Method for analyzing shedding kinetics of pegylated lipids and use thereof

Abstract

The application discloses a method for analyzing shedding kinetics of polyethylene glycolized lipids and application thereof, and belongs to the technical field of nano-drug analysis and pharmacokinetics. The technical problem to be solved is to provide a non-labeled and high-sensitivity in-vivo shedding kinetics direct analysis method. The technical solution points to a shedding kinetics analysis method, which comprises the following steps: S1, pretreating a biological sample to obtain a corresponding to-be-detected sample; S2, activating and pre-protecting a solid-phase extraction column; S3, mixing the to-be-detected sample with an internal standard working solution and PBS to prepare a sample loading solution, loading and centrifuging, step-by-step selective elution, and obtaining a shedding PEG-lipid component; S4, ultrasonic extraction of the to-be-detected sample to obtain a total PEG-lipid component; S5, detection, obtaining a shedding PEG-lipid concentration and a total PEG-lipid concentration; and S6, calculating a shedding percentage of the PEG-lipid at each time point and drawing a shedding kinetics curve.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine analysis and pharmacokinetics technology, specifically relating to a method for analyzing the shedding kinetics of polyethylene glycol-modified lipids and its application. Background Technology

[0002] Lipid nanocarriers are nanoparticles ranging in size from 10 to 1000 nm, primarily composed of lipids. They are multifunctional drug delivery systems (DDS) that can encapsulate various drugs and selectively target cells / tissues by modifying external components. They can even penetrate cell barriers to achieve highly efficient drug delivery. The most important types of nanocarriers in this category are liposomes and lipid nanoparticles (LNPs), which offer advantages such as protecting drugs from degradation, reducing toxicity, enhancing the solubility and bioavailability of hydrophobic molecules, as well as biocompatibility and biodegradability.

[0003] Polyethylene glycol-modified lipids (PEG-lipids) are a key component of lipid nanoparticles (LNPs) and liposomes. PEG-lipids are anchored to the carrier surface by hydrophobic alkyl chains, while the hydrophilic PEG chains extend outward to form steric hindrance, reducing the carrier's clearance by the mononuclear phagocytic system and prolonging the circulation time.

[0004] The role of PEG-lipids in vivo presents a dilemma, often referred to as the PEG dilemma: on the one hand, the PEG layer must be sufficiently stable to maintain long-term circulation; on the other hand, once the carrier reaches the target tissue or enters the cell, the PEG-lipids need to dissociate and detach from the carrier at an appropriate rate to expose the internal functional lipids, promoting endocytosis and drug release. Too slow detachment hinders the interaction between the carrier and target cells, reducing efficacy; too rapid detachment leads to premature clearance of the carrier. Therefore, the rate and extent of PEG-lipid detachment from the carrier surface over time (i.e., detachment kinetics) is a core parameter determining the carrier delivery efficiency.

[0005] In vivo, PEG-lipids exist in two forms: an "assembled state" anchored to a carrier surface and a "detached state" free in body fluids. Currently, the industry generally faces a technical bottleneck: achieving label-free separation and quantification of these two forms in complex biological matrices is difficult. Existing detection methods have the following problems: (1) Fluorescent labeling method: Exogenous labeling alters the physicochemical properties of PEG-lipids, affecting their natural shedding behavior; (2) Nuclear magnetic resonance method: low sensitivity, unable to detect low concentration samples in vivo; (3) Conventional total analysis method: The sample processing process damages the carrier structure, and the assembled and detached states are mixed together and cannot be distinguished; (4) Physical separation methods such as ultracentrifugation, dialysis, and gel permeation chromatography: have problems such as artificially induced secondary shedding, long time consumption, serious dilution, and large matrix interference.

[0006] Therefore, developing a label-free, highly sensitive direct analysis method for in vivo exfoliation kinetics is key to overcoming the PEG dilemma. Summary of the Invention

[0007] The purpose of this invention is to provide a method for analyzing the shedding kinetics of polyethylene glycol-modified lipids and related technologies, in order to solve the problem that existing technologies are unable to achieve label-free separation and quantification of two forms of PEG-lipids in complex biological matrices, or a combination thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms, the definition provided in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Pharmacopoeia of the People's Republic of China (2025 Edition), Application of Lipid-Based Nanocarriers in Drug Delivery and Diagnostics" by Pakistani scholar Muhannad Raza Shah, translated by Liu Ying, published by Science Press in September 2019 (ISBN 9787030613486)

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as the detection of detached PEG-lipid concentration, the detection of total PEG-lipid concentration, the plotting of standard curves, the calculation of detachment half-life and detachment rate constant, and the detection of intact LNP recovery, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] In this article, the term "lipid nanocarrier" refers to nanoparticles with a lipid backbone and a particle size of 10-1000 nm, including two main categories: lipid nanoparticles and PEGylated liposomes. These nanoparticles can encapsulate nucleic acids and small molecule chemotherapy drugs for in vivo delivery.

[0015] In this article, the term "lipid nanoparticle" (LNP) refers to the mainstream mRNA delivery carrier, which is formed by the co-assembly of cationic lipids, auxiliary lipids, cholesterol, and PEG-lipids.

[0016] In this article, the term "liposome" refers to a vesicle-type nanocarrier formed by a phospholipid bilayer.

[0017] In this article, the term "PEG-lipid" (polyethylene glycol-modified lipid) refers to amphiphilic lipids with a hydrophobic alkyl chain at one end and a hydrophilic PEG long chain at the other end. The hydrophobic end is anchored on the surface of the nanocarrier, and the PEG chain forms steric hindrance to prolong the cycling time. It is divided into two categories: assembled PEG-lipids and detached PEG-lipids.

[0018] In this article, the term "assembled PEG-lipid" refers to PEG-lipids that are stably anchored to the surface of intact LNPs / liposomes through hydrophobic interactions.

[0019] In this article, the term "exfoliated PEG-lipids" refers to PEG-lipids that have detached from the carrier surface and exist freely in the biological matrix.

[0020] In this article, the term "solid phase extraction column" (SPE column) refers to the sample pretreatment separation packing material.

[0021] In this article, the term "complete LNP recovery" refers to a core indicator for evaluating the protection capability of an SPE column, and its calculation formula is: Complete LNP recovery = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0022] In this article, the term "PBS" refers to phosphate buffer solution, a commonly used buffer solution in biological experiments. It is prepared by mixing disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), and other components in a specific ratio, and the pH value is usually maintained at 7.2-7.4.

[0023] In this article, the term "DDVP" refers to O,O-dimethyl-O-(2,2-dichlorovinyl) phosphate, which can act as a potent esterase inhibitor to ensure the stability of the PEG-lipid system.

[0024] In this article, the term "blank test sample" refers to a test sample that does not contain lipid nanocarriers.

[0025] In this article, the term "glucose" refers to a glucose buffer standard solution with a concentration of 10 mmol / L.

[0026] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides: a method for analyzing the shedding kinetics of polyethylene glycol-modified lipids, comprising the following steps: S1. Collect biological samples containing lipid nanocarriers at multiple different sampling time points, preprocess the biological samples, and obtain the corresponding test samples; S2. The solid-phase extraction column is activated and pre-protected to obtain a pretreated solid-phase extraction column; S3. Mix the sample to be tested with the internal standard working solution and PBS to prepare the loading solution. Load the sample onto the pretreated solid phase extraction column, centrifuge, and selectively elute stepwise to obtain the detached PEG-lipid fraction. S4. Take the sample to be tested and extract it by ultrasonic extraction to obtain the total PEG-lipid fraction; S5. The exfoliated PEG-lipid fraction and the total PEG-lipid fraction were detected separately to obtain the concentration of exfoliated PEG-lipid and the concentration of total PEG-lipid. S6. Calculate the percentage of PEG-lipids detached at each time point and plot the detachment kinetic curve; In some embodiments, the lipid nanocarrier described in step S1 includes liposomes or lipid nanoparticles. This invention is universally applicable to the lipid nanocarrier, and preferably contains C. 12 -C 18 Lipid nanocarriers composed of alkyl groups and PEG with a concentration of 550-5000 Da.

[0027] In some implementations, the biological sample described in step S1 includes at least one of plasma, serum, whole blood, tissue homogenate, and intercellular fluid.

[0028] In some implementations, the sampling time points in step S1 are at least 3, preferably 5, 6, 7, 8 or 9.

[0029] In some implementations, the pretreatment described in step S1 involves removing proteins under mild conditions. Specifically, this involves adding 0.5%-2.5% DDVP and 0.8%-1.2% heparin sodium to the biological sample, mixing well, centrifuging, and obtaining the sample to be tested.

[0030] Preferably, the final concentration of DDVP can be selected as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, or a range derived thereof; and the final concentration of heparin sodium is 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, or a range derived thereof.

[0031] Preferably, the centrifugation is performed at 4-10°C with a centrifugation force of 2000-5000 g for 5-10 min; for example, the relative centrifugal force can be selected as 2000 g, 3000 g, 4000 g, 5000 g, or a range derived therefrom; the centrifugation time can be selected as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or a range derived therefrom; more preferably, the centrifugation temperature is 4-8°C, and more preferably 4-6°C.

[0032] In some embodiments, the solid-phase extraction column in step S2 is a polymer column or a C1-C column. 18 The solid-phase extraction column is a reversed-phase silica column and an HLB column; preferably, the solid-phase extraction column is a C1-C8 reversed-phase silica column; more preferably, the solid-phase extraction column is a C1-C4 reversed-phase silica column; even more preferably, the solid-phase extraction column is a C1-C2 reversed-phase silica column.

[0033] In some implementations, the solvents used for activation in step S2 are methanol and water, respectively.

[0034] In some implementations, the preprotection solution used in step S2 is a blank test sample and PBS or a blank test sample and glucose.

[0035] Preferably, the volume fraction of the blank test sample in the pre-protection solution is 5%-9%, for example, the volume fraction can be selected as 5%, 6%, 7%, 8%, 9%, or a range derived therefrom.

[0036] In some embodiments, the internal standard working solution in step S3 is an aqueous solution of an amphiphilic lipid with a concentration of 1-100 μg / mL. For example, the concentration of the aqueous solution of the amphiphilic lipid is 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, or a range derived therefrom. Preferably, the amphiphilic lipid contains C... 12 -C 18 Amphiphilic lipids of alkyl groups and PEG (1000-5000 Da); more preferably DMG-PEG2000 or DOPE-PEG2000.

[0037] In some implementations, the volume ratio of the test sample, internal standard working solution, and PBS in step S3 is 1:0.8-1.2:16-20. For example, the volume ratio can be selected from 1:1:16, 1:1:17, 1:1:18, 1:1:19, 1:1:20, or a range derived therefrom.

[0038] In some embodiments, the centrifugation in step S3 is performed at 500-1500 rpm for 1-5 minutes. For example, the centrifugation speed can be selected from 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm, or a range derived therefrom; the centrifugation time can be selected from 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or a range derived therefrom. Preferably, centrifugation is performed at 700-900 rpm for 1-3 minutes; more preferably, centrifugation is performed at 800 rpm for 2 minutes.

[0039] In some implementations, the stepwise selective elution in step S3 is as follows: the stepwise selective elution in step S3 is as follows: after rinsing with PBS and water in sequence, the product is eluted with isopropanol, the eluent is collected, dried, and reconstituted to obtain the detached PEG-lipid component.

[0040] In some implementations, the stepwise selective elution in step S3 is as follows: after rinsing with glucose and water in sequence, elution is performed with isopropanol, the eluent is collected, dried, and reconstituted to obtain the detached PEG-lipid component.

[0041] Preferably, the drying process in step S3 is carried out by nitrogen blowing.

[0042] Preferably, the resolution solvent in step S3 is a mixture of acetonitrile containing formic acid and water.

[0043] More preferably, the volume ratio of acetonitrile to water in the mixture is 2-4:2, and the volume fraction of formic acid is 0.05%-0.15%. For example, the volume ratio can be selected as 1:1, 3:2, 2:1, or a range derived therefrom; the volume fraction of formic acid can be selected as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, or a range derived therefrom.

[0044] In some implementations, the ultrasonic extraction in step S4 is as follows: after adding internal standard working solution and formic acid methanol solution to the sample to be tested and mixing, ultrasonication is performed for 20-40 min. After mixing twice, solid-liquid separation is performed to obtain the liquid phase, which is the total PEG-lipid component.

[0045] Preferably, the volume percentage of formic acid in the formic acid methanol solution is 0.08%-0.12%. For example, the volume percentage can be selected as 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, or a range derived therefrom.

[0046] Preferably, the volume ratio of the test sample, the internal standard working solution, and the formic acid methanol solution is 1:0.8-1.2:70-90. For example, the volume ratio can be selected as 1:1:70, 1:1:75, 1:1:80, 1:1:85, 1:1:90, or a range derived therefrom.

[0047] In some implementations, the detection method described in step S5 is liquid chromatography-tandem mass spectrometry; the mass spectrometry detection uses source fragmentation combined with multiple reaction monitoring scanning mode to select characteristic ion pairs of intact PEG-lipids; a standard curve is prepared, and quantitative analysis is performed using the ratio of characteristic ion peak area to internal standard peak area.

[0048] Preferably, the detection conditions are: Chromatographic column: C18 column (50×4.6 mm, 5 μm, Zorbax SB300); mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid-isopropanol-acetonitrile; gradient: starting with 40% organic phase, increasing to 90% in 1-5 min, holding for 5 min, then returning to the starting ratio; flow rate: 1 mL / min; column temperature: 40℃; injection: 20 μL.

[0049] Mass spectrometry: Tandem quadrupole mass spectrometry, ESI positive ion mode, ion ejection voltage 5500 V, temperature 500℃. Intrasource fragmentation (IS-CID) and multiple reaction monitoring (MRM) modes were used to select characteristic ion pairs of intact PEG-lipids (e.g., ALC-0159: m / z 177.2→88.9, tolerance ±0.5 Da).

[0050] Preferably, the detection conditions are: Reversed-phase C8 column (50 × 4.6 mm, 5 μm, Zorbax SB300); mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid isopropanol-water (95:5); gradient: starting with 30% organic phase, increasing to 90% in 3-6 min, holding for 3 min, then returning to the initial ratio; flow rate 1 mL / min; column temperature 60℃; injection 20 μL. Mass spectrometry: Tandem quadrupole mass spectrometry, ESI positive ion mode, ion ejection voltage 5500 V, temperature 500℃. Intrasource fragmentation (IS-CID) and multiple reaction monitoring (MRM) modes were used to select characteristic ion pairs of intact PEG-lipids (e.g., DSPE-PEG2000 characteristic ion pair m / z 607.1→71.2, with a tolerance of ± 0.5 Da).

[0051] In some implementations, the formula for calculating the detachment percentage in step S6 is: detachment percentage = detached PEG-lipid concentration / total PEG-lipid concentration × 100%.

[0052] In some implementations, the shedding kinetic curve in step S6 is a shedding percentage-time curve.

[0053] In some implementations, step S6 further includes obtaining the shedding half-life and shedding rate constant based on the shedding kinetics curve.

[0054] Secondly, this invention provides the application of the above-mentioned polyethylene glycol-modified lipid shedding kinetic analysis method in the evaluation of lipid nanocarrier drugs or the development of novel PEG-lipid materials.

[0055] In some implementations, the application includes at least one of the following: (a) Evaluating the stability of lipid nanocarriers in blood circulation; (b) Analyze the effect of PEG-lipid shedding on the efficiency of target cell endocytosis and endosome escape; (c) Predicting the risk of immunogenicity caused by the accumulation of free PEG-lipids; (d) Design PEG-lipid derivatives.

[0056] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred option is the packing material for a solid-phase extraction column. This technical solution addresses the technical problem of separating exfoliated PEG-lipids from assembled PEG-lipids, and further improves the separation selectivity of exfoliated PEG-lipids.

[0057] The second preferred option: pretreatment temperature in biological samples. This technical solution addresses the technical problem of separating exfoliated PEG-lipids from assembled PEG-lipids, and further improves the separation selectivity of exfoliated PEG-lipids.

[0058] The third preferred option is a pre-protected solution in the solid-phase extraction column pretreatment. This technical solution addresses the technical problem of separating exfoliated PEG-lipids from assembled PEG-lipids, and further improves the separation selectivity of exfoliated PEG-lipids.

[0059] The fourth preferred option: the type of eluent. This technical solution, based on solving the technical problem of separating exfoliated PEG-lipids from assembled PEG-lipids, further improves the separation selectivity of exfoliated PEG-lipids.

[0060] The fifth preferred option: the type of eluent. This technical solution, based on solving the technical problem of separating exfoliated PEG-lipids from assembled PEG-lipids, further improves the separation selectivity of exfoliated PEG-lipids.

[0061] The sixth preferred option: the type of anticoagulant. This technical solution, based on solving the technical problem of separating exfoliated PEG-lipids and assembled PEG-lipids, further improves the separation selectivity of exfoliated PEG-lipids.

[0062] Examples 1-9 of this invention at least support the protection scope of "method for analysis of the shedding kinetics of polyethylene glycol-modified lipids".

[0063] The technical feature "method for the detachment kinetics of polyethylene glycol-modified lipids" is summarized from the foregoing explanation and / or the corresponding steps in Examples 1-9. Therefore, those skilled in the art can reasonably infer that the method for the detachment kinetics of polyethylene glycol-modified lipids, its subordinate concepts, its essentially equivalent technical means, and technical means based on existing technology within the scope of conventional and common knowledge should all fall within the protection scope of this invention.

[0064] The beneficial effects of this invention are as follows: Compared with the prior art, the present invention has the following beneficial effects: 1. Label-free direct separation avoids the drawbacks of labeling methods that alter the properties of PEGylation. The entire process requires no exogenous fluorescent or isotopic labeling, preserving the natural physicochemical properties of PEG-lipids. The test results completely reproduce the actual in vivo shedding behavior, solving the industry pain point of fluorescent labeling methods interfering with PEG shedding kinetics.

[0065] 2. Wide range of applications, supporting the entire chain of nanomedicine research and evaluation. This invention can be used for evaluating the blood circulation stability of mRNA-LNP and PEG liposomes, as well as for screening novel detachable PEG-liposome materials, predicting immunogenicity risks, and optimizing carrier structures, covering the entire R&D process from drug formulation screening, in vivo pharmacokinetics, and safety evaluation.

[0066] 3. Multi-matrix compatible, suitable for plasma, whole blood, serum, tissue homogenate, and intercellular fluid. This invention is adaptable to a variety of biological matrices, eliminating the need to develop a complete set of analytical methods for different matrices, and significantly reducing the R&D cost of in vivo kinetic evaluation of nanocarriers. Attached Figure Description

[0067] Figure 1 The standard curve for the detached state of ALC-0159 in Example 1 is shown.

[0068] Figure 2 The standard curve for total ALC-0159 in Example 1.

[0069] Figure 3 This is a graph showing the PEG-lipid concentration-time curve in the plasma sample of rats injected with mRNA-LNP via intramuscular injection, as shown in Example 1.

[0070] Figure 4 Statistical chart of solid phase extraction column screening in Example 2.

[0071] Figure 5 Example 3: Screening statistics on pretreatment temperature in biological samples.

[0072] Figure 6 This is a statistical chart showing the screening of the preprotection solution in the solid-phase extraction column pretreatment in Example 4.

[0073] Figure 7 Statistical chart of the screening of eluent in Example 5.

[0074] Figure 8 Statistical chart of eluent screening in Example 6.

[0075] Figure 9 This is a statistical chart showing the screening of anticoagulants in biological samples in Example 7.

[0076] Figure 10 The standard curve for the detached DSPE-PEG2000 in Example 8 is shown.

[0077] Figure 11 The standard curve for total DSPE-PEG2000 in Example 8.

[0078] Figure 12This is a concentration-time curve of free lipid material and liposome particles in plasma samples after intravenous injection of doxorubicin hydrochloride liposomes into rats in Example 8.

[0079] Figure 13 Statistical chart of solid phase extraction column screening in Example 9.

[0080] Figure 14 Example 10: Statistical chart of pretreatment temperature screening in biological samples.

[0081] Figure 15 Statistical chart of eluent screening in Example 11. Detailed Implementation

[0082] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0083] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in this invention shall be understood to be modified by the word "about" in all cases. When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10%, preferably within 5%, of the specified value or range.

[0084] The present invention will be further described below with reference to specific embodiments. All instruments, devices, equipment, reagents, and products used in the embodiments of the present invention, unless otherwise specified, are obtained through conventional commercial channels. The mRNA-LNP in this embodiment can be a commercially available product or can be prepared in-house. Doxorubicin hydrochloride liposomes (Doxorubicin®, containing DSPE-PEG2000).

[0085] The preparation method of mRNA-LNP is as follows: Ionizable lipids ALC-0315, PEG-lipid ALC-0159, DSPC, and cholesterol were dissolved in anhydrous ethanol at a molar ratio of 50 / 1.5 / 10 / 38.5 to prepare a lipid solution with a total lipid concentration of 10 mg / mL. mRNA-eGFP was dissolved at 0.2 mg / mL in lipid-free citrate buffer (pH 4.5, 50 mM) to obtain an aqueous nucleic acid solution. Using a microfluidic device, the volumetric flow rate of the nucleic acid aqueous solution was controlled at 3:1 to the lipid solution, and the two were mixed at a flow rate of 12 mL / min. The initial product was diluted 5-fold with citrate buffer (pH 4.5, 50 mM). Dialysis with PBS (pH 7.4) for at least 16 h yielded mRNA-LNP.

[0086] Example 1 Methods for analyzing the shedding kinetics of ALC-0159 in mRNA-LNP S1. Sampling and Pretreatment: SD rats were used, and approximately 200 μL of mRNA-LNP was injected intramuscularly. Blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h.

[0087] Add DDVP (final concentration 2wt%, Aladdin, D114153) and heparin sodium (final concentration 1wt%, Aladdin, H758710) to the collected blood sample, mix well, and centrifuge (4℃, 2000 g, 10 min) to separate the plasma, which is the sample to be tested.

[0088] S2. Solid-phase extraction column pretreatment: A C1 solid-phase extraction column (Agilent, 12102004) was selected. After activation with 1 mL of methanol and 1 mL of water, 500 μL of preprotection solution (5% rat blank plasma PBS) was added for preprotection.

[0089] S3, Exfoliated PEG-lipid fraction: Take 25 μL of plasma, add 25 μL of internal standard working solution (10.0 μg / mL DMG-PEG2000, Jiankai, A3294) and 450 μL of PBS, mix well, and load onto a solid-phase extraction column. Centrifuge at 800 rpm for 2 min. Add 2 mL of PBS to wash, centrifuge at 800 rpm for 2 min; then add 2 mL of water to wash, centrifuge, and the assembled PEG-lipid fraction is separated from the plasma at this time. Elute with 2 mL of isopropanol, collect the eluent, dry with nitrogen, and reconstitute with a mixture of 200 μL of acetonitrile and water (3:2, v / v, containing 0.1% formic acid) to obtain the detached PEG-lipid fraction.

[0090] S4. Total PEG-lipid fraction: Take 20 μL of plasma, add 20 μL of internal standard working solution (20.0 μg / mL DMG-PEG2000, Jiankai, A3294) and 1.6 mL of 0.1% formic acid methanol solution, vortex for 1 min, sonicate for 30 min, vortex for 1 min, centrifuge at 13300 rpm for 5 min at 4℃, and take the supernatant, which is the total PEG-lipid fraction, used to determine the total PEG-lipid concentration.

[0091] S5. Concentration detection: The exfoliated PEG-lipid fraction and the total PEG-lipid fraction were detected separately to obtain the concentration of exfoliated PEG-lipid and the concentration of total PEG-lipid. LC-MS / MS conditions: Column: C 18Chromatographic column (50×4.6 mm, 5 μm, Zorbax SB300); Mobile phase A: 0.1% formic acid in water; Mobile phase B: a solution of isopropanol and acetonitrile containing 0.1% formic acid (volume ratio of isopropanol to acetonitrile is 3:1). Gradient: The initial volume percentage of mobile phase B is 40%, which is increased to 90% in 1-5 min, maintained for 5 min, and then reduced to 40%. Flow rate: 1 mL / min; column temperature: 40℃; injection volume: 20 μL.

[0092] Mass spectrometry: Tandem quadrupole mass spectrometry, ESI positive ion mode, ion ejection voltage 5500 V, temperature 500℃. Intrasource fragmentation (IS-CID) and multiple reaction monitoring (MRM) modes were used to select characteristic ion pairs of intact PEG-lipids, specifically: ALC-0159 characteristic ion pair m / z 177.2→88.9, with an allowable deviation of ±0.5 Da; Standard curves for exfoliated ALC-0159 and total ALC-0159 were prepared using blank plasma. Quantification was performed using the ratio of characteristic ion peak area to internal standard peak area. Results are as follows: Figure 1 and Figure 2 As shown.

[0093] S6. Plot the curve: Based on the measured concentrations of exfoliated PEG-lipids and total PEG-lipids in plasma, plot the concentration-time curve of exfoliated PEG-lipids.

[0094] The results are as follows Figure 3 As shown.

[0095] Example 2 Solid phase extraction column screening The preservation capabilities of different solid-phase extraction columns (C1, C2, C8, C18 and HLB) for mRNA-LNP (LNP for short) were investigated.

[0096] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0097] The results are as follows Figure 4 As shown.

[0098] Example 3 Screening of pretreatment temperatures in biological samples The preservation ability of mRNA-LNP (LNP for short) at different centrifugation temperatures (4℃, 8℃, 15℃ and 25℃) in step S1 was investigated.

[0099] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0100] The results are as follows Figure 5 As shown.

[0101] Example 4 Screening of preprotected solutions in solid phase extraction column pretreatment The preservation ability of different preprotection solutions (1% rat blank plasma PBS, 3% rat blank plasma PBS, 5% rat blank plasma PBS, 7% rat blank plasma PBS and 9% rat blank plasma PBS) in step S2 was investigated for mRNA-LNP (LNP).

[0102] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0103] The results are as follows Figure 6 As shown.

[0104] Example 5 Eluent Screening The preservation ability of different rinsing solutions (PBS, water, methanol, acetonitrile and isopropanol, with the same rinsing solution used for both rinsings) on mRNA-LNP (LNP for short) was investigated.

[0105] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0106] The results are as follows Figure 7 As shown.

[0107] Example 6 Elution Buffer Screening The ability of different eluents (isopropanol, ethyl acetate, methanol, acetonitrile, n-hexane and petroleum ether) to preserve exfoliated PEG-lipids was investigated.

[0108] The fraction of detached PEG was calculated according to the detachment kinetic analysis method in Example 1; the recovery rate of free PEG-lipids was calculated according to the formula: Free recovery rate = (fraction of detached PEG measured by SPE) / (fraction of detached PEG from gold standard by ultracentrifugation) × 100%.

[0109] The results are as follows Figure 8 As shown.

[0110] Example 7 Screening of anticoagulants in biological samples The preservation ability of different anticoagulants (sodium heparin, EDTA salt, and citrate) on mRNA-LNP (LNP for short) was investigated.

[0111] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0112] The results are as follows Figure 9 As shown.

[0113] Example 8 kinetic analysis method for the exfoliation of PEG-lipids in liposomes S1. Sampling and Pretreatment: SD rats were collected and intravenously injected with liposomes (doxorubicin hydrochloride liposomes, doxorubicin). ® Approximately 500 μL of blood was collected (containing DSPE-PEG2000). Blood was collected at 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 10, and 24 hours.

[0114] Add DDVP (final concentration 2wt%, Aladdin, D114153) and heparin sodium (final concentration 1wt%, Aladdin, H758710) to the collected blood sample, mix well, and centrifuge (4℃, 2000 g, 10 min) to separate the plasma, which is the sample to be tested.

[0115] S2. Solid-phase extraction column pretreatment: A C1 solid-phase extraction column (Agilent, 12102004) was selected. After activation with 1 mL of methanol and 1 mL of water, 500 μL of preprotection solution (5% rat blank plasma glucose) was added for preprotection.

[0116] S3, Exfoliated PEG-lipid fraction: Take 25 μL of plasma, add 25 μL of internal standard working solution (10.0 μg / mL DOPE-PEG2000, Ruixi Biotechnology, R-DO-0991-1g) and 450 μL of PBS, mix well, and load onto a solid-phase extraction column. Centrifuge at 800 rpm for 2 min. Add 2 mL of glucose and wash, centrifuge at 800 rpm for 2 min; then add 2 mL of water and wash, centrifuge, and the assembled DSPE-lipid fraction is separated from the plasma at this time. Elute with 2 mL of isopropanol, collect the eluent, dry with nitrogen, and reconstitute with 200 μL of a mixture of acetonitrile and water (3:2, v / v, containing 0.1% formic acid) to obtain the exfoliated PEG-lipid fraction.

[0117] S4. Total PEG-lipid fraction: Take 20 μL of plasma, add 20 μL of internal standard working solution (100.0 μg / mL DOPE-PEG2000, Ruixi Biotechnology, R-DO-0991-1 g) and 1.6 mL of 0.1% formic acid methanol solution, vortex for 1 min, sonicate for 30 min, vortex for 1 min, centrifuge at 13300 rpm for 5 min at 4℃, and take the supernatant, which is the total PEG-lipid fraction, used to determine the total PEG-lipid concentration.

[0118] S5. Concentration detection: The exfoliated PEG-lipid fraction and the total PEG-lipid fraction were detected separately to obtain the concentration of exfoliated PEG-lipid and the concentration of total PEG-lipid. LC-MS / MS conditions: Chromatographic column: Reversed-phase C8 column (50×4.6 mm, 5 μm, Zorbax SB300); Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid isopropanol-water (volume ratio 95:5). Gradient: The initial mobile phase B volume percentage is 30%, which is increased to 90% in 3-6 min, maintained for 3 min, and then reduced to 30%. Flow rate: 1 mL / min; column temperature: 60℃; injection volume: 20 μL.

[0119] Mass spectrometry: Tandem quadrupole mass spectrometry, ESI positive ion mode, ion ejection voltage 5500 V, temperature 500℃. Intrasource fragmentation (IS-CID) and multiple reaction monitoring (MRM) modes were used to select characteristic ion pairs of intact PEG-lipids, specifically: DSPE-PEG2000 characteristic ion pair m / z 494.6→298.4, with an allowable deviation of ±0.5 Da; Standard curves for detached DSPE-PEG2000 and total DSPE-PEG2000 were prepared using blank plasma, and quantification was performed by the ratio of characteristic ion peak area to internal standard peak area. Results are as follows: Figure 10 and Figure 11 As shown.

[0120] S6. Plot the curve: Based on the measured concentrations of exfoliated PEG-lipids and total PEG-lipids in plasma, plot the concentration-time curve of exfoliated PEG-lipids.

[0121] The results are as follows Figure 12 As shown.

[0122] Example 9 Solid phase extraction column screening The preservation capabilities of different solid-phase extraction columns (C1, C8 and HLB) for liposomes (LNP) were investigated.

[0123] According to the shedding kinetics analysis method in Example 8, the assembled PEG-lipid component was obtained, and the proportion of detached PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0124] The results are as follows Figure 13 As shown.

[0125] Example 10 Screening of pretreatment temperatures in biological samples The preservation ability of mRNA-LNP (LNP for short) at different centrifugation temperatures (4℃, 10℃ and 25℃) in step S1 was investigated.

[0126] According to the shedding kinetics analysis method in Example 1, the assembled PEG-lipid component was obtained, and the proportion of shedding PEG-lipid in the assembled PEG-lipid component was determined by ultracentrifugation. The recovery rate of LNP is calculated using the following formula: Complete LNP recovery rate = (1 (SPE measured the fraction of exfoliated PEG) / (1) The recovery rate is calculated as follows: (fraction of PEG detached from gold standard by ultracentrifugation) × 100%; the closer the recovery rate is to 100%, the more it indicates that there is no LNP breakage or artificial PEG detachment during the SPE process.

[0127] The results are as follows Figure 14 As shown.

[0128] Example 11 Elution Buffer Screening The preservation capabilities of different eluents (isopropanol, ethyl acetate, methanol, acetonitrile, n-hexane, petroleum ether) for free PEG-lipids were investigated.

[0129] The fraction of detached PEG was calculated according to the detachment kinetic analysis method in Example 1; the recovery rate of free PEG-lipids was calculated according to the formula: Free recovery rate = (fraction of detached PEG measured by SPE) / (fraction of detached PEG from gold standard by ultracentrifugation) × 100%.

[0130] The results are as follows Figure 15 As shown.

[0131] Verification of technical effectiveness and / or analysis of technical problem solving 1. This invention can directly separate without labeling, avoiding the defects of fluorescent labeling and isotope labeling that change the physicochemical properties of PEG, and the detection results completely restore the real in vivo shedding behavior; 2. The combination of LC-MS / MS with intrasource lysis and MRM mode has high sensitivity and can accurately detect extremely low concentration samples in the late 24 hours after drug administration, making up for the lack of sensitivity of nuclear magnetic resonance and gel chromatography. 3. The pretreatment time for a single sample is less than 30 minutes, which is far superior to traditional separation methods such as ultracentrifugation and dialysis, and can achieve batch simultaneous processing of hundreds of pharmacokinetic samples. 4. Multi-matrix compatibility, compatible with plasma, whole blood, serum, tissue homogenate, and intercellular fluid, eliminating the need to develop a complete set of analytical methods for different matrices, thus reducing the R&D cost of in vivo kinetic evaluation of nanocarriers; 5. It has a wide range of applications, including evaluation of carrier blood circulation stability, research on PEG exfoliated cell uptake, prediction of immunogenicity risk of free PEG, and screening of novel exfoliable PEG-lipid molecules, supporting the development of nanomedicine formulations and the entire chain of research and development on in vivo efficacy and safety.

[0132] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for analyzing the shedding kinetics of polyethylene glycol-modified lipids, characterized in that, Includes the following steps: S1. Collect biological samples containing lipid nanocarriers at multiple different sampling time points, preprocess the biological samples, and obtain the corresponding test samples; S2. The solid-phase extraction column is activated and pre-protected to obtain a pretreated solid-phase extraction column; S3. Mix the sample to be tested with the internal standard working solution and PBS to prepare the loading solution. Load the sample onto the pretreated solid phase extraction column, centrifuge, and selectively elute stepwise to obtain the detached PEG-lipid fraction. S4. Take the sample to be tested and extract it by ultrasonic extraction to obtain the total PEG-lipid fraction; S5. The exfoliated PEG-lipid components and total PEG-lipid components were detected separately to obtain the concentrations of exfoliated PEG-lipids and total PEG-lipids. S6. Calculate the percentage of PEG-lipids detached at each time point and plot the detachment kinetic curve; The pretreatment described in step S1 is as follows: add DDVP with a final concentration of 0.5%-2.5% and heparin sodium with a final concentration of 0.8%-1.2% to the biological sample, mix well, and centrifuge at 4-10℃ to obtain the sample to be tested. The solvents used for activation in step S2 are methanol and water, respectively; the pre-protection solution used for pre-protection in step S2 is either a blank test sample and PBS or a blank test sample and glucose. The internal standard working solution mentioned in step S3 is an amphiphilic lipid aqueous solution with a concentration of 1-100 μg / mL.

2. The shedding kinetics analysis method according to claim 1, characterized in that, The biological sample mentioned in step S1 includes at least one of plasma, serum, whole blood, tissue homogenate, and intercellular fluid.

3. The shedding kinetics analysis method according to claim 1, characterized in that, The centrifugation temperature in step S1 is 4-8℃.

4. The shedding kinetics analysis method according to claim 1, characterized in that, The solid-phase extraction column mentioned in step S2 is C1-C 18 Reversed-phase silica gel column and HLB column.

5. The shedding kinetics analysis method according to claim 1, characterized in that, The volume fraction of the blank test sample in the pre-protection solution in step S2 is 5%-9%.

6. The shedding kinetics analysis method according to claim 1, characterized in that, The volume ratio of the test sample, internal standard working solution and PBS in step S3 is 1:0.8-1.2:16-20.

7. The shedding kinetics analysis method according to claim 1, characterized in that, The stepwise selective elution described in step S3 is as follows: after rinsing with PBS and water in sequence, elute with isopropanol, collect the eluent, dry and reconstitute to obtain the detached PEG-lipid component; Alternatively, the mixture can be washed sequentially with glucose and water, then eluted with isopropanol. The eluent can be collected, dried, and reconstituted to obtain the detached PEG-lipid fraction.

8. The shedding kinetics analysis method according to claim 7, characterized in that, The drying in step S3 is nitrogen blowing, and the resolution solvent is a mixture of acetonitrile containing formic acid and water. The volume ratio of acetonitrile to water in the mixture is 2-4:2, and the volume fraction of formic acid is 0.05%-0.15%.

9. The shedding kinetics analysis method according to claim 1, characterized in that, The ultrasonic extraction described in step S4 is as follows: add internal standard working solution and formic acid methanol solution to the sample to be tested, mix well, sonicate for 20-40 min, mix twice, and then separate solid and liquid to obtain the liquid phase, which is the total PEG-lipid component.

10. The shedding kinetics analysis method according to claim 1, characterized in that, The detection method described in step S5 is: liquid chromatography-tandem mass spectrometry, with mass spectrometry detection using source fragmentation combined with multiple reaction monitoring scanning mode, selecting characteristic ion pairs of intact PEG-lipids; Prepare a standard curve and perform quantitative analysis using the ratio of the characteristic ion peak area to the internal standard peak area.

11. The shedding kinetics analysis method according to claim 1, characterized in that, The formula for calculating the detachment percentage in step S6 is: detachment percentage = detached PEG-lipid concentration / total PEG-lipid concentration × 100%.

12. The shedding kinetics analysis method according to claim 1, characterized in that, The shedding kinetic curve mentioned in step S6 is a shedding percentage-time curve.

13. The application of the shedding kinetic analysis method according to any one of claims 1-12 in the evaluation of lipid nanocarrier drugs or the development of novel PEG-lipid materials.

14. The application according to claim 13, characterized in that, Includes at least one of the following: (a) Evaluating the stability of lipid nanocarriers in blood circulation; (b) Analyze the effect of PEG-lipid shedding on the efficiency of target cell endocytosis and endosome escape; (c) Predicting the risk of immunogenicity caused by the accumulation of free PEG-lipids; (d) Design PEG-lipid derivatives.

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