A method for identification and quantification of nanoparticle surface binding to cell membrane receptors

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a method for identifying and quantifying cell membrane receptors combined with nanoparticle surfaces. The method comprises the following steps: fixing the nanoparticle to be detected on the surface of a biosensor; incubating the nanoparticle to be detected fixed on the surface of the biosensor in a cell / cell membrane lysate containing sodium alginate, or first incubating the nanoparticle to be detected with a body fluid such as blood plasma to form a protein corona, and then incubating the protein corona with the cell / cell membrane lysate containing sodium alginate; monitoring the combination of the nanoparticle and the protein by using a biological membrane layer interference technology; eluting the protein; identifying the cell membrane receptor component in the protein; and detecting the affinity between the cell membrane receptor component and the nanoparticle. The method provided by the present application can greatly reduce the non-specific adsorption of the sensor and various impurity components in the lysate by adding a blocking component (sodium alginate) in the cell / cell membrane lysate, thereby reducing the interference and improving the accuracy of the detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a method for identifying and quantifying cell membrane receptors bound to the surface of nanoparticles. BACKGROUND

[0002] Nanomaterials have shown great potential in biomedical fields such as drug delivery, disease diagnosis and treatment, tissue engineering, and vaccine development due to their unique physicochemical properties. However, the interaction mechanisms between nanoparticles and biological systems are complex and diverse, involving multiple levels such as body fluids, organs, cells, and organelles. Among them, the interaction between nanoparticles and cell membrane receptors directly determines the efficiency of nanoparticle uptake by the biological system. Therefore, studying the interaction between nanoparticles and cell membrane receptors is crucial for optimizing the design of nanomaterials and improving their effectiveness and safety in clinical applications.

[0003] Currently, experimental methods for studying the interaction between nanoparticles and cell membrane receptors require specific cell membrane receptor proteins that interact with nanoparticles. Fluorescent labeling and confocal microscopy imaging can confirm the co-localization of nanoparticles and cell membrane receptors. Techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and isothermal titration calorimetry (ITC) can be used to calculate the affinity between nanoparticles and cell membrane receptors.

[0004] For unknown cell membrane receptor proteins that interact with nanoparticles, the mixture after incubation of nanoparticles and cell / cell membrane lysate can be separated by centrifugation or size exclusion chromatography to obtain nanoparticle-protein complexes. Sodium dodecyl sulfate solution is added to denature and strip the proteins on the surface of the nanoparticles, and then the nanoparticles are separated by centrifugation to collect the protein components, which are identified by mass spectrometry. However, for small-sized and low-density nanoparticles, it is difficult to separate and elute them by centrifugation. The size exclusion chromatography method requires a large amount of sample and takes a very long time (several hours for one sample). In addition, identifying cell membrane receptors that interact with nanoparticles can be studied by screening specific receptors involved in nanoparticle uptake using gene screening libraries. However, due to the high cost, time-consuming, and complexity of gene editing tools and screening techniques, this method still has high challenges. Therefore, there is an urgent need to develop more efficient, accurate, in-situ, and real-time monitoring techniques for studying the interaction between nanoparticles and cell membrane receptors.

[0005] In view of the above, the present application is proposed. SUMMARY

[0006] To solve the above technical problems, the present application provides a method for identifying and quantifying cell membrane receptors bound to the surface of nanoparticles.

[0007] Specifically, the technical scheme of the present application is as follows:

[0008] Firstly, the present application provides a method for detecting the interaction between nanoparticles and cell membrane receptors, comprising: fixing the nanoparticles to be detected on the surface of a biosensor; incubating the nanoparticles to be detected fixed on the surface of the biosensor in a solution of cell membrane receptors to be detected; detecting the interaction between the nanoparticles and the cell membrane receptors by using a biological membrane layer interference technology; and the solution of the cell membrane receptors to be detected contains 0.01-1 mg / mL of sodium alginate.

[0009] Preferably, the fixing method is selected from at least one of the following:

[0010] (1) directly adsorbing the nanoparticles based on the carboxyl contained on the surface of the biosensor.

[0011] (2) activating the carboxyl on the surface of the biosensor by using EDC / NHS, fixing the nanoparticles by chemical reaction with the activated carboxyl, and then blocking with ethanolamine.

[0012] (3) fixing the nanoparticles on the surface of the biosensor by electrostatic force, van der Waals force, hydrogen bond, π-π or hydrophobic interaction.

[0013] Preferably, the cell membrane receptors to be detected are configured into a solution with PBS.

[0014] Further preferably, the concentration of the solution of the cell membrane receptors to be detected is preferably 1 nM-5 uM, and the pH is preferably 1-9.

[0015] Preferably, the cell membrane receptors to be detected are prepared into different concentration gradients as analytes to detect the affinity between the nanoparticles and the cell membrane receptors to be detected.

[0016] Further preferably, at least 4 concentration gradients of analytes are configured.

[0017] Preferably, the interaction between the nanoparticle-protein crown complex and the cell membrane receptors can also be studied by using a surface plasmon resonance instrument (SPR) in a continuous sample feeding mode.

[0018] The present application also provides a method for in-situ identification and quantification of the cell membrane receptor components on the surface of nanoparticles, comprising: fixing the nanoparticles to be detected on the surface of a biosensor; incubating the nanoparticles to be detected fixed on the surface of the biosensor in a cell / cell membrane lysate; monitoring the binding between the nanoparticles and the proteins by using a biological membrane layer interference technology, then eluting the proteins by using an eluent, and identifying the cell membrane receptor components by using a mass spectrometry technology; and the cell / cell membrane lysate contains 0.01-1 mg / mL of sodium alginate.

[0019] Preferably, the immobilization method is selected from at least one of the following:

[0020] (1) Direct adsorption of nanoparticles based on carboxyl contained in the biosensor surface.

[0021] (2) Using EDC / NHS to activate the carboxyl of the biosensor surface, fixing the nanoparticles by chemical reaction with the activated carboxyl, and then using ethanolamine for blocking.

[0022] (3) Fixing the nanoparticles on the biosensor surface by electrostatic, van der Waals force, hydrogen bond, π-π or hydrophobic interaction.

[0023] Preferably, the concentration of the cell / cell membrane lysate is 0.1-5 mg / mL.

[0024] Further preferably, the cell lysate is obtained by lysing the cells with NP-40 or RIPA solution, and then taking the supernatant by centrifugation; and / or, the cell membrane lysate is obtained by extracting with GPCR extraction and stabilization reagent, and then taking the supernatant by centrifugation.

[0025] Preferably, the nanomaterial immobilized on the biosensor directly interacts with the cell membrane receptor; or, the nanomaterial immobilized on the biosensor first interacts with the body fluid to form a protein corona, and then interacts with the cell membrane receptor; the dilution multiple of the body fluid is 5-20 times; the body fluid can be selected from plasma and lymph.

[0026] Preferably, the eluent is trifluoroacetic acid with different concentrations; further preferably, the eluent is 0.001%-0.5% trifluoroacetic acid aqueous solution; wherein 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute proteins with weak binding to the surface of the nanomaterial, and 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute proteins with strong binding to the surface of the nanomaterial.

[0027] Preferably, after identifying the cell membrane receptor components by mass spectrometry, the affinity between the cell membrane receptor and the nanoparticles is detected by the above-mentioned method for detecting the interaction between the nanoparticles and the cell membrane receptor. Preferably, the interaction between the nanoparticle-protein corona complex and the cell membrane receptor can also be studied by surface plasmon resonance (SPR) continuous sample injection. Finally, the biological function of the cell membrane receptor is verified by detecting the amount of nanoparticles taken up by the cells after knocking down the cell membrane receptor by flow cytometry.

[0028] Advantages:

[0029] The application provides a method for identifying and quantifying the binding of nanoparticle surface to cell membrane receptors. The method comprises the following steps: fixing the to-be-detected nanoparticle on the surface of a biosensor; incubating the to-be-detected nanoparticle fixed on the surface of the biosensor in a cell / cell membrane lysate; monitoring the binding of the nanoparticle to the protein by using a biological membrane layer interference technology, then eluting the protein by using an eluent, and identifying the cell membrane receptor components in the protein by using a mass spectrometry technology; and finally detecting the interaction between the nanoparticle or the nanoparticle-protein crown complex and the cell membrane receptor by using a biological membrane layer interference and a surface plasmon resonance instrument. The cell / cell membrane lysate contains 0.01-1 mg / mL of sodium alginate. The method provided by the application can greatly reduce the non-specific adsorption of the sensor to various impurity components in the lysate by adding a blocking component (sodium alginate) in the cell / cell membrane lysate, thereby reducing the interference and improving the accuracy of the detection result. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be described below.

[0031] Figure 1 The lipid nanoparticle is fixed by a carboxyl reaction in Example 1 of the application.

[0032] Figure 2 The P of the LNP before and after the reaction of the modification component sodium alginate on the surface of the AR2G sensor 31 NMR spectrum.

[0033] Figure 3 The scanning electron microscope image after the LNP is fixed.

[0034] Figure 4 The gold nanorod is fixed by electrostatic interaction in Example 1 of the application.

[0035] Figure 5 The black phosphorus nanosheet material is fixed by hydrogen bond, van der Waals force, π-π, hydrophobic interaction and the like in Example 1 of the application.

[0036] Figure 6 The biosensor on which the lipid nanoparticle is fixed is incubated with a cell lysate and eluted in Example 2 of the application.

[0037] Figure 7 The biosensor on which the lipid nanoparticle is fixed is first incubated with plasma, then incubated with a cell membrane lysate and eluted in Example 2 of the application.

[0038] Figure 8Effect comparison of different blocking components in Example 3 of the present application for reducing non-specific adsorption of cell lysate to the empty sensor.

[0039] Figure 9 Significant reduction of non-specific adsorption of cell lysate to the empty sensor after adding sodium alginate in Example 3 of the present application.

[0040] Figure 10 Proteomic data results in Example 4 of the present application.

[0041] Figure 11 Detection of affinity of lipid nanoparticles to cell membrane receptors in Example 5 of the present application.

[0042] Figure 12 Detection of interaction of lipid nanoparticle-protein corona complex to cell membrane receptors in Example 6 of the present application.

[0043] Figure 13 Significant reduction of nanoparticle uptake by cells after knocking down cell membrane receptor genes in Example 7 of the present application. DETAILED DESCRIPTION

[0044] The present application provides a method for detecting interaction of nanoparticles to cell membrane receptors, and a method for in situ identification and quantification of cell membrane receptor components on the surface of nanoparticles.

[0045] Firstly, the present application provides a method for detecting interaction of nanoparticles to cell membrane receptors, which comprises: fixing the nanoparticles to be detected on the surface of a biosensor, incubating the nanoparticles to be detected fixed on the surface of the biosensor in a solution of cell membrane receptors to be detected, and detecting the binding of the nanoparticles to the cell membrane receptors by using a biological membrane layer interference (BLI) technology; wherein the solution of the cell receptors to be detected contains 0.01-1 mg / mL of sodium alginate.

[0046] The present application has no special limitation on the type of biosensor, and all biosensors that can be used for biological membrane layer interference technology detection can be used, including commercial or self-made amino propyl silane sensor (APS sensor), second generation amino coupling sensor (AR2G sensor), streptavidin sensor (SA sensor), super streptavidin sensor (SSA sensor), Protein A sensor (ProA sensor), NTA sensor, etc.

[0047] In some embodiments of the present application, the biosensor surface contains carboxyl groups, which can directly adsorb the nanoparticles, or the surface thereof is first activated with EDC / NHS, then the nanoparticles are fixed by chemical reaction with the activated carboxyl groups, and then blocked with ethanolamine, so as to fix the nanoparticles on the biosensor surface. In addition, the nanoparticles can also be fixed on the biosensor surface through electrostatic, hydrogen bond, van der Waals force, π-π, hydrophobic and other interactions.

[0048] The kind of the nanoparticles to be detected is not particularly limited in the present application, and includes inorganic nanomaterials, metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials, organic-inorganic hybrid composite nanomaterials, and various nanodrugs, etc.

[0049] In the above method, the cell membrane receptors to be detected are configured with PBS, and the concentration is preferably 1 nM-5 uM, and the pH is 1-9.

[0050] In the above method, the cell membrane receptors to be detected are respectively configured into different concentration gradients (usually at least 4 concentrations) as analytes, and the affinity of the nanoparticles to the cell membrane receptors to be detected is detected.

[0051] In the above method, the detection by the biological membrane layer interference technology can be realized by a biological membrane layer interference analyzer.

[0052] In the above method, the addition of sodium alginate can greatly reduce the non-specific adsorption of the sensor and the cell receptor solution, thereby reducing the interference and improving the accuracy of the detection results. The concentration of sodium alginate is preferably 0.01-1 mg / mL, and more preferably 0.05 mg / mL.

[0053] Further, the interaction of the nanoparticle-protein crown complex and the cell membrane receptor can also be studied by a surface plasmon resonance instrument (SPR) in a continuous sample feeding mode.

[0054] Further, the present application develops an in-situ identification and quantification method of the cell membrane receptor components on the surface of the nanoparticles based on the biological membrane layer interference technology. Compared with the existing detection methods, the detection method based on the biological membrane layer interference technology of the present application can realize the in-situ enrichment, elution and collection of the cell membrane receptors, and has the advantages of high throughput, small sample consumption, rapidness and real-time detection.

[0055] Specifically, the present application provides a method for in-situ identification and quantification of cell membrane receptor components on the surface of nanoparticles, which comprises: fixing the nanoparticles to be tested on the surface of a biosensor, incubating the nanoparticles to be tested fixed on the surface of the biosensor in a cell / cell membrane lysate, monitoring the binding of the nanoparticles and proteins by using a biological membrane layer interference technology, then eluting the proteins by using an eluent, and identifying the cell membrane receptor components by using a mass spectrometry technology; the cell / cell membrane lysate contains 0.01-1 mg / mL of sodium alginate.

[0056] The cell lysate can be obtained by lysing the cells by using NP-40, RIPA, etc., and then taking the supernatant by using a centrifugal method. The cell membrane lysate can be obtained by extracting the GPCR by using a GPCR extraction and stabilization reagent, and then taking the supernatant by using a centrifugal method. The centrifugal condition is 4°C, 16000 g centrifugation for 20 minutes. The obtained cell / cell membrane lysate is stored at-20°C for no more than 2 weeks.

[0057] Considering that the nanoparticles interact with body fluids such as plasma after entering the organism to form a "protein crown", and then interact with cell membrane receptors, the nanoparticles to be tested can be fixed on the surface of a biosensor, the nanoparticles to be tested fixed on the surface of the biosensor are first incubated in a body fluid such as plasma, and then incubated in a cell / cell membrane lysate, and then the proteins are eluted by using an eluent, and the cell membrane receptor components are identified by using a mass spectrometry technology.

[0058] The concentration of the cell / cell membrane lysate to be tested is preferably 0.1-5 mg / mL, and the dilution multiple of the body fluid such as plasma is 5-20 times.

[0059] The present application has explored the blocking components, and found that the addition of sodium alginate can greatly reduce the non-specific adsorption of the sensor and other impurity components in the cell / cell membrane lysate, when 0.01-1 mg / mL of sodium alginate (preferably 0.05 mg / mL of sodium alginate) is used as a blocking component, the non-specific adsorption of the proteins and the biosensor can be effectively reduced, and the interaction between the nanoparticles and the cell membrane receptors will not be adversely affected, thereby improving the accuracy of the detection results.

[0060] The type of the biosensor is not particularly limited in the present application, and all biosensors that can be used for biological membrane interference technology detection can be used, including commercial or self-made amino propyl silane sensors (APS sensors), second-generation amino coupling sensors (AR2G sensors), streptavidin sensors (SA sensors), super streptavidin sensors (SSA sensors), Protein A sensors (ProA sensors), NTA sensors, etc.

[0061] In some embodiments of the present application, the biosensor surface contains carboxyl groups, which can directly adsorb the nanoparticles, or the surface is first activated with EDC / NHS, then the nanoparticles are fixed by chemical reaction with the activated carboxyl groups, and finally blocked with ethanolamine, so as to fix the nanoparticles on the biosensor surface. In addition, the nanoparticles can also be fixed on the biosensor surface through electrostatic, hydrogen bond, van der Waals force, π-π, hydrophobic and other interactions.

[0062] The kind of the nanoparticles to be detected is not particularly limited in the present application, including inorganic nanomaterials, metal and metal oxide nanomaterials, metal sulfide nanomaterials, organic nanomaterials, organic-inorganic hybrid composite nanomaterials, and various nanodrugs, etc.

[0063] Further, in order to better analyze the proteins bound on the surface of the nanoparticles, the composition and concentration of the eluent for eluting the proteins are explored in the present application. Preferably, the eluent is 0.001%-0.5% trifluoroacetic acid aqueous solution, wherein 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute the proteins (soft protein crown) weakly bound on the surface of the nanomaterials, and 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute the proteins (hard protein crown) strongly bound on the surface of the nanomaterials.

[0064] It is found in the present application that the use of the above eluents with different concentrations can respectively simply and efficiently elute the proteins on the outer layer and the inner layer of the nanoparticles, so as to realize the relatively accurate differentiation and identification of the soft protein crown and the hard protein crown.

[0065] During the elution process, the protein solution eluted is collected for subsequent mass spectrometric identification of the protein components.

[0066] For the biological sample solution to be detected, the concentration of total proteins is preferably 0.1-10 mg / mL, and the pH is 1-9.

[0067] Controlling the concentration of total proteins in the biological sample solution within the above range is beneficial to further reduce non-specific adsorption.

[0068] In the above method, the time for co-incubation of the biosensor with the nanoparticles fixed thereon and the biological sample solution is determined according to the real-time dynamic monitoring of the bound proteins by the biofilm layer interference technology, and the incubation time is determined according to the monitoring results.

[0069] Generally, the incubation time can be from 1 second to 7 days, and from the perspective of protein crown formation time and protein stability, the incubation time is preferably 1 second to 48 hours.

[0070] In the above method, the temperature of the incubation can be adjusted from 6-40℃. Preferably, the temperature of the incubation is 37℃, which is consistent with the temperature of the human body.

[0071] During the incubation, in order to make the biological sample fully contact with the nanoparticles, the sample plate can be set to a vibration mode, and the vibration speed can be adjusted from 0-5000 rpm, preferably, the vibration speed is 500-2000 rpm.

[0072] In the above method, for the mass spectrometric identification of the protein corona components, preferably, the LC-MS / MS method is used. Before the mass spectrometric identification, the collected protein corona elution solution usually needs to be concentrated and solution exchanged. The concentration and solution exchange methods include but are not limited to the use of ultrafiltration tubes, desalting columns, freeze-drying, polyacrylamide gel electrophoresis (SDS-PAGE), etc.

[0073] Preferably, the above-mentioned in-situ identification and quantification method of the cell membrane receptor components on the surface of the nanoparticles further comprises: after the cell membrane receptor components are identified by using the mass spectrometry technology, the above-mentioned method for detecting the interaction between the nanoparticles and the cell membrane receptors is used to prepare the cell membrane receptor proteins to be detected for the affinity, the affinity between the cell membrane receptor components and the nanoparticles is detected by using the biological membrane layer interference technology, and the interaction between the nanoparticle-protein corona complex and the cell membrane receptor can also be studied by using the surface plasmon resonance instrument (SPR) in a continuous sample feeding mode, so that the combination of the nanoparticles and the cell membrane receptors is verified and evaluated.

[0074] The detection method provided by the present application has the advantages of high throughput and small sample consumption, fills the gap of the real-time dynamic, in-situ detection and high-efficiency and rapid separation method of the nanoparticles and the cell membrane receptors, has a very broad application prospect, and provides an effective method for further exploring the interaction (including cell recognition, cell uptake, and cytotoxicity) between the nanoparticles and the organisms and the rational design of functional nanodrugs.

[0075] In the detection method of the present application, the technical advantage of high throughput is achieved, and at least 8 groups of samples can be analyzed simultaneously, thereby improving the detection efficiency.

[0076] The detection method of the present application can be applied to complex biological samples, including cells and cell membrane lysates, and the sample consumption is small.

[0077] The detection method of the present application also has the advantages of simple and easy operation, rapidity and convenience, and strong popularization.

[0078] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0079] The endpoints of the ranges and any values disclosed in the specification are not limited to the precise values recited. The endpoints of the ranges and the values are approximations that are used in the specification to enable the reader to understand the scope of the disclosure. Any range of values, whether explicit or implicit, in the specification, defines the outer limits of a range to include all values between the upper and lower limits whether addressed or unaddressed. Any numerical value, however, inherently contains certain errors necessarily resulting from the round-off or measurement of its components. Also, the endpoints of the ranges are included in the ranges themselves.

[0080] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "specific embodiments", or "some specific embodiments" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0081] In the embodiments provided in the present specification, the specific techniques or conditions not specified are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used without specifying the manufacturer are conventional products that can be purchased through regular channels.

[0082] Embodiment 1

[0083] The present embodiment provides three methods for fixing nanomaterials on a biosensor.

[0084] (1) Fixing lipid nanoparticles by carboxyl reaction.

[0085] Lipid nanoparticles (LNPs) were prepared using microfluidic systems or ultrasound. The AR2G biosensor was immersed in a pre-wetted plate for 10 minutes, then activated for 300 seconds with a 1:1 mixture of 100 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). LNPs were prepared at a concentration of 0.1 mg / mL using 10 mM sodium acetate at pH 4.5. The AR2G biosensor was then immersed in the LNP solution for 300 seconds. Finally, it was blocked for 300 seconds with a 1 M ethanolamine aqueous solution at pH 8.5. Figure 1 The process of immobilizing LNPs on a biosensor is shown. Figure 2 It is the P before and after the reaction of LNP with sodium alginate. 31 NMR spectrum. The AR2G sensor surface component is sodium alginate, which has many carboxyl groups. (The last part, "P," appears to be unrelated and likely refers to a different topic.) 31 The changes in the NMR spectrum prove that the P element on the LNP reacted chemically with the AR2G sensor, that is, the phosphate ion underwent an esterification reaction with sodium alginate. Figure 3 This is a scanning electron microscope image after the LNP was fixed. The results show that the LNP remained uniform and well dispersed after being fixed on the sensor.

[0086] (2) Gold nanorod materials are fixed by electrostatic and other interactions.

[0087] Trimethylhexadecylammonium bromide-modified gold nanorods (product number YM-NM-002) were purchased from Changsha Yimo Biotechnology Co., Ltd. The AR2G biosensor was immersed in a pre-wetted plate for 10 minutes. Gold nanorod nanomaterials were prepared using ultrapure water at a concentration of 20 ug / mL. The AR2G biosensor was immersed in ultrapure water for 60 seconds, then in the gold nanorod solution for 200 seconds. Finally, the AR2G biosensor was immersed in ultrapure water to wash away unbound gold nanorods. Figure 4 The process of immobilizing gold nanorods on a biosensor is shown.

[0088] (3) Black phosphorus nanosheets are fixed by interactions such as hydrogen bonds, van der Waals forces, π-π, and hydrophobicity.

[0089] Black phosphorus nanosheet dispersion (CAS No.: 7723-14-0) was purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. The SA biosensor was immersed in a pre-wetted plate for 10 minutes. Black phosphorus nanosheet material was prepared with water at a concentration of 50 ug / mL. The SA biosensor was first immersed in water for 60 seconds, then immersed in the black phosphorus nanosheet solution for 200 seconds, and finally immersed in water again to wash away unbound black phosphorus nanosheet material. Figure 5The process of immobilizing black phosphorus nanosheets on a biosensor is shown.

[0090] Example 2

[0091] The present example provides a method for detecting the interaction of lipid nanoparticles with cells / cell membrane lysate based on biofilm layer interferometry.

[0092] The method provided in the present example specifically comprises the following steps:

[0093] 1) Immobilization of lipid nanoparticles on a biosensor:

[0094] LNP nanoparticles were immobilized on the AR2G biosensor surface following the method of Example 1 (1) (AR2G biosensor surface with immobilized LNP nanoparticles). Figure 1 ).

[0095] 2) Extraction of cell membrane proteins:

[0096] Neutrophils (HL-60; 1 x 10 7 ) were washed twice with ice-cold PBS and then 1 mL of cold GPCR extraction and stabilization reagent (Cat. No. A43436) was added to the cell pellet. After obtaining a homogenized cell suspension using a Dounce homogenizer (25-50 strokes), the cells were incubated at 4°C for 60 minutes with continuous mixing. The supernatant was then collected as the cell membrane lysate after centrifugation at 16000 g for 20 minutes at 4°C.

[0097] 3) Incubation of the biosensor with cell lysate:

[0098] As shown in Figure 6 , the AR2G biosensor with immobilized LNP was immersed in a 0.05 mg / mL aqueous sodium alginate solution for 60 seconds and then incubated in the cell membrane lysate for 1800 seconds, where the cell membrane lysate concentration was 1 mg / mL containing 0.05 mg / mL sodium alginate. Then 0.005% and 0.5% trifluoroacetic acid were used to elute the soft and hard protein crowns bound on the surface of LNP, respectively.

[0099] In addition, the AR2G biosensor with immobilized LNP can also be pre-incubated with plasma before being combined with the cell / cell membrane lysate. Specifically, as shown in Figure 7As shown, the AR2G biosensor fixed with LNP was immersed in 0.05 mg / mL aqueous sodium alginate solution for 60 s, then incubated in 10% plasma (containing 0.05 mg / mL sodium alginate) for 120 s, and then incubated with cell membrane lysate for 120 s, wherein the cell membrane lysate had a concentration of 1 mg / mL and contained 0.05 mg / mL sodium alginate. Then, 0.001% and 0.5% trifluoroacetic acid were used to elute, respectively, to obtain the soft protein crown and the hard protein crown bound to the surface of the LNP.

[0100] 4) The collected protein sample was subjected to protein spectrum detection:

[0101] The composition of the soft protein crown and the hard protein crown eluted by gradient were identified by LC-MS / MS mass spectrum, respectively.

[0102] Example 3

[0103] The present example provides a method for reducing non-specific adsorption of cell lysate to an empty sensor, which is achieved by adding sodium alginate.

[0104] First, the effects of different chemical components on reducing non-specific adsorption of cell lysate to an empty sensor were compared. As shown in Figure 8 (1) is the non-specific adsorption of cell lysate itself to the AR2G sensor; (2) 0.05% Tween 20 is added to the cell lysate; (3) 0.5 mg / mL polyethylene glycol (molecular weight 2000 Da) is added to the cell lysate; none of them can reduce the non-specific adsorption of the cell lysate itself to the AR2G sensor; (4) 0.01 mg / mL sodium alginate is added to the cell lysate, which can effectively reduce the non-specific adsorption of the cell lysate to the AR2G sensor.

[0105] The present application further optimizes the concentration of sodium alginate. As shown in Figure 9 (1) the cell lysate itself has a certain non-specific adsorption to the empty AR2G sensor; (2) 0.001 mg / mL sodium alginate is added to the cell lysate; (3) 0.01 mg / mL sodium alginate is added to the cell lysate; (4) 0.05 mg / mL sodium alginate is added to the cell lysate; as the concentration of sodium alginate increases, the non-specific adsorption becomes smaller and smaller, and the effect of 0.05 mg / mL sodium alginate is the best.

[0106] Example 4

[0107] The present example detects and identifies the cell membrane receptors on the surface of the nanoparticles.

[0108] The protein sample collected in Example 2 was subjected to protein spectrum detection. The experimental results are as follows: Figure 10As shown, by plotting p-value against logFc, the biological function of the binding protein was identified as cell membrane receptor CSF2RB.

[0109] Example 5

[0110] This example provides a method and results for verifying the affinity of nanoparticle binding to cell membrane receptor proteins.

[0111] To verify the binding of cell membrane receptors to nanoparticles, the affinity of LNP to cell membrane receptor CSF2RB was detected using a bio-layer interferometry analyzer, comprising the following steps:

[0112] 1) Fix LNP on a biosensor:

[0113] According to the method of Example 1 (1), LNP nanoparticles were fixed on the surface of an AR2G biosensor (GE Healthcare, USA) Figure 1 ).

[0114] 2) Prepare cell membrane receptor CSF2RB solution

[0115] Cell membrane receptor CSF2RB was prepared in PBS at different concentration gradients: 50 nM, 100 nM, 200 nM, 400 nM. 0.05 mg / mL sodium alginate was added to PBS.

[0116] 3) Detect the affinity of LNP to CSF2RB using a bio-layer interferometry analyzer:

[0117] The affinity of LNP to CSF2RB was detected using a bio-layer interferometry analyzer with a double subtraction method. The results are shown in Figure 11 LNP binding to CSF2RB has obvious kinetic characteristics and strong binding ability, with an affinity K D of 20 nM and R 2 of 0.9917.

[0118] Example 6

[0119] This example provides a method and results for detecting the interaction of nanoparticle-complexes with cell membrane receptor proteins.

[0120] First, the lipid nanoparticles are immobilized on the surface plasmon resonance instrument CM5 chip by carboxyl reaction. Specifically, the CM5 chip is activated for 1200 seconds using a 1:1 mixture of 100 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). The LNP is configured with 10 mM sodium acetate at pH 4.5, with a concentration of 0.1 mg / mL, and is injected for 300 seconds. Then, 1 M aqueous ethanolamine at pH 8.5 is used for blocking for 1200 seconds.

[0121] Then, by the method of continuous injection, the chip surface is first flowed through 200 nM of the protein corona component anti-PEG-IgG, and then flowed through 200 nM of the cell membrane receptor CSF2RB. The surface plasmon resonance instrument is used to detect the binding of the lipid nanoparticles to the cell membrane receptor CSF2RB after the lipid nanoparticles bind to the protein corona component anti-PEG-IgG to form a protein corona. Figure 12 )。

[0122] Example 7

[0123] This example provides the influence of cells with knocked down cell membrane receptor genes on the uptake of nanoparticles, to verify the biological function of the cell membrane receptor.

[0124] The HL-60 cells are subjected to cell membrane receptor CSF2RB gene knockdown. Then, DiR dye-labeled lipid nanoparticles are prepared. The DiR dye-labeled lipid nanoparticles are incubated with the HL-60 cells for 1.5 hours, and then the uptake of the lipid nanoparticles by the HL-60 cells is detected by flow cytometry. Figure 13 As shown in FIG. 8, the HL-60 cells with the CSF2RB gene knocked down have a significantly reduced uptake of the lipid nanoparticles compared to the wild-type HL-60 cells, indicating that the cell membrane receptor CSF2RB does play an important role in the cellular uptake of the lipid nanoparticles.

[0125] In summary, the method of the present application can be used to quickly and conveniently detect, identify and verify the cell membrane receptor proteins bound to the surface of the nanoparticles.

[0126] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for in situ identification and quantification of surface membrane receptor components of nanoparticles, characterized in that, The application relates to a method for detecting the interaction between a nanomaterial and a cell membrane receptor. The method comprises the following steps: fixing the nanomaterial to be detected on a biosensor surface; placing the nanomaterial to be detected fixed on the biosensor surface in a cell / cell membrane lysate for incubation; monitoring the combination of the nanomaterial and the protein by using a biological membrane layer interference technology, then eluting the protein by using an eluent, and identifying the cell membrane receptor component by using a mass spectrometry technology; the cell / cell membrane lysate contains 0.01-1 mg / mL of sodium alginate; the concentration of the cell / cell membrane lysate is 0.1-5 mg / mL. The fixing method is selected from at least one of the following methods:

2. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 1, characterized in that, (1) directly adsorbing the nanomaterial based on the carboxyl contained in the biosensor surface; (2) activating the carboxyl of the biosensor surface by using EDC / NHS, fixing the nanomaterial by chemically reacting with the activated carboxyl, and then blocking by using ethanolamine; (3) fixing the nanomaterial on the biosensor surface by electrostatic force, van der Waals force, hydrogen bond, pi-pi or hydrophobic interaction. The cell lysate is obtained by lysing the cells by using NP-40 or RIPA solution, and then taking the supernatant by using a centrifugal method; and / or, the cell membrane lysate is obtained by extracting the GPCR by using a GPCR extraction and stabilization reagent, and then taking the supernatant by using a centrifugal method.

3. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 1 or 2, characterized in that, The nanomaterial fixed on the biosensor directly interacts with the cell membrane receptor; or the nanomaterial fixed on the biosensor first interacts with a body fluid to form a protein crown, and then interacts with the cell membrane receptor; the dilution multiple of the body fluid is 5-20; the body fluid is selected from plasma, lymph.

4. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 1, characterized in that, The eluent is trifluoroacetic acid with different concentrations.

5. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 1, characterized in that, The eluent is 0.001%-0.5% trifluoroacetic acid aqueous solution; wherein 0.001-0.1% trifluoroacetic acid aqueous solution is used to elute the protein with weak binding on the surface of the nanomaterial, and 0.1-0.5% trifluoroacetic acid aqueous solution is used to elute the protein with strong binding on the surface of the nanomaterial.

6. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 5, characterized in that, After identifying the cell membrane receptor component by using the mass spectrometry technology, the affinity between the cell membrane receptor and the nanomaterial is detected by using a method for detecting the interaction between the nanomaterial and the cell membrane receptor.

7. The method for in situ identification and quantification of surface cell membrane receptor components of nanoparticles according to claim 1, characterized in that, The method for detecting the interaction between the nanomaterial and the cell membrane receptor comprises the following steps: fixing the nanomaterial to be detected on a biosensor surface; placing the nanomaterial to be detected fixed on the biosensor surface in a cell membrane receptor solution to be detected for incubation; and detecting the interaction between the nanomaterial and the cell membrane receptor by using a biological membrane layer interference technology; the cell membrane receptor solution to be detected contains 0.01-1 mg / mL of sodium alginate.

8. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 7, characterized in that, The fixing method in the method for detecting the interaction between the nanomaterial and the cell membrane receptor is selected from at least one of the following methods:

9. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 8, characterized in that, (1) directly adsorbing the nanomaterial based on the carboxyl contained in the biosensor surface; (2) activating the carboxyl of the biosensor surface by using EDC / NHS, fixing the nanomaterial by chemically reacting with the activated carboxyl, and then blocking by using ethanolamine; (3) fixing the nanomaterial on the biosensor surface by electrostatic force, van der Waals force, hydrogen bond, pi-pi or hydrophobic interaction. The cell membrane receptor to be detected is configured into a solution by using PBS.

10. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 8 or 9, characterized in that, ​ 11. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 10, characterized in that, The concentration of the cell membrane receptor solution to be tested is 1 nM-5 uM, and the pH is 1-9.

12. The method of in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 8, characterized in that, The cell membrane receptor to be tested is prepared into different concentration gradients as analytes, and the affinity of the nanoparticles to the cell membrane receptor to be tested is detected.

13. The method for in situ identification and quantification of the surface cell membrane receptor components of nanoparticles according to claim 12, characterized in that, At least 4 concentration gradients of analytes are configured.

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