, The engineered exosome has a retention rate of the functional modification molecule still greater than or equal to 80% after incubation in a buffer containing 50% plasma at 37°C for 4 hours.

10. An amphiphilic functional modification molecule, characterized in that, The engineered exosome has a general structure L-S-T, wherein: L is a hydrophobic anchoring group; S is a hydrophilic polymer spacer arm; T is a targeting functional ligand connected to the end of S.

11. The amphiphilic functional modification molecule according to claim 10, characterized in that, said hydrophobic anchor group L is selected from: (i) a cholesterol derivative with rigid steroidal structure; or (ii) a multi-tailed hydrophobic structure formed by C14-C22 straight chain or branched chain saturated fatty acid chains covalently linked to the same backbone and number ≥3; or (iii) a hydrophobic group with rigid planar or polycyclic aromatic structure, and can stably insert into the lipid bilayer of biological membrane through hydrophobic interaction.

12. The amphiphilic functional modification molecule of claim 10 or 11, for use in the preparation of the engineered exosome of claim 1 or 3.

13. A method of purifying an exosome surface-modified with an exogenous amphiphilic molecule, characterized by, The method utilizes the critical micelle concentration property of the amphiphilic molecule for purification, comprising the following steps: (1) providing a mixed reaction system containing exosomes and excess amphiphilic molecules, wherein the concentration of the amphiphilic molecules is higher than the critical micelle concentration, and they exist in the form of micelles; (2) thermodynamically driven micelle disassembly: adjusting the physical and chemical environment of the mixed reaction system in step (1) so that the environmental concentration of free amphiphilic molecules not anchored on the surface of the exosome membrane is maintained or reduced to below the critical micelle concentration threshold, thereby breaking the thermodynamic equilibrium of the micelles, inducing the dissociation of free micelles and converting them into monomers or oligomers to expand the hydrodynamic size difference between them and the exosomes; (3) size exclusion separation: based on the size difference between molecules, the exosomes are separated from the small molecule components after disassembly, and purified surface-modified exosomes are obtained.

14. The method of claim 13 for the preparation of the engineered exosome of any one of claims 1 to 9, characterized in that, The amphiphilic molecule is an amphiphilic functional modification molecule of the general structure L-S-T, and before step (1) further comprises: providing an amphiphilic functional modification molecule L-S-T, dispersing it in an aqueous medium to form a micellar solution; mixing the micellar solution with an exosome suspension and incubating at 30-42°C and below the denaturation temperature of the surface proteins of the exosome membrane to transfer L-S-T molecules from micelles to the exosome membrane using thermodynamic driving force.

15. The method of claim 13, wherein, In step (2), the physical and chemical environment adjustment method is selected from one or more of the following groups and combinations thereof: (i) direct dilution method: adding solvent or buffer to the reaction system to reduce the volume molar concentration of free amphiphilic molecules; (ii) medium replacement method: continuously removing free amphiphilic molecules by dialysis, ultrafiltration, tangential flow filtration or elution of the mobile phase during chromatography to maintain their concentration below CMC; (iii) critical point regulation method: by adjusting the temperature, pH or ionic strength of the system, the CMC threshold of the amphiphilic molecule is increased, so that it changes to a monomer state at the current concentration.

16. A lyophilized formulation of an engineered exosome, characterized in that, It is prepared by freeze-drying of a pre-freeze-drying solution containing the following components: (a) the engineered exosome of any one of claims 1 to 9; (b) a non-permeable freeze-drying protective agent selected from sucrose or trehalose at a concentration of 5% ~ 10% (w / v); (c) a membrane stabilizer and a supporting agent selected from human serum albumin or recombinant human serum albumin at a concentration of 1% ~ 5% (w / v); The average particle size change rate of the exosome is less than or equal to 15% after the preparation is reconstituted, and the retention rate of the targeting functional ligand is greater than or equal to 85%; the freeze-dried preparation has a specific microstructure, wherein the engineered exosome particles are uniformly embedded and fixed in a continuous porous network matrix formed by the freeze-drying protective agent and the membrane stabilizer, the porosity of the matrix is between 60% and 90%, and the average pore size is in the range of 0.1 μm to 10 μm.

17. A pharmaceutical composition comprising, in combination, a compound of any one of claims 1-16 and a pharmaceutically acceptable carrier. The engineered exosome of any one of claims 1 to 9 or the freeze-dried preparation of claim 16, and a pharmaceutically acceptable carrier.

18. Use of the engineered exosome of any one of claims 1 to 9 or the freeze-dried preparation of claim 16 or the pharmaceutical composition of claim 17 in the preparation of a drug delivery system for treating an inflammatory disease, an autoimmune disease or a malignant tumor characterized by high expression of VCAM-1.

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