A method for identifying in vivo targeting of lipid nanoparticles and uses thereof

Abstract

This invention discloses a method for identifying the in vivo targeting ability of lipid nanoparticles (LNPs) and its application. The method involves mixing aqueous phases containing nucleic acid barcodes with different core coding regions and random error correction regions with lipid phases composed of different lipid formulations to prepare lipid nanoparticles. These lipid nanoparticles are then exposed in experimental animals, and DNA is extracted from different tissues and organs for sequencing to obtain sequencing data. Based on the number of sequencing reads corresponding to the nucleic acid barcodes, the relative abundance spectra of various lipid nanoparticles in different organ or tissue samples are obtained. The targeting ability of various lipid nanoparticle formulations in experimental animals is determined based on the relative abundance spectra. This method not only allows for a simultaneous parallel comparison of the in vivo distribution of multiple LNP formulations but also aims to produce reliable and quantitative data, thereby directly guiding the rational design of LNPs with specific targeting functions for clinical development or the preparation of drug delivery products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for identifying the in vivo targeting of lipid nanoparticles and its application. Background Technology

[0002] Lipid nanoparticles (LNPs), as a class of efficient and safe non-viral delivery carriers, have shown great potential in the clinical translation of nucleic acid drugs (such as mRNA vaccines and siRNA therapies). The in vivo delivery efficiency and organ targeting of these materials are highly dependent on the complex lipid formulation composition, including the types and proportions of ionizable lipids, cofactor lipids, cholesterol, and functional lipids (Nat. Rev. Drug Discov., 2021, 20, 101). However, traditional screening techniques for assessing the in vivo distribution of LNPs (such as single labeling based on radioisotopes or fluorescent dyes) have inherent limitations: low throughput, high cost, difficulty in parallel comparison of multiple formulations in a single experiment, and inability to perform simultaneous quantitative analysis of multi-organ distribution (J. Radioanal. Nucl. Chem., 2014, 302, 837; J. Control. Release. 2014, 188, 31). These limitations severely hinder the systematic analysis of the structure-activity relationship between formulation composition and in vivo distribution, posing a significant challenge to the rational design of organ-specific LNPs.

[0003] Developing an integrated, nucleic acid barcode-based high-throughput screening method and system for LNPs in vivo to achieve parallel and quantitative evaluation of the distribution characteristics of multiple LNP formulations in multiple organs is of great significance for promoting the innovative development of nucleic acid drug delivery technology. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to perform integrated high-throughput screening of the in vivo targeted distribution of lipid nanoparticles (LNPs) and / or how to perform parallel and quantitative evaluation of the distribution characteristics of multiple lipid nanoparticle (LNP) formulations in multiple organs and / or how to efficiently and systematically resolve the quantitative structure-activity relationship between the complex formulation composition of lipid nanoparticles (LNPs) and their in vivo organ targeting.

[0005] To address the aforementioned technical problems, this invention first provides a method for identifying the in vivo targeting ability of lipid nanoparticles, the method comprising the following steps:

[0006] 1) Prepare a mixture containing A types of lipid nanoparticles, where A is a natural number greater than or equal to 2; each type of lipid nanoparticle in the mixture is a nanoscale particle obtained by phase mixing an aqueous phase containing a nucleic acid barcode P with a lipid phase containing a lipid mixture; the nucleic acid barcode P is a single-stranded DNA molecule containing a core coding region and a random error correction region; the core coding region and the lipid mixture are different between each pair of lipid nanoparticles in the mixture, and the mass ratio of each lipid mixture to the nucleic acid barcode P mixed with it is the same; The random error correction region is a sequence randomly composed of N, where N is A, T, C, or G; 2) The mixture is used to administer drugs to experimental animals to obtain exposed experimental animals. Samples of different organs or tissues of the exposed experimental animals are obtained. DNA is extracted from the samples of different organs or tissues to obtain sequencing libraries for each organ or tissue. The sequencing libraries of each organ or tissue are mixed in equal amounts and then sequenced to obtain sequencing data. The sequencing data is analyzed, and the relative abundance spectrum of the lipid nanoparticle A in the samples of different organs or tissues is obtained based on the number of sequencing reads corresponding to the nucleic acid barcode P. The targeting of the lipid nanoparticle A in vivo is determined based on the relative abundance spectrum. The equal mass mixing is based on the mass of DNA.

[0007] In one specific embodiment of the present invention, the equal quantity mentioned in 2) refers to equal mass.

[0008] The diameter of the nanoscale particles mentioned above can be 60-200 nm.

[0009] The lipid nanoparticles (LNPs) described above can be nanoscale (60-200 nm in diameter) delivery systems composed of lipid molecules.

[0010] In the above method, the administration may be non-oral. Specifically, the administration may be by injection. In one specific embodiment of the present invention, the administration is by intravenous injection.

[0011] In one specific embodiment of the present invention, the phase mixing is achieved by microfluidic mixing technology.

[0012] In the above method, the lipid mixture may consist of four parts: ionizable lipids, PEG-modified lipids, phospholipids, and cholesterol. The ionizable lipids may be MC3 and DOTAP, or other lipids with ionizable properties or targeting functions.

[0013] In one specific embodiment of the present invention, the lipid mixture may consist of methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester (MC3), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), distearate phosphatidylcholine (DSPC), cholesterol (Chol), and 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2k).

[0014] In the above method, the sequence length of the core coding region can be 6-12 nucleotides, and the sequence length of the random error correction region can be 6-10 nucleotides.

[0015] In one specific embodiment of the present invention, the sequence length of the core coding region may be 10 nucleotides, and the sequence length of the random error correction region may be 8 nucleotides.

[0016] In the above method, the mass ratio of the total lipids of the lipid phase to the nucleic acid barcode P during the phase mixing process can be from 20:1 to 40:1.

[0017] In the above method, the volume mixing ratio of the lipid phase and the aqueous phase during the phase mixing process can be 1:3. The total concentration of lipids in the lipid phase is 12 mM.

[0018] In the above method, the 5' end of the nucleic acid barcode P may contain sequence P1, in which each nucleoside in P1 is linked by a phosphate thioester bond; the 3' end of the nucleic acid barcode P may contain sequence P2, in which each nucleoside in P2 is linked by a phosphate thioester bond.

[0019] P1 can be composed of 3-5 nucleotides. P2 can be composed of 3-5 nucleotides.

[0020] In the above method, the nucleic acid barcode P may include a universal site 1 sequence, a core coding region connected to the universal site 1 sequence, a random error correction region connected to the core coding region, and a universal site 2 sequence connected to the random error correction region; the 5' end of the universal site 1 sequence includes P1, and the 3' end of the universal site 2 sequence includes P2.

[0021] In the above method, the nucleic acid barcode P may further include a sequencing adapter sequence, which is used to complement the adapter sequence of the sequencing platform corresponding to the sequencing.

[0022] The method described above may further include a step of PCR amplification of the DNA, wherein the primers for the PCR may contain organ barcode region sequences, which are used to distinguish the different organ or tissue samples.

[0023] The method described above may further include a step of purifying and dialysis the lipid nanoparticles MP before the equal-volume mixing, wherein the purification and dialysis includes purifying and dialysis the lipid nanoparticles MP using a buffer solution at 4°C.

[0024] In one specific embodiment of the present invention, the buffer solution may be a phosphate buffer solution with a pH of 7.4.

[0025] In one specific embodiment of the present invention, the injection is a tail vein injection, and the injection dose may be 0.5 mg / kg body weight.

[0026] The time for extracting DNA from samples of different organs or tissues can be 6 hours after the injection.

[0027] To address the aforementioned technical problems, the present invention also provides lipid nanoparticles, wherein the lipid nanoparticles are those described above.

[0028] To address the aforementioned technical problems, the present invention also provides a DNA molecule, characterized in that: the DNA molecule is the nucleic acid barcode P described above.

[0029] Any of the following applications of the lipid nanoparticles described above and / or the DNA molecules described above: 1) Application in the development or preparation of in vivo targeted screening products for lipid nanoparticles; 2) Applications in the development or preparation of drug delivery products.

[0030] The application of the methods described above in the development or preparation of drug delivery products also falls within the scope of protection of this invention.

[0031] The drugs mentioned above may be nucleic acids.

[0032] The lipid phase described above can be an ethanol solution containing a mixture of lipids.

[0033] A long-standing core challenge in lipid nanoparticle (LNP) development is the efficient and systematic analysis of the quantitative structure-activity relationship (QSAR) between their complex formulations and their in vivo organ targeting. Existing screening technologies are limited by throughput, cost, and standardization constraints. This invention aims to overcome this bottleneck by providing an innovative integrated high-throughput screening method and system. This method not only aims to compare the in vivo distribution of multiple LNP formulations in parallel in a single operation but also strives to generate reliable and quantitative data, thereby directly guiding the rational design of LNPs with specific targeting functions and accelerating their translation to clinical applications.

[0034] This invention provides an integrated high-throughput method for in vivo targeted screening of lipid nanoparticles. It includes the following steps: (1) Design and synthesize nucleic acid barcodes, which are single-stranded DNA structures containing a core coding region, a random error correction region, and a sequencing adapter sequence, and modify both ends with thiophosphate bonds to enhance their stability. After synthesis, purification and quality control are performed.

[0035] (2) Prepare the reaction solution for the lipid nanoparticles carrying nucleic acid barcodes. Specifically, weigh methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (MC3), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), distearate phosphatidylcholine (DSPC), cholesterol (Chol), and 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2k) according to the preset molar ratios in the lipid nanoparticle formulation library, and dissolve them in anhydrous ethanol to prepare the lipid phase. Dissolve the nucleic acid barcodes in an acidic buffer solution to prepare the aqueous phase.

[0036] (3) Lipid nanoparticles loaded with nucleic acid barcodes were prepared using microfluidic mixing technology. The lipid phase and aqueous phase prepared in step (2) were loaded into separate injection systems driven by an injection pump and instantaneously mixed via a microfluidic mixing chip. The volume ratio of the lipid phase to the aqueous phase was controlled at 1:3, and the total flow rate was set at 12 mL / min. The mixing process was controlled by adjusting the flow rate ratio of the two phases and the total flow rate to form a primary product of lipid nanoparticles with uniform particle size, which was then collected in a sterile container.

[0037] (4) The initial product of lipid nanoparticles loaded with nucleic acid barcodes obtained in step (3) was purified and characterized. First, a small amount of the initial product was diluted and subjected to preliminary physicochemical determination. Then, the remaining initial product was transferred to a dialysis apparatus and purified by dialysis using phosphate buffer at pH 7.4 at 4°C. After dialysis, the final product was subjected to comprehensive physicochemical characterization, including average particle size, polydispersity index (PDI), zeta potential, and nucleic acid encapsulation efficiency.

[0038] (5) The purified lipid nanoparticle formulations from step (4) were subjected to in vivo exposure and sample collection in animals, along with the naked nucleic acid barcode control. Equal volumes of lipid nanoparticles loaded with different nucleic acid barcodes were mixed and injected into experimental animals via the tail vein, along with the naked nucleic acid barcode control. Target tissue samples were collected at preset time points, flash-frozen in liquid nitrogen, and stored at -80 °C for subsequent nucleic acid extraction and analysis.

[0039] (6) Extract total DNA from the tissue sample obtained in step (5) and construct a PCR amplification library containing organ-specific barcodes. After the tissue was disrupted by liquid nitrogen grinding, nucleic acid was extracted using a rapid DNA extraction kit, and the extracted products were purified and concentrated. The extracted nucleic acids were amplified by PCR using a forward primer with an Illumina sequencing adapter and a reverse primer with an 8-nucleotide organ barcode. The amplification products were separated by gel electrophoresis, the target fragments were recovered by gel excision and purified to obtain a DNA library for high-throughput sequencing.

[0040] (7) High-throughput sequencing and data analysis were performed on the DNA library constructed in step (6) to quantify the relative abundance of each lipid nanoparticle in different tissues. The purified library was quality checked, and after passing the quality check, deep sequencing was performed using the Illumina paired-end sequencing platform. The raw data after sequencing was subjected to quality control, filtering, and normalization, and finally the relative abundance of each nucleic acid barcode in a specific tissue was calculated. Furthermore, in order to quantify the targeting selectivity of different lipid nanoparticle formulations for specific organs, this invention introduces a targeting index as an evaluation indicator. The targeting index is defined as the ratio of the nucleic acid barcode reading of the formulation in the target organ to its total reading in all tested organs. By comparing the targeting indices of different formulations, the superior formulations with specific organ targeting advantages can be screened.

[0041] Furthermore, the total length of the nucleic acid barcode described in step (1) is 59 nucleotides, of which the core coding region is 10 nucleotides at the center of the sequence, and the random error correction region is 8 random nucleotides at the 3' end of the core coding region, used for error correction in high-throughput sequencing. Each end contains 5 consecutive phosphate-thioester bonds and integrates an Illumina sequencing platform adapter sequence. The synthesized nucleic acid barcode needs to be purified. As a specific implementation method, the ULTRAPAGE method can be used for purification to obtain a high-purity product.

[0042] Further, the lipids MC3, DOTAP, DSPC, Chol, and DMG-PEG 2k used in step (2) were dissolved in anhydrous ethanol at standard atmospheric pressure and room temperature, and the total concentration of the lipid phase was kept constant at 12 mM. During the preparation process, the mass ratio of total lipids to nucleic acids was kept constant at 30:1, and the required concentration of nucleic acid barcodes was calculated accordingly. The aqueous phase solution was 50 mM citrate buffer with a pH of 4.0. The mass ratio of total lipids to nucleic acids can be adjusted from 20:1 to 40:1. The ionizable lipids in the formulation library are not limited to MC3 and DOTAP, and other lipids with ionizable properties or targeting functions can also be used for replacement or combination.

[0043] Furthermore, the core of the microfluidic mixing technology described in step (3) lies in using a microfluidic chip to achieve rapid and uniform mixing of the lipid phase and the aqueous phase, thereby forming nanoparticles with a concentrated particle size distribution. As a preferred embodiment, this step can be performed using an automated synthesis system integrating an injection pump and a microfluidic mixing chip (e.g., the FluidicLab LNP intelligent synthesizer). Before mixing, the microfluidic system needs to be pre-rinsed: for the lipid phase pipeline, ethanol is used to rinse to dissolve and remove organic residues; for the aqueous phase pipeline, deionized water and a buffer solution with a pH value close to that of the aqueous phase to be mixed are used sequentially to clean to remove impurities and wet the channels. After the mixing reaction is completed, it is recommended to use buffer solution, deionized water, and ethanol again to sequentially clean the entire system (including the chip and common channels) to prevent blockage or cross-contamination caused by lipid or nucleic acid residues. The channel size and structure of the microfluidic mixing chip can be selected according to the target particle size.

[0044] Further, step (4) includes the following specific procedures: 1. Preliminary determination: Within 10 minutes of collecting the initial product, it is diluted with an acidic buffer (e.g., 50 mM citrate buffer, pH 4.0) with the same pH as the aqueous phase used for preparation, and its hydrated particle size and polymer polydispersity index (PDI) are determined using dynamic light scattering technology to quickly assess the mixing effect. 2. Dialysis purification: The initial product is placed in a dialysis device with an appropriate molecular weight cutoff (e.g., using a... A MINI dialysis apparatus (10K MWCO) was immersed in 1× PBS buffer (pH 7.4) and dialyzed on a shaker at 4 °C. The dialysate was changed after 2 hours, and dialysis was continued for at least 12 hours (e.g., overnight). 3. Characterization of the final product. The Zeta potential was determined using a dynamic light scattering instrument (e.g., Malvern Zetasizer Nano ZS) on the dialyzed product. The nucleic acid encapsulation efficiency was determined using a fluorescence quantitative method based on nucleic acid dyes. For example, a highly sensitive single-stranded DNA fluorescent dye kit (e.g., ...) was used. The LNP sample was analyzed using an ssDNA quantitative kit. First, the standards provided in the kit were diluted with 1×PBS buffer to create a concentration gradient, and two standard curves were prepared: one without Triton X-100, used to calculate the free nucleic acid concentration; the other with 2% Triton X-100 added (consistent with the Triton concentration in the demulsified sample system), used to calculate the total nucleic acid concentration. The fluorescence intensity of each concentration of standard was measured after incubation with OliGreen working solution, and two standard curves were plotted. Subsequently, the LNP sample was diluted 50-fold with 1×PBS buffer, and two equal volumes of the diluted solution were prepared: one was directly incubated with OliGreen working solution in the dark, and the fluorescence intensity (F) of the free nucleic acid was measured. free Substitute the values ​​into the standard curve without Triton to calculate the free nucleic acid concentration (C). free In another sample, an equal volume of Triton X-100 (final concentration 4%) was added and mixed. The mixture was then incubated at room temperature for 5 minutes to break the emulsion and release the encapsulated nucleic acid. At this point, the final Triton concentration in the system was 2%. The mixture was then incubated with OliGreen working solution, and the total nucleic acid fluorescence intensity (F) was measured. total Substitute the values ​​into the standard curve containing 2% Triton to calculate the total nucleic acid concentration (C). total Encapsulated nucleic acid concentration (Cencapsulated) is calculated according to C. total - C free Encapsulation efficiency (EE) is calculated using the following formula: Formula 1, The target encapsulation efficiency should be no less than 80%. Fluorescence measurement can be performed using a multi-functional microplate reader.

[0045] Further, the experimental animals described in step (5) are 7-10 week old female C57BL / 6J mice, with at least 3 mice in each group. The total injection dose of the lipid nanoparticle mixture is 0.5 mg / kg based on the total amount of nucleic acid barcodes. Six hours after administration via tail vein injection, liver, spleen, lung, and other required organ tissue samples are collected. The collected tissue samples are rapidly frozen in liquid nitrogen and then stored at -80 °C for subsequent nucleic acid extraction and high-throughput sequencing analysis. The exposure time can be adjusted within 4 to 24 hours according to the research objectives; the injection dose can vary from 0.1 to 2 mg / kg; and the selected animal model can be replaced with other strains or species according to research needs.

[0046] Further, the DNA extraction in step (6) specifically includes: taking approximately 20 mg of tissue sample ground with liquid nitrogen and extracting it according to the instructions of the selected DNA extraction kit (e.g., Rapid DNA Extraction Kit, CWbio). The extracted DNA is purified and concentrated using a nucleic acid purification and concentration reagent column (e.g., Oligo Clean & Concentrator, Zymo Research) to improve the efficiency of subsequent amplification. The PCR amplification uses forward primers containing Illumina adapter sequences and a set of reverse primers containing different 8-nucleotide organ barcodes. The PCR reaction system uses a high-fidelity PCR premixed reaction system (e.g., 2 × HiAmp PCR Master Mix, Cwbio). The amplification program is set as follows: ① 95 ℃ pre-denaturation for 2 minutes; ② Cyclic amplification: 95 ℃ for 15 seconds, 67 ℃ for 20 seconds, 72 ℃ for 15 seconds, for a total of 30 cycles; ③ 72 ℃ final extension for 5 minutes. The product purification process involved the following steps: PCR products were separated by electrophoresis on a 3.0% agarose precast gel (Solarbio) in 1×TAE buffer at 150 mV for 40 min. The target band (approximately 142 bp) was excised and purified using an agarose gel DNA recovery kit (e.g., Gel DNA Recovery Kit, Zymo Research). The purified DNA library can be stored at -20 °C for later use.

[0047] Further, the library quality control in step (7) can be performed using a bioanalyzer (such as the Agilent Bioanalyzer) to confirm that the library fragment size and concentration meet the requirements for sequencing. The high-throughput sequencing is performed on the Illumina NovaSeq6000 platform, using paired-end sequencing mode (e.g., 2×150 bp), with a sequencing depth of no less than 1 Gb (Gigabases) per library to meet the statistical requirements for subsequent quantitative analysis. The data analysis includes the following main steps: 1. Raw data quality assessment and preprocessing: The raw FASTQ format data obtained after sequencing is assessed for quality, including the number of reads, base count, GC content, and Q20 / Q30 base ratio, with a target Q30 base ratio greater than 80%. Data filtering is performed using quality control software (such as Fastp) to remove low-quality reads and adapter sequences, and tools such as R language can be used to perform visualization analysis of sequencing error rate distribution. 2. Nucleic Acid Barcoding Quantification and Normalization: Quality-controlled sequencing reads are categorized and counted based on their core coding regions and organ-specific barcodes. To remove background and non-specific signals, readings from a naked b-DNA control can be used for correction. Subsequently, the relative abundance (RA) of each lipid nanoparticle formulation (corresponding to a specific nucleic acid barcode) delivered to a given tissue is calculated using the following formula: Equation 2, In the formula, Count barcode,tissue For the reading of a specific nucleic acid barcode in this organization, Count total,tissue This represents the total reading of all nucleic acid barcodes in the tissue. To quantify the targeting selectivity of different LNP formulations for specific organs, this invention introduces a targeting index as an evaluation metric. The targeting index is defined as the ratio of the nucleic acid barcode reading of the LNP formulation in the target organ to its total reading in all tested organs, calculated using the following formula: Formula 3, The data processing and calculation can be implemented through sequencing analysis software (such as PilotEdit Lite) or custom scripts (such as those based on Python or R language) to ultimately obtain the target distribution profile of each LNP formulation in different organs.

[0048] This invention relates to the fields of nanobiotechnology and drug delivery, and more specifically, to an innovative high-throughput methodology and dedicated platform for the parallel and quantitative evaluation of the organ distribution characteristics of multiple lipid nanoparticle formulations in a single in vivo experiment, thereby enabling rapid and targeted screening and optimization.

[0049] The advantages of this invention compared to the prior art are as follows: (1) Highly efficient parallel screening capability: This invention achieves simultaneous in vivo evaluation of multiple LNP formulations under the same physiological environment by designing and synthesizing unique nucleic acid barcodes for different LNP formulations. This technical feature revolutionizes the traditional single-threaded mode of "in vitro screening, in vivo injection one by one, and analysis one by one" into a high-throughput parallel mode of "one experiment, multiple comparisons", which improves the screening efficiency by several orders of magnitude, while significantly reducing the error caused by the number of experimental animals used and individual differences.

[0050] (2) Precise Quantification and Structure-Activity Relationship Analysis Capability: This invention transforms complex biological distribution information in vivo into precisely quantifiable sequencing reads through nucleic acid extraction, library construction, and high-throughput sequencing of tissue samples. By calculating the relative abundance of each formulation in specific organs and the organ targeting index, it can quantitatively reveal the structure-activity relationship between lipid composition (such as the proportion of cationic lipids) and organ targeting efficiency (such as targeting of the lung, liver, and spleen) with unprecedented sensitivity and accuracy, providing direct and reliable data support for the rational design of LNPs with specific targeting functions.

[0051] (3) Standardized and reproducible preparation process: This invention uses microfluidic mixing technology as the core preparation method. By precisely controlling key parameters such as the flow rate ratio of the lipid phase to the aqueous phase and the total flow rate, stable control of LNP particle size, polydispersity index (PDI), and encapsulation efficiency is achieved. This standardized "on-chip synthesis" process overcomes the batch-to-batch variation of traditional manual preparation methods, ensuring the consistency of the basic physicochemical properties of different batches and different formulations of LNP in the screening experiment, thereby ensuring that the final in vivo distribution differences mainly stem from formulation design rather than process fluctuations.

[0052] (4) Complete and scalable technology platform: This invention provides a complete solution from nucleic acid labeling, lipid nanoparticle preparation, in vivo exposure to data analysis. This technology platform has excellent modularity and scalability: the barcode library can be easily expanded as needed; the microfluidics and characterization modules are universal; and the analysis process can be automated. Therefore, the method described in this invention is not only applicable to LNP formulation screening based on selective organ targeting strategies, but can also be directly extended to the screening of carriers carrying other nucleic acid drugs such as siRNA and mRNA, as well as the broader field of in vivo nanomedicine fate research, demonstrating strong versatility and broad application prospects. Attached Figure Description

[0053] Figure 1 This is a schematic diagram illustrating the overall technical route and workflow of the integrated high-throughput screening system described in this invention.

[0054] Figure 2This is a schematic diagram of the molecular structure design of the nucleic acid barcode (DNA) used in this invention.

[0055] Figure 3 Characterization data for lipid nanoparticles loaded with different nucleic acid barcodes prepared using microfluidic technology, including hydrated particle size, polymer dispersibility index (PDI), and zeta potential.

[0056] Figure 4 The encapsulation performance data of lipid nanoparticles prepared using microfluidic technology are presented, showing the encapsulation DNA concentration ( Figure 4 a) and nucleic acid encapsulation rate ( Figure 4 b).

[0057] Figure 5 To demonstrate the feasibility of high-throughput parallel screening and the differences in formulation distribution among different organs, a heatmap of the relative abundance distribution of LNP formulations with six different DOTAP ratios in mouse lungs, liver, spleen, heart, and kidneys was generated to illustrate the application of the method described in this invention.

[0058] Figure 6 A bar chart showing the lung targeting index analysis of LNP formulations with different DOTAP ratios. The figure shows… "The lung targeting index of LNP 5 (40% DOTAP) was significantly different from that of the baseline formulation LNP 1 (0% DOTAP) (p<0.01).

[0059] Figure 7 The target index stacking bar charts for LNP formulations with different DOTAP ratios in multiple organs (lung, liver, spleen, heart, and kidney) demonstrate how formulation changes reshape the overall in vivo distribution profile, further revealing the structure-activity relationship between formulation and distribution. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] The following examples used statistical software to process the data. The experimental results are expressed as mean ± standard deviation. GraphPad software was used for graph analysis, and a one-way ANOVA test was performed. P < 0.05 ( () indicates a significant difference, P < 0.01. () indicates a highly significant difference, P < 0.001. () indicates a highly significant difference.

[0063] Example 1. Integrated high-throughput method flow for in vivo targeted screening of lipid nanoparticles 1. Design, synthesis, and quality control of nucleic acid barcodes (b-DNA).

[0064] First, seven single-stranded DNA strands, each 59 nucleotides in length, were designed and synthesized as nucleic acid barcodes. The structure of the nucleic acid barcodes is as follows: Figure 1 As shown, the structure of each nucleic acid barcode is uniform: the 5' end is a universal site 1 (sequence), containing 5 consecutive phosphate-thiocarboxyl bonds (indicated by " "). The sequence is defined by a 10-nucleotide core coding region (sequence), followed by an 8-nucleotide random sequence region (random error correction region, used for sequencing error correction), and finally a universal site 2 (sequence) with five consecutive phosphate-thioester bonds at the 3' end. The core coding region sequences of the seven nucleic acid barcodes are different, as shown in Table 1. Barcodes 1 to 6 are used to label six different formulations LNP 1 to LNP 6 (as shown in Table 2 in this embodiment), while Barcode 7 serves as a naked nucleic acid barcode control. All nucleic acid barcodes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and purified using the ULTRAPAGE method.

[0065] Table 1. Nucleic acid barcode sequences

[0066] Note: Bold nucleotide sequences in the table represent core coding regions, and underlined nucleic acid sequences represent random sequence regions. "" indicates that each nucleoside link is a phosphate thioester bond. Nucleotides 1-21 of each nucleic acid barcode sequence are universal site 1 sequences, and nucleotides 40-59 are universal site 2 sequences.

[0067] 2. Construction of lipid nanoparticle formulation library and lipid phase formulation.

[0068] First, a LNP formulation library with a DOTAP molar percentage gradient from 0% to 50% was established, and its specific lipid composition is shown in Table 2. All lipids (MC3, DOTAP, Chol, DSPC, DMG-PEG 2000) were derived from AvantiResearch with a purity >99%. Subsequently, according to the molar percentage of each lipid in Table 2, each component was dissolved in anhydrous ethanol to prepare six lipid-ethanol stock solutions with a total concentration of 12 mM, which constitute the lipid phase.

[0069] Table 2. Lipid composition (molar percentage) of the LNP formulation library

[0070] 3. Calculate the nucleic acid barcode concentration and prepare the aqueous phase.

[0071] 3.1 Calculation of nucleic acid barcode concentration First, based on the total lipid to nucleic acid mass ratio (30:1), the lipid phase to aqueous phase volume mixing ratio (1:3), and the total lipid phase concentration (12 mM), the required nucleic acid barcode concentration for each formulation was calculated. For example, for LNP 4 (30% DOTAP formulation), based on its lipid composition (the molar percentages of MC3, DOTAP, DSPC, Choli, and DMG-PEG 2k are 35%, 30%, 7%, 26.9%, and 1.05%, respectively) and the molecular weight of each component, the mass concentration of the 12 mM lipid phase was calculated to be approximately 7336.61 mg / L. Therefore, the working concentration of its corresponding nucleic acid barcode (Barcode 4) should be determined to be 81.52 mg / L. The barcode concentrations for other formulations are calculated according to this principle, resulting in working solution concentrations of 76.57 mg / L, 78.26 mg / L, 79.89 mg / L, 81.52 mg / L, 83.20 mg / L, and 84.75 mg / L for Barcode 1, Barcode 2, Barcode 3, Barcode 4, Barcode 5, and Barcode 6, respectively, for labeling LNP 1, LNP 2, LNP 3, LNP 4, LNP 5, and LNP 6.

[0072] 3.2 Preparation of the aqueous phase The solution used to dissolve the nucleic acid barcodes is a 50 mM citrate buffer (pH 4.0). Its preparation method is as follows: In a clean bench, prepare 100 mM anhydrous citric acid (C805019, Macklin) and 100 mM sodium citrate dihydrate (S818273, Macklin) solutions using nuclease-free water. Mix 16.5 mL of the citric acid solution with 8.5 mL of the sodium citrate solution, precisely adjust the pH to 4.0 with 1 mol / L sodium hydroxide solution, and finally bring the volume to 50 mL with nuclease-free water. Finally, dissolve each nucleic acid barcode separately in this buffer solution to prepare an aqueous solution of the corresponding concentration.

[0073] 4. Prepare nucleic acid barcode-labeled lipid nanoparticles using microfluidic mixing technology.

[0074] The FluidicLab LNP-S1 intelligent synthesizer and its accompanying LNP lipid nanoparticle volume mixing COC microfluidic chip (with Luer port) were used. The specific process is as follows: First, perform system preparation and pre-rinsing. Rinse the lipid phase tubing with ethanol, and then rinse the aqueous phase tubing with deionized water and 50mM citrate buffer (pH 4.0).

[0075] Subsequently, sample loading and mixing were carried out: each of the six lipid phases (ethanol solutions) prepared in step 2 and the aqueous phase containing the corresponding nucleic acid barcode prepared in step 3 were loaded into an independent injection system driven by an injection pump.

[0076] The total flow rate was controlled at 12 mL / min, and the flow rate ratio of the lipid phase to the aqueous phase was adjusted to 1:3 (corresponding to a volume mixing ratio of 1:3). The mixing program was started, and the initial lipid nanoparticle product was collected in a sterile container. After each preparation, the entire flow path system was cleaned sequentially with citrate buffer, deionized water, and ethanol to prevent cross-contamination.

[0077] By repeating the above process, six lipid nanoparticles loaded with different nucleic acid barcodes (Barcode 1 to Barcode 6) can be prepared sequentially (LNP1-Barcode1, LNP2-Barcode2, LNP3-Barcode3, LNP4-Barcode4, LNP5-Barcode5 and LNP6-Barcode6).

[0078] 5. Purification and characterization of LNP.

[0079] 5.1 Preliminary characterization: Within 10 minutes of collecting each LNP primary product (LNP-Barcode), 100 μL of the primary product was added to 400 μL of 50 mM citrate buffer (pH 4.0) and diluted 5-fold. The average hydrated particle size (Z-average) and polymer polydispersity index (PDI) were immediately determined using a Malvern Zetasizer Nano ZS dynamic light scattering instrument.

[0080] 5.2 Dialysis purification: The remaining primary products were quickly transferred to Add 14 mL of 1×PBS buffer (pH 7.4) to the MINI dialysis apparatus (10K MWCO) and place it on a shaker at 4 ℃. Change the dialysis buffer after 2 hours and continue dialysis overnight (>12 hours).

[0081] 5.3 Characterization of the final product: After dialysis, the Zeta potential of the LNP final product was measured using a dynamic light scattering instrument. The concentration and encapsulation efficiency of the encapsulated nucleic acid were determined using... The OliGreen ssDNA quantification kit was used for assay. First, the standards provided in the kit were diluted to a concentration gradient using 1×PBS buffer to prepare two standard curves: one without Triton X-100, used to calculate the free nucleic acid concentration; the other with a final concentration of 2% Triton X-100 (consistent with the Triton concentration in the demulsified sample system), used to calculate the total nucleic acid concentration. The fluorescence intensity of each concentration of standard was measured after incubation with OliGreen working solution, and two standard curves were plotted. Subsequently, the LNP sample to be tested was diluted 50-fold with 1×PBS buffer, and two equal volumes of the dilution were taken: one was directly incubated with OliGreen working solution in the dark, and the fluorescence intensity (F) of the free nucleic acid was measured. free Substitute the values ​​into the standard curve without Triton to calculate the free nucleic acid concentration (C). free In another sample, an equal volume of Triton X-100 (final concentration 4%) was added and mixed. The mixture was then incubated at room temperature for 5 minutes to break the emulsion and release the encapsulated nucleic acid. At this point, the final Triton concentration in the system was 2%. The mixture was then incubated with OliGreen working solution, and the total nucleic acid fluorescence intensity (F) was measured. total Substitute the values ​​into the standard curve containing 2% Triton to calculate the total nucleic acid concentration (C). total Encapsulated nucleic acid concentration (Cencapsulated) is calculated according to C. total - C free Encapsulation efficiency (EE) is calculated using the following formula: Formula 1, Fluorescence signals were detected using a multi-functional microplate reader with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0082] The six prepared LNPs (LNP1-Barcode1 to LNP6-Barcode6) were systematically characterized, and detailed results on their hydration particle size, polydispersity index (PDI), zeta potential, and nucleic acid encapsulation performance are as follows: Figure 3 and Figure 4 As shown.

[0083] like Figure 3 As shown, the LNPs prepared in this invention all exhibit excellent colloidal properties. Specifically, the particle size of all formulations is distributed within a suitable range of 75-115 nm, and the PDI value is below 0.15, indicating that uniformly sized and concentrated nanoparticles were successfully prepared using microfluidic technology. Figure 3 (Top-middle figure). Zeta potential measurements showed that the surface charge of LNPs exhibited a regular change with the adjustment of the proportion of cationic lipid DOTAP in the formulation. LNP 1 (MC3 baseline formulation) without DOTAP had a particle size of approximately 79 nm and exhibited a near-neutral surface charge (+1.51 mV). As the DOTAP proportion increased from 10% to 30% (LNP 2 to LNP 4), the particle size underwent a process of first increasing and then significantly decreasing, while the Zeta potential gradually increased from +1.32 mV to +5.52 mV, confirming that the introduction of cationic lipids effectively enhanced the positive charge of the particle surface. When the DOTAP proportion continued to increase to 40% and 50% (LNP 5 and LNP 6), the particle size stabilized in the 80-90 nm range, while the surface positive charge remained at a relatively high level of approximately +6 mV. It is worth noting that LNP 3 (20% DOTAP) had the largest particle size (~113 nm), and its PDI (0.08) and Zeta potential (+2.18 mV) also fluctuated relatively greatly. This suggests that under this specific ratio, there may be a transitional state between the uniformity and stability of lipid assembly. Figure 3 (Lower middle image).

[0084] At the same time, such as Figure 4 As shown, all LNPs achieved efficient nucleic acid encapsulation. The nucleic acid encapsulation efficiency ranged from 83.75% to 97.24%. Figure 4 (b) Furthermore, with the increase in the proportion of DOTAP, the encapsulation efficiency generally showed an upward trend, with encapsulation efficiencies of LNP 4 to LNP 6 all exceeding 94%. The encapsulation nucleic acid concentrations of each LNP ranged from 28.20 ng / μL to 48.66 ng / μL (b). Figure 4(a) This provides sufficient drug loading for subsequent in vivo delivery. Overall, the composition of lipid nanoparticles (especially the proportion of cationic lipids DOTAP) significantly affects their hydrated particle size, surface charge, colloidal uniformity, and nucleic acid encapsulation efficiency. The method of this invention successfully prepared a library of LNP formulations with gradient physicochemical properties and excellent encapsulation performance. This series of LNPs possesses suitable nanoparticle size, low PDI, tunable surface potential, and extremely high nucleic acid encapsulation efficiency, meeting the requirements for high-throughput parallel screening in vivo based on nucleic acid barcoding.

[0085] 6. Pool-style mixed animal exposure and sample collection.

[0086] Animal experiments were conducted with the approval of the relevant ethics committee. Female C57BL / 6J mice aged 7-10 weeks were randomly divided into groups of three. The six types of nucleic acid barcode-labeled lipid nanoparticles (LNP1-Barcode1 to LNP6-Barcode6) purified and characterized in step 5, along with the naked Barcode 7 control, were mixed according to their encapsulated nucleic acid concentrations and equal nucleic acid masses to prepare a pooled exposure mixture. Mice were exposed by tail vein injection at a dose of 0.5 mg / kg (approximately 150 μL per mouse) based on the total nucleic acid mass. Six hours after injection, the mice were euthanized, perfused with pre-cooled 1×PBS buffer (pH 7.4), and immediately dissected to collect tissue samples from the liver, spleen, lungs, heart, and kidneys. The collected tissue samples were rapidly flash-frozen in liquid nitrogen and then transferred to a -80 °C cryopreservation system for subsequent analysis.

[0087] 7. Tissue DNA extraction and sequencing library construction.

[0088] 7.1 DNA Extraction and Purification: The frozen tissue sample obtained in step 6 was homogenized in liquid nitrogen. Approximately 20 mg of the homogenate was weighed and total DNA was extracted using a commercial DNA extraction kit (such as the Rapid DNA Extraction Kit, Cwbio). Subsequently, the extracted DNA was purified and concentrated using a nucleic acid purification and concentration column (such as Oligo Clean & Concentrator, Zymo Research) to improve the efficiency of subsequent amplification.

[0089] 7.2 PCR amplification: Using purified DNA as a template, amplification was performed using a high-fidelity PCR premixed reaction system (such as 2×HiAmp PCR MasterMix, Cwbio) to construct sequencing libraries.

[0090] The forward primers contained Illumina P5 sequencing adapter sequences with the structure: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT CTTCCGATCT-3'. To distinguish different tissue origins, a set of reverse primers containing 8-nucleotide organ barcode sequences (as shown in Table 3) were used to amplify DNA samples from different organs independently. The PCR amplification program was set as follows: 95 ℃ pre-denaturation for 2 minutes; cyclic amplification: 95 ℃ for 15 seconds, 67 ℃ for 20 seconds, 72 ℃ for 15 seconds, for a total of 30 cycles; final extension at 72 ℃ for 5 minutes.

[0091] 7.3 Product purification and library homogenization: PCR products were separated by electrophoresis on a 3.0% agarose gel (Solarbio) in 1×TAE buffer at 150 mV for 40 min. The target band (approximately 142 bp) was excised and purified using an agarose gel DNA recovery kit (e.g., Gel DNA Recovery Kit, Zymo Research). The purified organ libraries were then mixed according to the principle of equal quality to form the final sequencing library pool, which was stored at -20 °C.

[0092] Table 3. Reverse primer sequences

[0093] Note: The bolded nucleotide sequences in the table are organ barcode region sequences.

[0094] 8. High-throughput sequencing, data analysis, and in vivo distribution results.

[0095] The library quality control described in step 7 can be performed using a bioanalyzer (such as the Agilent Bioanalyzer) to confirm that the library fragment size and concentration meet the requirements for sequencing. After passing the quality control, paired-end sequencing (2×150 bp) is performed on the Illumina NovaSeq 6000 platform. The sequencing depth of each library is no less than 1 Gb (Gigabases) to meet the statistical requirements of subsequent quantitative analysis. After the sequencing data is quality controlled by Fastp (v0.23.1) (Q30>85%), the organ index and nucleic acid barcodes are parsed using data analysis scripts or software (in this embodiment, a self-written Python script combined with PilotEdit Lite software) to obtain the raw readings of all nucleic acid barcodes in each organ library.

[0096] To obtain comparable target distribution data, the following steps were performed: ① Background subtraction: The raw readings of each nucleic acid barcode (Barcode 1-6) in each organ library were subtracted from the readings of the naked DNA control (Barcode 7) in the same library to obtain the background-corrected readings of each nucleic acid barcode (the number of sequencing reads corresponding to each nucleic acid barcode). ② Organ abundance normalization: To eliminate systemic differences in DNA extraction, PCR amplification, and sequencing depth among samples from different organs, the background-corrected readings were converted into relative abundance within that organ. The calculation formula is as follows: Equation 2, In the formula, Count barcode,tissue Count is the reading of a specific nucleic acid barcode in that organ. total,tissue This represents the total reading of all nucleic acid barcodes in the organ. Taking the Lung_1 organ library as an example, after background correction, the readings of each nucleic acid barcode are as follows: Barcode 1: 61, Barcode 2: 106, Barcode 3: 549, Barcode 4: 277, Barcode 5: 117, Barcode 6: 625. Based on the above formula, the relative abundances of the corresponding LNPs in the Lung_1 organ library are LNP 1: 3.49%, LNP 2: 6.11%, LNP 3: 31.66%, LNP 4: 15.94%, LNP 5: 6.77%, and LNP 6: 36.02%. Through the above analysis, the relative abundance spectra of all LNP formulations in different organs can be obtained and displayed in heatmap form. Figure 5 .

[0097] 9. Targeted assessment.

[0098] To quantify the targeting selectivity of different LNP formulations for specific organs, this study introduced the targeting index as an evaluation metric. The targeting index is defined as the ratio of the nucleic acid barcode readings of the LNP formulation in the target organ to its total readings across all tested organs, calculated using the following formula: Formula 3, This index directly reflects the relative selectivity of LNP delivery to a specific tissue: a higher targeting index value indicates a stronger enrichment capacity of the LNP in that tissue, i.e., superior targeting. Focusing on this core indicator of lung targeting index, this invention observes that the introduction of the cationic lipid DOTAP has a non-linear effect on lung targeting with a clearly defined optimal window. Figure 6As shown, the lung targeting index exhibits a non-linear change as the DOTAP ratio increases from 0% to 50%. Crucially, when the DOTAP ratio is 40% (corresponding to LNP 5), the lung targeting index reaches a peak of 31.29% ± 3.04%, showing a highly significant statistical difference (p<0.01) compared to the baseline formulation LNP 1 (14.87% ± 3.93%) without DOTAP. Formulations with other DOTAP ratios also show a fluctuating increase in the lung targeting index (12.20% - 21.78%). This result clearly demonstrates that in the LNP system constructed in this invention, there exists an optimal ratio window centered around approximately 40% DOTAP, which can maximally and specifically enhance the lung tissue targeting ability of nanoparticles. In addition to lung targeting, the introduction of DOTAP systematically alters the overall in vivo distribution pattern of LNPs. Figure 7 As the proportion of DOTAP increases, liver uptake initially increases and then decreases, dropping to 16% at LNP 5, suggesting that this formulation may have better liver escape properties. Spleen uptake reaches its lowest value (10%) at LNP 5, indicating a potential risk of low activation of the immune system. The distribution in the heart and kidneys also adjusts accordingly, reflecting a rebalancing of the systemic distribution. Notably, while achieving the highest lung targeting (31.29%), LNP 5 has a relatively low total liver and spleen accumulation (26%) among all formulations, exhibiting an ideal distribution characteristic of "increased lung targeting and decreased liver and spleen accumulation." It not only has a statistically significant improvement in lung targeting but also a more optimized overall distribution spectrum.

[0099] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for identifying the in vivo targeting ability of lipid nanoparticles, characterized in that: The method includes the following steps: 1) Prepare a mixture containing A types of lipid nanoparticles, where A is a natural number greater than or equal to 2; each type of lipid nanoparticle in the mixture is a nanoscale particle obtained by phase mixing an aqueous phase containing a nucleic acid barcode P with a lipid phase containing a lipid mixture; the nucleic acid barcode P is a single-stranded DNA molecule containing a core coding region and a random error correction region; the core coding region and the lipid mixture are different between each pair of lipid nanoparticles in the mixture, and the mass ratio of each lipid mixture to the nucleic acid barcode P mixed with it is the same; The random error correction region is a sequence randomly composed of N, where N is A, T, C, or G; 2) The mixture is used to administer drugs to experimental animals to obtain exposed experimental animals. Samples of different organs or tissues of the exposed experimental animals are obtained. DNA is extracted from the samples of different organs or tissues to obtain sequencing libraries of each organ or tissue. The sequencing libraries of each organ or tissue are mixed in equal amounts and then sequenced to obtain sequencing data. The sequencing data is analyzed, and the relative abundance spectrum of the lipid nanoparticles A in the samples of different organs or tissues is obtained based on the number of sequencing reads corresponding to various nucleic acid barcodes P. The targeting of lipid nanoparticles A in vivo is determined based on the relative abundance spectrum.

2. The method according to claim 1, characterized in that: The core coding region has a sequence length of 6-12 nucleotides, and the random error correction region has a sequence length of 6-10 nucleotides.

3. The method according to claim 1 or 2, characterized in that: The 5' end of the nucleic acid barcode P contains sequence P1, in which each nucleoside is linked by a phosphate thioester bond; the 3' end of the nucleic acid barcode P contains sequence P2, in which each nucleoside is linked by a phosphate thioester bond.

4. The method according to any one of claims 1-3, characterized in that: The nucleic acid barcode P also includes a sequencing adapter sequence, which is used to complement the adapter sequence of the sequencing platform corresponding to the sequencing.

5. The method according to any one of claims 1-4, characterized in that: The method further includes a step of performing PCR amplification on the DNA, wherein the primers for the PCR contain organ barcode region sequences, which are used to distinguish the different organ or tissue samples.

6. The method according to any one of claims 1-5, characterized in that: The method further includes a step of purifying and dialysis the lipid nanoparticles MP before the equal-volume mixing, wherein the purification and dialysis includes purifying and dialysis the lipid nanoparticles MP using a buffer solution at 4°C.

7. Lipid nanoparticles, characterized in that: The lipid nanoparticles are those described in claim 1.

8. A DNA molecule, characterized by: The DNA molecule is the nucleic acid barcode P as described in claim 1.

9. Any of the following applications of the lipid nanoparticles of claim 7 and / or the DNA molecule of claim 8: 1) Application in the development or preparation of in vivo targeted screening products for lipid nanoparticles; 2) Applications in the development or preparation of drug delivery products.

10. Use of the method according to any one of claims 1-6 in the development or preparation of drug delivery products.

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