A method for detecting the number and density of surface modified molecules of a nano drug delivery carrier

Abstract

The present invention provides a method for measuring the density of surface modified molecules of a nano drug delivery carrier, characterized in that the measuring method comprises: (1) preparing a fluorescent marker of a nano delivery carrier; (2) measuring the particle size distribution and surface area of ​​a nano delivery carrier sample; (3) calculating the number of surface modified molecules of the nano delivery carrier sample; and (4) calculating the density of surface modified molecules of the nano delivery carrier. The method of the present invention is accurate, efficient, easy to operate, requires a small amount of sample, and can analyze the number and density of surface modified molecules of a nano drug delivery carrier from a single particle level.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for detecting the number and density of surface modified molecules of a nano drug delivery carrier. Background Art

[0002] Nanomedicine has the following advantages due to the scale effect of nanostructure: (1) increase the solubility of drugs, improve drug absorption, or eliminate inter-individual differences; (2) improve the in vivo and in vitro stability of drugs, or improve the dissolution or release behavior of drugs by encapsulating or compounding drugs; (3) improve the selectivity of drugs for tissues, organs or cells, improve drug efficacy and / or reduce drug adverse reactions; (4) change the final formulation form, storage conditions or administration method of drugs, reduce storage and transportation costs, and improve the convenience of drug production and use. Functional modification on the surface of nanomedicine may improve its biocompatibility, increase the circulation time in the body, and achieve targeted delivery. Using appropriate characterization techniques to analyze the surface structure of nanomedicine can provide evaluation information. Related research methods include X-ray photoelectron spectroscopy (XPS), X-ray energy dispersive spectroscopy (EDS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), nuclear magnetic resonance (NMR), etc. The above methods have the disadvantages of being cumbersome to operate and highly equipment-dependent, and these methods can only obtain the average number of modified molecules on each nanocarrier, and cannot fully reflect the distribution of the number of modified molecules on the surface of nano drug delivery carriers and the heterogeneity between them.

[0003] Therefore, there is still an urgent need for a method that is accurate, efficient, easy to operate, requires little sample, and can analyze the number and density of surface modified molecules on nano-drug delivery carriers at the single particle level. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for measuring the density of surface modified molecules of a nano drug delivery carrier, the measuring method comprising:

[0005] (1) preparing a fluorescent marker of a nano drug delivery carrier: mixing the nano drug delivery carrier with a molecular capture agent labeled with an optical reagent having a known number of fluorescent molecules n to prepare a fluorescent marker of the nano drug delivery carrier;

[0006] (2) measuring the surface area of ​​a single particle of a nano drug delivery carrier sample; the nano drug delivery carrier sample comprises a nano drug delivery carrier and / or a fluorescent marker of the nano drug delivery carrier;

[0007] (3) Calculating the number of molecules modified on the surface of the nano drug delivery carrier: Based on the fluorescence intensity of the fluorescent quantitative microspheres and the number of fluorescent molecules, a linear relationship between the number of fluorescent molecules and the fluorescence intensity is obtained. Combined with the known number of fluorescent molecules of each optical agent molecule on the surface of the nano drug delivery carrier after labeling, the number of fluorescent molecules on the surface of the fluorescent marker of the nano drug delivery carrier is obtained. The number of molecules modified on the surface of the nano drug delivery carrier is then calculated based on the number of molecules modified on the surface of the nano drug delivery carrier = the number of fluorescent molecules on the surface of the fluorescent marker of the nano drug delivery carrier θ / the number of fluorescent molecules n;

[0008] (4) Based on the single particle surface area of ​​the nanodrug delivery carrier sample obtained in step (2) and the number of surface modified molecules of the nanodrug delivery carrier obtained in step (3), the surface modified molecule density of the nanodrug delivery carrier is calculated according to density = number of surface modified molecules of the nanodrug delivery carrier / single particle surface area of ​​the nanodrug delivery carrier sample.

[0009] In some embodiments, in step (1), a molecular capture agent labeled with an optical agent containing a known number of fluorescent molecules is mixed with a nano drug delivery carrier, so that the optical agent specifically binds to the modified molecules on the surface of the nano drug delivery carrier to obtain a fluorescent marker of the nano drug delivery carrier.

[0010] In some embodiments, the molecular capture agent labeled with an optical agent containing a known number of fluorescent molecules is a capture agent that can bind to the surface modification molecules of the nano drug delivery carrier, so that the optical agent and the surface modification molecules of the nano drug delivery carrier are specifically bound through the capture agent.

[0011] In some embodiments, step (2) includes: preparing a particle size standard solution of particle size standards of different particle sizes; using a flow particle detection device to detect the particle size standard solution, recording the scattered light channel detection data and / or the fluorescence channel detection data of the particle size standard solution, and fitting a curve based on the scattered light channel detection data and / or the fluorescence channel detection data and the particle size value of the particle size standard; measuring the particle size distribution of the scattered light channel detection data and / or the fluorescence channel detection data of the nanodelivery carrier sample based on the fitting curve, and recording the particle size D of the nanodrug delivery carrier sample; according to the particle size of the nanodrug delivery carrier sample, calculating the surface area S=π(D)2 of the nanodelivery carrier sample with a positive scattering and / or fluorescence signal.

[0012] In some embodiments, step (3) comprises:

[0013] (3.1) using a flow particle detection device to detect the fluorescence intensity γ of the fluorescent marker of the labeled nano drug delivery carrier;

[0014] (3.2) using the flow particle detection device described in (3.1) and the same fluorescence signal collection conditions to detect the fluorescence intensity f of the fluorescence quantitative microspheres, and to establish a linear relationship between the fluorescence intensity f and the number of fluorescent molecules; the fluorescence quantitative microspheres are fluorescent microspheres whose number of fluorescent molecules m is known;

[0015] (3.3) According to the linear relationship described in step (3.2), based on the fluorescent marker and fluorescence intensity data of the nano drug delivery carrier, the number of fluorescent molecules on the surface of the fluorescent marker of the nano drug delivery carrier θ is calculated; and then the number of modified molecules on the surface of the nano drug delivery carrier Z = θ / n is calculated.

[0016] In some embodiments, the optical agent on the fluorescent marker of the nanodelivery carrier is the same as or different from the optical agent on the standard fluorescent microsphere.

[0017] In some embodiments, step 4) calculates the surface modification molecular density of the nanodelivery carrier as ρ = Z / π(D) 2 .

[0018] The fluorescence intensity f is linearly related to the number of fluorescent molecules, wherein the number of fluorescent molecules can be expressed by the value of equivalent reference fluorescence (ERF) or the value of equivalent soluble fluorescent molecules (MESF). The fluorescent quantitative microspheres expressed by the MESF value contain the same fluorescent molecules as the fluorescent markers to be tested. The fluorescent quantitative microspheres expressed by the ERF value contain different fluorescent molecules from the fluorescent markers to be tested. The number of fluorescent molecules in the microspheres is converted to the number of fluorescent molecules on the corresponding fluorescent markers after optical measurement and calculation. The method of using fluorescent quantitative microspheres is to mix fluorescent quantitative microspheres coated with different numbers of molecules to form mixed fluorescent quantitative microspheres containing different numbers of fluorescent molecules and detect and calculate the excitation light source and fluorescent channel used by the fluorescent molecules. For example, if the fluorescent marker to be tested contains AF488, and the fluorescent quantitative microspheres labeled with AF488 dye are used, in the FITC fluorescent channel, the average fluorescence intensity (x) and the MESF value (y) of each mixed fluorescent quantitative microsphere are linearly regressed, and the formula y=a+bx is obtained. For example, if the fluorescent marker to be tested contains AF647, and the fluorescent quantitative microspheres labeled with CY5 dye are used and the corresponding number of AF647 molecules is known, in the APC fluorescent channel, the average fluorescence intensity (x') and the ERF value (y') of each mixed fluorescent quantitative microsphere are linearly regressed, and the formula y'=a+bx' is obtained.

[0019] In some embodiments, the flow-type particle detection device is a particle analysis detection device capable of achieving directional flow of a sample flow; and / or

[0020] The directional fluid system consists of a loading unit and a flow unit; and / or

[0021] The particle analysis and detection equipment comprises an optical system and a particle detector; and / or

[0022] The particle detector is composed of a photoelectric sensor and a signal conditioning circuit with a band-limiting filtering high-frequency noise function.

[0023] In some embodiments, the molecular capture agent comprises at least one selected from polypeptides, proteins, antibodies, ligands and / or receptors, nucleic acid aptamers, enzymes, growth factors, glycolipids, polysaccharides, nucleic acids, or a combination thereof.

[0024] In some embodiments, the molecular capture agent is at least one of an antibody, an antigen, a receptor, a secondary antibody, a ligand, or a combination thereof.

[0025] The molecular capture agent is a capture agent that can bind to the surface modification molecules of the nano drug delivery carrier. Specifically, if the surface modification molecules are antigens, the molecular capture agent is an antibody. In addition, for example, the relationship between several more representative molecular capture agents and the surface modification molecules of the nano drug delivery carrier is: primary antibody-secondary antibody; enzyme-substrate; enzyme-inhibitor; hormone-receptor; ligand-receptor; electron donor-electron acceptor; protein-aptamer, etc.

[0026] By mixing and incubating the nano drug delivery carrier with a molecular capture agent labeled with an optical agent having a known number of fluorescent molecules n, the optical agent can specifically bind to the surface modification molecules of the nano drug delivery carrier through the capture agent. In some embodiments, the nano drug delivery carrier includes a viral vector and a non-viral vector.

[0027] In some embodiments, the viral vector includes a lentiviral vector, a retroviral vector, a herpes simplex virus vector, a recombinant coronavirus vector, an adenoviral vector, an adeno-associated virus vector, and the like.

[0028] In some embodiments, the non-viral vectors include exosomes, extracellular vesicles, lipid nanoparticles, liposomes, mRNA vaccines, cationic polymers, cationic lipids, inorganic nanoparticles, nucleic acid conjugates, etc.

[0029] In some embodiments, the optical agent includes at least one of organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, and Polymer Dots, or a combination thereof.

[0030] In some embodiments, the present invention relates to step (3.2) in which other collection conditions other than the same fluorescence signal collection conditions when using a flow particle detection device for detection can be the same or different. For example, the certain injection pressure used in detecting fluorescent quantitative microspheres in step (3.2) can be the same as or different from the certain injection pressure used in the fluorescent marker of the nanodrug delivery carrier in step (3.1).

[0031] Terminology

[0032] Certain embodiments of the present invention are now described in detail. The present invention is intended to cover all substitutions, modifications and equivalent technical solutions, which are all included in the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the combined documents, patents and similar materials are different from or contradictory to the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0033] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0034] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0035] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0036] In the following, all numbers disclosed herein are approximate, regardless of whether the words "about" or "approximately" are used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number having a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% is explicitly disclosed, where "+ / -" means plus or minus.

[0037] The “particle size standard solution” is a microsphere particle of known particle size, which may or may not contain one or more fluorescent dyes on its surface and / or inside.

[0038] "Fluorescence quantitative microspheres" are microspheres with a known number of fluorescent molecules on their surface or inside, including Equivalent Reference Fluorophores (ERF) microspheres or Equivalent Soluble Fluorochrome (MESF) microspheres. The microspheres can be commercially provided with a calibrated number of fluorescent molecules or microspheres whose fluorescent molecules are obtained by optical measurement and calculation.

[0039] An "optical reagent with a known number of fluorescent molecules" can be a commercially available optical reagent with a calibrated number of fluorescent molecules or an optical reagent whose number of fluorescent molecules is obtained by calculation through optical measurement.

[0040] The term "aptamer" refers to a nucleic acid molecule that has been engineered to bind to a target molecule by repeated cycles of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment). An aptamer can be a DNA or RNA molecule. An aptamer can include modifications, for example, modified nucleotides, such as 2'-fluoro-substituted pyrimidines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the particle size distribution histogram of the exosome sample in Example 1.

[0042] Figure 2 It is the linear regression equation for calculating the ERF standard value in Example 1.

[0043] Figure 3 It is a statistical histogram of the density of CD9 molecules on the surface of exosomes in Example 1.

[0044] Figure 4 This is the distribution diagram of the particle size values ​​of LNP single particles in Example 2.

[0045] Figure 5 This is a diagram of the NGR copy number on the LNP surface in Example 2.

[0046] Figure 6 It is a statistical histogram of the NGR molecular density on the LNP surface in Example 2.

[0047] Figure 7 This is a diagram of the GM-CSF copy number on the Lipsome surface in Example 3.

[0048] Figure 8 It is a statistical histogram of the density of GM-CSF molecules on the Lipsome surface in Example 3.

[0049] Fig. 9 It is the linear regression equation for calculating the ERF standard value in Example 4.

[0050] Fig.10 This is a diagram of the VSVG copy number on the surface of the lentivirus in Example 4.

[0051] Fig.11 It is a statistical histogram of the density of VSVG molecules on the surface of the lentivirus in Example 4.

[0052] Fig.12 This is the HSV-1 surface gB copy number diagram in Example 5.

[0053] Fig.13 It is a statistical histogram of the density of gB molecules on the surface of HSV-1 in Example 5.

[0054] Fig.14 This is a graph showing the number of gD copies on the surface of HSV-1 in Comparative Example 1.

[0055] Fig.15 It is a statistical histogram of the gD molecule density on the surface of HSV-1 in Comparative Example 1.

[0056] Fig.16 This is a graph showing the number of gE copies on the surface of HSV-1 in Comparative Example 1.

[0057] Fig.17 It is a statistical histogram of the density of gE molecules on the surface of HSV-1 in Comparative Example 1.

[0058] Fig.18 This is a graph of the CD5 antibody copy number on the LNP surface in Example 6.

[0059] Fig.19 It is a statistical histogram of the CD5 molecule density on the LNP surface in Example 6.

[0060] Fig. 20 This is a graph of ApoE copy numbers on the LNP surface in Example 7.

[0061] Fig.21 It is a statistical histogram of the ApoE molecule density on the LNP surface in Example 7.

[0062] Fig. 22 This is a graph of the CD5 antibody copy number on the LNP surface in Example 8.

[0063] Fig.23 It is a statistical histogram of the CD5 molecule density on the LNP surface in Example 8. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0065] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0066] The detection method of the present invention:

[0067] (1) Preparation of fluorescent markers for nano-delivery vectors:

[0068] Mixing an optical reagent-labeled molecular capture agent containing a known number of fluorescent molecules n with a sample k of a nano-delivery carrier, so that the optical reagent specifically binds to the modified molecules on the surface of the nano-delivery carrier to obtain a mixture c;

[0069] The molecular capture agent comprises at least one selected from polypeptides, proteins, antibodies, ligands and / or receptors, enzymes, growth factors, glycolipids, polysaccharides, nucleic acids, or a combination thereof.

[0070] The nano drug delivery carriers include viral carriers and non-viral carriers.

[0071] The viral vectors include lentiviral vectors, retroviral vectors, herpes simplex virus vectors, recombinant coronavirus vectors, adenoviral vectors, and adeno-associated virus vectors; the non-viral vectors include exosomes, extracellular vesicles, lipid nanoparticles, liposomes, mRNA vaccines, cationic polymers, cationic lipids, inorganic nanoparticles, and nucleic acid conjugates.

[0072] The optical agent may be at least one of organic fluorescent molecules, fluorescent proteins, nucleic acid dyes, lipid membrane dyes, quantum dots, polymer dots or a combination thereof.

[0073] (2) Measurement of particle size distribution and surface area of ​​nano-delivery carrier samples:

[0074] Prepare particle size standard solutions of particle size standards of different particle sizes; use a flow particle detection device to detect the particle size standard solution, record the scattered light channel detection data and / or the fluorescence channel detection data of the particle size standard solution, and fit a curve based on the scattered light channel detection data and / or the fluorescence channel detection data and the particle size value of the particle size standard; measure the particle size distribution based on the scattered light channel detection data and / or the fluorescence channel detection data of the nano delivery carrier sample based on the fitting curve, and record the particle size D of the nano drug delivery carrier sample; calculate the surface area S=π(D)2 of the nano delivery carrier sample with positive scattering and / or fluorescence signals according to the particle size of the nano drug delivery carrier sample.

[0075] (3) Calculate the number of surface modified molecules of the nanodelivery carrier sample:

[0076] (3.1) using a flow particle detection device to detect the fluorescence intensity γ of the fluorescent marker of the labeled nano drug delivery carrier;

[0077] (3.2) using the flow particle detection device described in (3.1) and the same fluorescence signal collection conditions to detect the fluorescence intensity f of the fluorescence quantitative microspheres, and to establish a linear relationship between the fluorescence intensity f and the number of fluorescent molecules; the fluorescence quantitative microspheres are fluorescent microspheres whose number of fluorescent molecules m is known;

[0078] (3.3) According to the linear relationship described in step (3.2), based on the fluorescent marker and fluorescence intensity data of the nano drug delivery carrier γ , calculate the number of fluorescent molecules θ on the surface of the fluorescent marker of the nano drug delivery carrier; and then calculate the number of modified molecules Z=θ / n on the surface of the nano drug delivery carrier.

[0079] (4) Calculation of surface modified molecular density of nanodelivery carriers

[0080] Based on the surface area of ​​a single particle of the nano drug delivery carrier sample obtained in step (2) and the number of surface modified molecules of the nano drug delivery carrier obtained in step (3), the surface modified molecule density of the nano drug delivery carrier is calculated according to density = number of surface modified molecules of the nano drug delivery carrier / surface area of ​​a single particle of the nano drug delivery carrier sample; the surface modified molecule density of the nano drug delivery carrier is calculated as ρ = Z n / π(D n ) 2 .

[0081] Example 1: Density of CD9 on the surface of exosomes

[0082] Exosomes have become a popular research object, and they have shown great application prospects in the field of liquid biopsy. Exosomes are widely present in a variety of complex biological environments such as cell culture fluid, blood, milk, urine, ascites, etc., and Exosomes from different sources or even the same source have great differences in size, morphology and biochemical characteristics. Effective antibodies and detection methods are the two major bottlenecks in the current research of Exosomes. The relative molecular mass of CD9 is 24kD. There are 4 hydrophobic regions in the molecule, which go back and forth through the cell membrane 4 times, and together with other various homologous membrane proteins, they form the "tetraspanin" superfamily. CD9 has a variety of biological functions, and it plays an important role in cell adhesion, cell movement, activation, differentiation, tumor metastasis, and sperm-egg fusion. Current studies have shown that CD9 is a marker for exosomes and plays an important role in interacting with receptor cells. This embodiment provides a method for detecting the density of CD9 on the surface of exosomes, which provides a feasibility analysis method for the study of exosomes.

[0083] Reagents:

[0084] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0085] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4and 1.76 mM KH 2 PO 4 Aqueous solution of

[0086] The particle size standard solution was purchased from NanoFCM Inc.;

[0087] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0088] AF488-CD9 antibody was purchased from NanoFCM Inc.

[0089] instrument:

[0090] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0091] Test method:

[0092] 1) Fluorescence labeling of exosome samples

[0093] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the exosomes (vector) 10 times to obtain solution A, take 50 μL of solution A and 20 μL of AF488-CD9 antibody (molecular capture agent modified with fluorescent molecules, it is known that one CD9 antibody can label 5 AF488 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain fluorescently labeled exosomes.

[0094] 2) Particle size distribution and surface area detection of exosome samples

[0095] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kpa; signal type: small signal, using the scattered light channel; fixed Sampling pressure at 1.0kPa, and under this condition, the data of the 68±2nm, 91±3nm, 113±3nm, and 155±3nm particle size standard mixture tested were used to generate a fitting curve based on the Median value and Events value of the particle size standard display peak.

[0096] For the data of the exosome concentrate diluted 10 times under the same detection parameters, the gate tool was used to circle the target particle size distribution range, and the number of particles within the gate, the percentage of the number of particles within the gate, and the median, mean, and standard deviation of the particle size of the particles within the gate were recorded. The single particle size value of the exosome was converted based on the fitting curve, and the single particle surface area could be calculated. After statistics, the particle size distribution histogram of the exosome sample can be generated ( Figure 1 ). It can be seen that the average surface area of ​​the exosome samples is 30589±1157.53nm 2 , and its surface area distribution. Here, the exosomes can be exosomes before or after fluorescence labeling.

[0097] 3) Using ERF fluorescent microspheres

[0098] ERF fluorescent microspheres (ERF fluorescent microspheres used in this experiment were purchased from NanoFCM Inc..) were used for detection on a nanoflow detector, and the detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kpa; signal type: small signal, using the FITC channel; fixing the sampling pressure at 1.0 kPa, obtaining the average fluorescence intensity (logarithm) of the blank microsphere (P0) and the five fluorescent microsphere peaks (P1-P5); plotting the fluorescence channel number of P1-P5 as the x-axis and the ERF (logarithm) of the AF488 molecule in the corresponding fluorescent microsphere provided in the manual as the y-axis, and obtaining the linear regression equation; using the ERF fluorescent microspheres to actually measure the average fluorescence intensity of each fluorescence peak of the AF488 dye in the FITC fluorescence channel, the linear regression equation was calculated by calibrating the ERF standard values ​​corresponding to each peak in the microsphere manual to obtain lgy=0.72613+1.07397*lg x, R-square=0.99551( Figure 2 ).

[0099] 4) Detection of CD9 density on the surface of fluorescently labeled exosomes

[0100] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the FITC channel; fixing the sampling pressure at 1.0kPa, analyzing the fluorescence signal of the FITC fluorescence channel of the fluorescently labeled exosomes in step 1) and substituting it into the linear regression equation obtained in step 3), calculating the number of surface fluorescent molecules θ, and according to the fact that one CD9 antibody can label 5 AF488 molecules in step 1), the number of surface modified molecules of the nanodrug delivery carrier was calculated based on the number of surface modified molecules of the nanodrug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nanodrug delivery carrier / the number of fluorescent molecules n; setting 5AF488 / copies as the statistical parameter, and the surface area data of each particle of the exosome sample obtained in step 2), obtaining the statistical histogram of the CD9 area on the exosome surface ( Figure 3 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier is calculated. 2 There are 0.2±0.1 CD9 molecules in the sample. Example 2: Density of NGR targeting peptides on the surface of LNP

[0101] "Lipid nanoparticles" or "LNPs" include liposomes, which are formed by one or more lipid layers surrounding a core containing molecules to be released into the body. NGR polypeptides refer to polypeptides containing an asparagine-glycine-arginine tripeptide sequence (asn-gly-arg), which can heterotropically bind to CD13 metallopeptidase in tumor cells and tumor neovascular endothelial cells, and can actively target tumor sites. NGR targeting peptides are widely used in the field of drug delivery. Connecting NGR polypeptides to liposomes to obtain NGR polypeptide-modified liposomes can improve the targeting effect of LNP drugs, thereby aggregating at tumor sites and improving the anti-tumor effect of LNP. Studies on the density of NGR targeting peptides on the surface of LNP can further evaluate the targeting ability of LNP drugs. This embodiment improves the detection and analysis method of the density of NGR targeting peptides on the surface of LNP, and provides a basis for improving the tumor targeting study of LNP drugs.

[0102] Reagents:

[0103] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0104] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO4 Aqueous solution of

[0105] The particle size standard solution was purchased from NanoFCM Inc.;

[0106] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0107] AF488-CD13 was purchased from NanoFCM Inc.

[0108] instrument:

[0109] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0110] Test method:

[0111] 1) Fluorescence labeling of LNP samples

[0112] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the LNP sample 10 times to obtain solution B, take 50 μL of solution B and 5 μL of AF488-CD13 (it is known that one CD13 can label 5 AF488 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain the fluorescently labeled LNP.

[0113] 2) Particle size distribution detection of LNP samples

[0114] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kpa; signal type: small signal, using the scattered light channel; fixed Sampling pressure at 1.0kPa, and under this condition, the data of the 68±2nm, 91±3nm, 113±3nm, and 155±3nm particle size standard mixture tested were used to generate a fitting curve based on the Median value and Events value of the particle size standard display peak.

[0115] For the data of the concentrated solution of the LNP sample tested under the same test parameters diluted 10 times, the gate tool was used to circle the target particle size distribution range, and the number of particles within the gate, the percentage of the number of particles within the gate, and the median, mean and standard deviation of the particle size of the particles within the gate were recorded, and the LNP single particle size distribution value ( Figure 4 ), and used as the calculated data for the density of the NGR targeting peptide of the LNP sample;

[0116] 3) Using ERF fluorescent microspheres

[0117] Same as step 3 of Example 1)

[0118] 4) Detection of the copy number and density of NGR targeting peptides on the surface of fluorescently labeled LNPs

[0119] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the FITC channel; fixing the sampling pressure at 1.0kPa, analyzing the fluorescence signal detected by the FITC fluorescence channel of the fluorescently labeled LNP in step 1) and substituting it into the linear regression equation obtained in step 3), calculating the number of surface fluorescent molecules θ, and according to the fact that one CD13 protein can label 5 AF488 molecules in step 1), the number of surface modified molecules of the nano drug delivery carrier was calculated based on the number of surface modified molecules of the nano drug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nano drug delivery carrier / the number of fluorescent molecules n; setting 5AF488 / copies as a statistical parameter, and converting the single particle size data of the LNP sample obtained in step 2) into the single particle surface area, obtaining the statistical histogram of the number and area of ​​NGR copies on the LNP surface ( Figure 5 , Figure 6 ). According to density = number of surface modified molecules of nano drug delivery carrier / surface area of ​​single particle of nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of NGR on the LNP surface is 19.5±28.9, with an average of 100nm 2 There are 0.1±0.1 NGR molecules in the sample.

[0120] Example 3: Density of GM-CSF on Lipsome Surface

[0121] GM-CSF (granulocyte-macrophage colony stimulating factor) is a hematopoietic growth factor that stimulates the proliferation of bone marrow cells derived from bone marrow progenitor cells, but it has a short half-life and is easily hydrolyzed, requiring multiple injections in clinical applications. Liposomes can be used as a transport carrier for aqueous and oily substances.

[0122] Reagents:

[0123] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0124] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0125] The particle size standard solution was purchased from NanoFCM Inc.;

[0126] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0127] AF488-CSF2 was purchased from NanoFCM Inc.

[0128] instrument:

[0129] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0130] Test method:

[0131] 1) Fluorescence labeling of lipsome samples

[0132] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the Lipsome sample 10 times to obtain solution C, take 50 μL of solution C and 5 μL of AF488-CSF2 (it is known that one CSF2 can label 5 AF488 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain the fluorescently labeled Lipsome.

[0133] 2) Particle size distribution detection of Lipsome samples

[0134] Same as step 2 of Example 2), the particle size and surface area data of a single particle are directly counted using FlowNanoAnalyzer analysis software in step 4 of this example;

[0135] 3) Using ERF fluorescent microspheres

[0136] Same as step 3 of Example 1)

[0137] 4) Detection of GM-CSF copy number and density on the surface of fluorescently labeled Lipsomes

[0138] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the FITC channel; fixing the sampling pressure at 1.0kPa, analyzing the fluorescence signal detected by the FITC fluorescence channel of the fluorescently labeled Lipsome in step 1) and substituting it into the linear regression equation obtained in step 3), calculating the number of surface fluorescent molecules θ, and according to the fact that one CSF2 can label 5 AF488 molecules in step 1), the number of surface modified molecules of the nanodrug delivery carrier was calculated based on the number of surface modified molecules of the nanodrug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nanodrug delivery carrier / the number of fluorescent molecules n; setting 5AF488 / copies as a statistical parameter, and converting the single particle size data of the Lipsome sample obtained in step 2) into the single particle surface area, obtaining the statistical histogram of the GM-CSF copy number and area on the Lipsome surface ( Figure 7 , Figure 8 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of GM-CSF on the surface of Lipsome is 38.2±51.1, with an average of 100nm 2 There are 0.1±0.1 GM-CSF molecules in the blood.

[0139] Example 4: Density of VSVG on the Surface of Lentivirus

[0140] The lentivirus vector is a single-stranded RNA virus that can randomly insert exogenous fragments into the cell genome, so it can express the target gene in vivo for a long time, and it has low immunogenicity and high safety, and is an important gene manipulation tool. VSVG is a viral membrane protein. VSVG protein is more stable than the env protein of retrovirus or lentivirus, so the virus "pseudotyped" by the VSVG protein can be ultracentrifuged to obtain a higher titer; and the receptor of VSV-G is phosphatidylserine widely expressed on the cell membrane, so compared with the env protein, the host range of VSV-G "pseudotyped" virus becomes wider. Therefore, the analysis of the density of VSVG protein on lentivirus is one of the foundations of this type of vector analysis. This embodiment provides a method for detecting the density of VSVG on the surface of lentivirus.

[0141] Reagents:

[0142] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0143] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0144] The particle size standard solution was purchased from NanoFCM Inc.;

[0145] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0146] AF647-VSVG antibody was purchased from NanoFCM Inc.

[0147] instrument:

[0148] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0149] Test method:

[0150] 1) Fluorescent labeling of lentiviral samples

[0151] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the concentrated solution of the lentiviral vector product 10 times to obtain solution D, take 50 μL of solution 1 and 20 μL of AF647-VSVG antibody (VSVG antibody conjugated with AF647) and incubate at 37°C for 30 min, centrifuge at 100000g at 4°C for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 100000g at 4°C for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain the labeled lentiviral vector.

[0152] 2) Particle size distribution detection of lentiviral samples

[0153] The particle size and surface area data of single lentiviral particles in step 2) of Example 2 are directly counted using Flow NanoAnalyzer analysis software in step 4) of this example;

[0154] 3) Using ERF fluorescent microspheres

[0155] ERF fluorescent microspheres (ERF fluorescent microspheres used in this experiment were purchased from NanoFCM Inc.) were used for detection on a nanoflow cytometry detector, and the detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the PC5 channel; fixing the sampling pressure at 1.0kPa, and obtaining the average fluorescence intensity (logarithm) of blank microspheres (P0) and 5 fluorescent microsphere peaks (P1-P5); plotting a graph with the fluorescence channel number of P1-P5 as the x-axis and the ERF (logarithm) of the AF647 molecules in the corresponding fluorescent microspheres provided in the manual as the y-axis, and obtaining a linear regression equation; using the ERF fluorescent microspheres to actually measure the average fluorescence intensity of each fluorescence peak of the AF647 dye in the PC5 fluorescence channel, the linear regression equation lg was calculated by calibrating the ERF standard values ​​corresponding to each peak in the microsphere manual. y=0.50146+0.96188*lg x,R-square=0.99237( Fig. 9 ).

[0156] 4) Detection of VSVG copy number and density on the surface of fluorescently labeled lentivirus

[0157] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, PC5 channel was used; the sampling pressure was fixed to 1.0kPa, and the fluorescence signal detection result of the PC5 fluorescence channel of the fluorescently labeled lentivirus in step 1) was substituted into the linear regression equation obtained in step 3) to calculate the number of surface fluorescent molecules θ, and according to the fact that one VSVG antibody can label 4 AF647 molecules in step 1), the number of surface modified molecules of the nanodrug delivery carrier was calculated based on the number of surface modified molecules of the nanodrug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nanodrug delivery carrier / the number of fluorescent molecules n; 5AF647 / copies was set as the statistical parameter, and the single particle size data of the lentivirus sample obtained in step 2) was converted into the single particle surface area, and the statistical histogram of the number and area of ​​VSVG copies on the lentivirus surface was obtained ( Fig.10 , Fig.11 ). According to density = number of surface modified molecules of nano drug delivery carrier / surface area of ​​single particle of nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of VSVG on the surface of lentivirus is 230.0±266.5, with an average of 100nm 2 There are 0.8±0.5 VSVG molecules in the sample.

[0158] Example 5: Density of gB on the surface of HSV-1

[0159] The gB of herpes simplex virus type 1 (HSV-1) is one of several glycoproteins on the viral envelope, which determines the infectivity of the virus. At the same time, the DII domain of HSV-1gB has highly conserved antigenic epitopes in sequence and structure, which is an important target for the development of broad-spectrum drugs and vaccines for herpes viruses. This embodiment provides a method for detecting the density of gB on the surface of HSV-1, which provides a basis for the analysis and research of broad-spectrum drugs and vaccines for herpes viruses.

[0160] Reagents:

[0161] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0162] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0163] The particle size standard solution was purchased from NanoFCM Inc.;

[0164] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0165] AF647-Anti-Glycoprotein B of HSV (gB) antibody was purchased from NanoFCM Inc.

[0166] instrument:

[0167] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0168] Test method:

[0169] 1) Fluorescent labeling of HSV-1 samples

[0170] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4) and 0.5% Tween 20 buffer to dilute the concentrated HSV-1 vector product 10 times to obtain solution E, take 50 μL of solution 1 and 20 μL AF647-Anti-Glycoprotein B ofHSV (gB) antibody (gB antibody conjugated with AF647) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL PBS buffer to resuspend the precipitate to obtain the labeled HSV-1 vector.

[0171] 2) Particle size distribution detection of HSV-1 samples

[0172] The particle size and surface area data of HSV-1 single particles in step 4) of Example 4 are directly counted using FlowNanoAnalyzer analysis software in step 4) of this Example;

[0173] 3) Using ERF fluorescent microspheres

[0174] Same as step 3 of Example 4)

[0175] 4) Detection of the copy number and density of fluorescently labeled HSV-1 surface gB

[0176] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using PC5 channel; fixing the sampling pressure at 1.0kPa, analyzing the fluorescence signal detected by the PC5 fluorescence channel of the fluorescently labeled HSV-1 in step 1) and substituting it into the linear regression equation obtained in step 3), calculating the number of surface fluorescent molecules θ, and according to the fact that one gB antibody can label 4 AF647 molecules in step 1), the number of surface modified molecules of the nanodrug delivery carrier was calculated based on the number of surface modified molecules of the nanodrug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nanodrug delivery carrier / the number of fluorescent molecules n; setting 5AF647 / copies as the statistical parameter, and converting the single particle size data of the HSV-1 sample obtained in step 2) into the single particle surface area, and obtaining the statistical histogram of the gB copy number and area on the surface of HSV-1 ( Fig.12 , Fig.13 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of gB on the surface of HSV-1 is 218.0±1049.5, with an average of 100nm 2 There are 0.3±1.0 gB molecules in it.

[0177] Comparative Example 1: Density of gD and gE on the surface of HSV-1

[0178] Reagents:

[0179] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0180] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0181] The particle size standard solution was purchased from NanoFCM Inc.;

[0182] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0183] AF647-Anti-Glycoprotein D of HSV (gD) antibody was purchased from NanoFCM Inc.

[0184] AF647-Anti-Glycoprotein E of HSV (gE) antibody was purchased from NanoFCM Inc.

[0185] instrument:

[0186] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0187] Test method: Same as Example 5, in the detection of HSV-1 surface gD, AF647-Anti-Glycoprotein DofHSV (gD) antibody was used to replace AF647-Anti-Glycoprotein B ofHSV (gB) antibody; in the detection of HSV-1 surface gE, AF647-Anti-Glycoprotein E ofHSV (gE) antibody was used to replace AF647-Anti-Glycoprotein B ofHSV (gB) antibody;

[0188] from Fig.14 , Fig.15 It can be seen that the average copy number of gD on the surface of HSV-1 is 85.5±199.3, with an average of 100nm 2 There are 0.1±0.3 gD molecules in Fig.16 , Fig.17 It can be seen that the average copy number of HSV-1 surface gE is 29.7±73.4, an average of 100nm2 There are 0.1±0.1 gE molecules in the sample.

[0189] Example 6: Density of CD5 Antibody Modified on LNP Surface

[0190] CD5 antibody (CD5 is mainly expressed on the surface of T cells) is modified on the surface of LNP, and mRNA encoding chimeric antigen receptor (CAR) is delivered to circulating T cells in the body and in situ CAR-T cells are generated in the body, which can effectively treat, for example, myocardial fibrosis and restore normal heart function. Therefore, it is of great significance to study the modification of CD5 antibody on the surface of LNP. This embodiment provides a method for detecting the density of CD5 antibody modified on the surface of LNP, which provides a basis for such antibody analysis research.

[0191] Reagents:

[0192] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0193] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0194] The particle size standard solution was purchased from NanoFCM Inc.;

[0195] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0196] AF488-CD5 was purchased from NanoFCM Inc.

[0197] instrument:

[0198] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0199] Test method:

[0200] 1) Fluorescence labeling of LNP samples

[0201] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4) and 0.5% Tween 20 buffer to dilute the LNP sample 10 times to obtain solution F, take 50 μL of solution F and 5 μL AF488-CD5 (it is known that one CD5 can label 5 AF488 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C 100000g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C 100000g for 20 min, discard the supernatant, add 100 μL PBS buffer to resuspend the precipitate to obtain the fluorescently labeled LNP.

[0202] 2) Particle size distribution detection of LNP samples

[0203] Same as step 2 of Example 2), the particle size and surface area data of a single particle are directly counted using FlowNanoAnalyzer analysis software in step 4 of this example;

[0204] 3) Using ERF fluorescent microspheres

[0205] Same as step 3 of Example 1)

[0206] 4) Detection of the copy number and density of CD5 antibodies modified on the surface of fluorescently labeled LNPs. The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal. Under the conditions, FITC channel was used; the sampling pressure was fixed to 1.0kPa, and the results of the fluorescence signal detected by the FITC fluorescence channel of the fluorescently labeled LNP in step 1) were substituted into the linear regression equation obtained in step 3) to calculate the number of surface fluorescent molecules θ, and according to the fact that one CD5 can label 5 AF488 molecules in step 1), the number of surface modified molecules of the nano drug delivery carrier was calculated based on the number of surface modified molecules of the nano drug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nano drug delivery carrier / the number of fluorescent molecules n; 5AF488 / copies was set as the statistical parameter, and the single particle size data of the LNP sample obtained in step 2) was converted into the single particle surface area, and the statistical histogram of the copy number and area of ​​CD5 antibodies on the LNP surface was obtained ( Fig.18 , Fig.19 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of CD5 antibody on the surface of LNP is 70.1±115.3, with an average of 100nm 2 There are 0.4±0.2 CD5 antibody molecules in the sample.

[0207] Example 7: Density of LNP surface modified apolipoprotein E (ApoE)

[0208] ApoE (Apolipoprotein E) modified plasmid-loaded cationic liposomes are a drug delivery system. By firmly combining the positive charge of cationic liposomes with the negatively charged plasmids, ApoE (Apolipoprotein E) is modified on the surface of liposomes to enhance in vivo targeting and bioavailability, prolong drug half-life, improve stability and reduce drug toxicity.

[0209] Reagents:

[0210] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0211] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0212] The particle size standard solution was purchased from NanoFCM Inc.;

[0213] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0214] AF488-ApoE was purchased from NanoFCM Inc.

[0215] instrument:

[0216] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0217] Test method:

[0218] 1) Fluorescence labeling of LNP samples

[0219] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the LNP sample 10 times to obtain solution G, take 50 μL of solution G and 5 μL of AF488-ApoE (it is known that one ApoE can label 5 AF488 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain the fluorescently labeled LNP.

[0220] 2) Particle size distribution detection of LNP samples

[0221] Same as step 2 of Example 2), the particle size and surface area data of a single particle are directly counted using FlowNanoAnalyzer analysis software in step 4 of this example;

[0222] 3) Using ERF fluorescent microspheres

[0223] Same as step 3 of Example 1)

[0224] 4) Detection of the copy number and density of ApoE modified on the surface of fluorescently labeled LNP

[0225] The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal, using the FITC channel; fixing the sampling pressure at 1.0kPa, analyzing the fluorescence signal detected by the FITC fluorescence channel of the fluorescently labeled LNP in step 1) and substituting it into the linear regression equation obtained in step 3), calculating the number of surface fluorescent molecules θ, and according to the fact that one ApoE can label 5 AF488 molecules in step 1), the number of surface modified molecules of the nano drug delivery carrier was calculated based on the number of surface modified molecules of the nano drug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nano drug delivery carrier / the number of fluorescent molecules n; setting 5AF488 / copies as a statistical parameter, and converting the single particle size data of the LNP sample obtained in step 2) into the single particle surface area, obtaining the statistical histogram of the number and area of ​​ApoE copies on the LNP surface ( Fig. 20 , Fig.21 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of ApoE on the surface of LNP is 106.3±167.9, with an average of 100nm 2 There are 0.5±0.3 ApoE molecules in the blood.

[0226] Example 8: Density of CD5 Antibody Modified on LNP Surface

[0227] Compared with Example 6, this example further provides a method for detecting the density of CD5 antibodies modified on the surface of LNP.

[0228] Reagents:

[0229] Unless otherwise specified, the following reagents were selected from the following manufacturers:

[0230] PBS buffer: contains 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2 HPO 4 and 1.76 mM KH 2 PO 4 Aqueous solution of

[0231] The particle size standard solution was purchased from NanoFCM Inc.;

[0232] ERF fluorescent microspheres were purchased from NanoFCM Inc.;

[0233] AF647-goat anti-rabbit IgG secondary antibody was purchased from NanoFCM Inc.

[0234] instrument:

[0235] FlowNanoAnalyzer was purchased from NanoFCM Inc.

[0236] Test method:

[0237] 1) Fluorescence labeling of LNP samples

[0238] The cells were fixed with PBS (containing 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na 2 HPO 4 and 1.76 mM KH 2 PO 4 ) and 0.5% Tween 20 buffer to dilute the LNP sample 10 times to obtain solution F, take 50 μL of solution F and 5 μL of AF647-goat anti-rabbit IgG secondary antibody (it is known that one goat anti-rabbit IgG secondary antibody can label 5 AF647 molecules) and incubate at 37°C for 30 min, centrifuge at 4°C and 100,000 g for 80 min, discard the supernatant, resuspend the precipitate with PBS buffer, centrifuge at 4°C and 100,000 g for 20 min, discard the supernatant, add 100 μL of PBS buffer to resuspend the precipitate to obtain the fluorescently labeled LNP.

[0239] 2) Particle size distribution detection of LNP samples

[0240] Same as step 2 of Example 2), the particle size and surface area data of a single particle are directly counted using FlowNanoAnalyzer analysis software in step 4 of this example;

[0241] 3) Using ERF fluorescent microspheres

[0242] Same as step 3 of Example 4)

[0243] 4) Detection of the copy number and density of CD5 antibodies modified on the surface of fluorescently labeled LNPs. The detection parameters were set as follows: laser detector 488nm+638nm; single laser channel detection Laser: 10 / 50mW 488; scattered light attenuation: 10%; detection pressure: 1kPa; signal type: small signal. Under the conditions, PC5 channel was used; the sampling pressure was fixed to 1.0kPa, and the result of the fluorescence signal detected by the PC5 fluorescence channel of the fluorescently labeled LNP in step 1) was substituted into the linear regression equation obtained in step 3) to calculate the number of surface fluorescent molecules θ, and according to the known fact that one goat anti-rabbit IgG secondary antibody can label 5 AF647 molecules in step 1), the number of surface modified molecules of the nano drug delivery carrier was calculated based on the number of surface modified molecules of the nano drug delivery carrier = the number of surface fluorescent molecules of the fluorescent marker of the nano drug delivery carrier / the number of fluorescent molecules n; 5AF647 / copies was set as the statistical parameter, and the single particle size data of the LNP sample obtained in step 2) was converted into the single particle surface area, and the statistical histogram of the copy number and area of ​​CD5 antibodies on the LNP surface was obtained ( Fig. 22 , Fig.23 ). According to density = number of surface modified molecules of nano drug delivery carrier / single particle surface area of ​​nano drug delivery carrier sample, the surface modified molecule density of nano drug delivery carrier was calculated. It can be seen that the average copy number of CD5 antibody on the surface of LNP is 19.5±28.9, with an average of 100nm 2 There are 0.1±0.1 CD5 antibody molecules in the sample.

[0244] The method of the present invention comprises the following steps: mixing an optical reagent-labeled molecular capture agent with a known number of fluorescent molecules with a sample of a nano drug delivery carrier to prepare a particle size standard solution and a linear standard fluorescent microsphere, and performing detection by using a flow particle detection device to determine the median value of the fluorescence intensity of the nano drug delivery carrier before optical labeling and the fluorescence intensity of each nano drug delivery carrier after optical labeling; calculating the fluorescence intensity of each standard fluorescent microsphere, statistically obtaining a linear relationship between the number of fluorescent molecules and the fluorescence intensity, and then converting the number of fluorescent molecules corresponding to the fluorescence intensity of each nano drug delivery carrier after optical labeling after deducting the median value of the fluorescence intensity, and combining the number of fluorescent molecules of each optical reagent molecule on the surface of the nano drug delivery carrier after known labeling, and then calculating the number of surface modified molecules of each nano drug delivery carrier; and simultaneously measuring the scattered light or fluorescence intensity of each particle size standard ball of the particle size standard solution, and then calculating the particle size value of each nano drug delivery carrier, and then calculating the surface area of ​​each nano drug delivery carrier, and finally obtaining the number of surface modified molecules per unit area of ​​each nano drug delivery carrier, that is, the surface modified molecule density of the nano drug delivery carrier.

[0245] The method provides an evaluable method for analyzing the biocompatibility of surface-modified nano drug delivery carrier samples, increasing the in vivo circulation time, achieving targeted delivery and other functions, and has guiding significance for evaluating the safety and stability of nano drug delivery carriers. The method of the present invention is accurate, efficient, easy to operate, uses a small amount of sample, and can analyze the number and density of surface-modified molecules of nano drug delivery carriers at the single particle level.

[0246] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for measuring the density of surface modified molecules of a nano drug delivery carrier, characterized in that: The measuring method comprises: (1) Preparing a fluorescent marker for a nano drug delivery carrier: mixing the nano drug delivery carrier with a molecular capture agent labeled with an optical agent having a known number of fluorescent molecules n, and connecting the optical agent to the surface of the nano drug delivery carrier through the specific binding of the molecular capture agent to the modified molecule on the surface of the nano drug delivery carrier, thereby preparing the fluorescent marker; (2) measuring the surface area of ​​a single particle of a nano drug delivery carrier sample; the nano drug delivery carrier sample comprises a nano drug delivery carrier; (3) Calculating the number of molecules modified on the surface of the nano drug delivery carrier: Based on the fluorescence intensity of the fluorescent quantitative microspheres and the number of fluorescent molecules, a linear relationship between the number of fluorescent molecules and the fluorescence intensity is obtained, and the number of fluorescent molecules on the surface of the fluorescent marker of the nano drug delivery carrier is obtained. Combined with the known number of fluorescent molecules n possessed by each optical agent molecule on the surface of the nano drug delivery carrier after labeling, the number of molecules modified on the surface of the nano drug delivery carrier is calculated based on the number of molecules modified on the surface of the nano drug delivery carrier = the number of fluorescent molecules on the surface of the fluorescent marker of the nano drug delivery carrier θ / the number of fluorescent molecules n; (4) Based on the single particle surface area of ​​the nanodrug delivery carrier sample obtained in step (2) and the number of surface-modified molecules of the nanodrug delivery carrier obtained in step (3), the surface-modified molecule density of the nanodrug delivery carrier is calculated according to density = number of surface-modified molecules of the nanodrug delivery carrier / single particle surface area of ​​the nanodrug delivery carrier sample.

2. The measuring method according to claim 1, characterized in that: Step (2) includes: preparing a particle size standard solution of a particle size standard of different particle sizes; using a flow particle detection device to detect the particle size standard solution, recording the scattered light channel detection data of the particle size standard solution, and fitting a curve based on the scattered light channel detection data and the particle size value of the particle size standard; measuring the particle size distribution of the scattered light channel detection data of the nano drug delivery carrier sample based on the fitting curve, and recording the particle size D of the nano drug delivery carrier sample; calculating the surface area S=πD of the nano drug delivery carrier sample with a positive scattering signal according to the particle size of the nano drug delivery carrier sample 2 .

3. The measuring method according to claim 1, characterized in that: Step (3) includes: (3.1) using a flow particle detection device to detect the fluorescence intensity γ of the fluorescent marker of the labeled nano drug delivery carrier; (3.2) using the flow particle detection device described in (3.1) and the same fluorescence signal collection conditions to detect the fluorescence intensity f of the fluorescence quantitative microspheres, and establish a linear relationship between the fluorescence intensity f and the number of fluorescent molecules m; the fluorescence quantitative microspheres are fluorescent microspheres whose number of fluorescent molecules m is known; (3.3) According to the linear relationship described in step (3.2), based on the fluorescence intensity γ of the fluorescent marker of the nanodrug delivery carrier, the number of fluorescent molecules θ on the surface of the fluorescent marker of the nanodrug delivery carrier is calculated; and then the number of modified molecules Z = θ / n on the surface of the nanodrug delivery carrier is calculated.

4. The measuring method according to any one of claims 2 to 3, characterized in that: The flow-type particle detection device is a particle analysis and detection device that can realize directional flow of sample flow.

5. The measuring method according to claim 4, characterized in that: The particle analysis and detection equipment comprises an optical system and a particle detector.

6. The measuring method according to claim 5, characterized in that: The particle detector is composed of a photoelectric sensor and a signal conditioning circuit with a band-limiting filtering high-frequency noise function.

7. The measuring method according to claim 1, characterized in that: The optical reagent on the fluorescent marker of the nano drug delivery carrier and the optical reagent on the fluorescent quantitative microsphere are of the same kind or different kinds.

8. The measuring method according to any one of claims 1 to 3, characterized in that: The molecular capture agent includes at least one selected from antibodies, ligands, receptors, nucleic acid aptamers, enzymes, glycolipids, and polysaccharides.

9. The measuring method according to any one of claims 1 to 3, characterized in that: The nano drug delivery carriers include viral carriers and non-viral carriers.

10. The measuring method according to claim 9, characterized in that: The viral vectors include lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors; the non-viral vectors include extracellular vesicles, lipid nanoparticles, liposomes, and mRNA vaccines.

11. The measuring method according to any one of claims 1 to 3, characterized in that: The optical agent includes at least one of organic fluorescent molecules, fluorescent proteins, and quantum dots.

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