Universal method for evaluating intracellular drug release performance of carrier drug conjugate

Abstract

The invention belongs to the technical field of drug release, and relates to a universal method for evaluating the intracellular drug release performance of a carrier drug conjugate. According to the method, a delivery carrier is used as a dynamic reference object, the total amount of drugs entering cells is corrected, quantitative monitoring of prototype drugs in different forms in the cells is completed, and then based on the law of conservation of substances, effective evaluation of the intracellular drug release rate of the CDC is finally achieved. According to the method, different forms of intracellular drugs can be effectively monitored by applying a common substance quantification technology, so that the tedious quantification process of potential metabolites and extracellular efflux drugs in a traditional method is avoided, the experiment efficiency and accuracy are greatly improved, the intracellular drug release process is easily monitored, and the method is suitable for large-scale popularization and application. The high-throughput screening of the carrier drug conjugate key connexon is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug release, and relates to a general method for evaluating the intracellular drug release performance of a carrier-drug conjugate. Background Art

[0002] Compared with traditional chemotherapy, the carrier-drug conjugation strategy represented by antibody-drug conjugates (ADCs) has significantly improved the efficacy and safety of chemotherapy drugs, and there is strong market demand. So far, 15 ADC products have been approved for marketing globally. According to market analysis, by 2030, the global ADC market is expected to reach $40 billion. However, after nearly three years of rapid development, the ADC track has now become saturated, and the homogenization of popular targets is extremely serious. How can we break through the circle? We must have differentiated core technologies.

[0003] A carrier-drug conjugate (CDC) is a drug delivery system formed by conjugating a drug to a tumor-targeting delivery carrier through a linker, which has the functions of improving the circulation stability of the drug and increasing the uptake of target cells. However, the stable conjugation effect poses a risk of reducing the effective exposure of the prototype drug inside the target cells. Therefore, evaluating whether the linker used has appropriate target cell-specific drug release performance is the key to CDC development. Investigating the drug release of CDC in an in vitro simulation medium is the main method at present, which can indeed reflect the essential chemical bond properties of the linker. However, a cell is a complex living organism, which contains various enzymes and active substances, and these factors may all affect drug release. Moreover, substances inside the cell basically exist in membrane-protected vesicle structures, such as lysosomes. However, there is currently no technology that can use these vesicles as the smallest research unit to quantitatively determine the concentration of active substances inside the vesicles. And our current in vitro simulation conditions are based on the overall situation of cell populations and even the distribution of active substances in tissues as a whole, which cannot accurately reflect the concentration of active substances in cells and inside vesicles. That is, the current in vitro simulation conditions may not be close to the real situation, especially for enzymes. The concentration of enzymes directly determines the magnitude of the maximum reaction rate value. Investigating the drug release situation on living cells is the most direct solution. However, living cells may metabolize or excrete the released free drug at any time. Even using the most advanced combined means of isotope labeling and mass spectrometry detection, it is still not possible to complete the work well. At least the amount excreted by cells is extremely low in the extracellular medium and is difficult to be quantified. Therefore, finding a simple, general and efficient method for monitoring intracellular drug release is the key point to break through the R & D bottleneck and achieve original technological innovation of CDC at present. Summary of the Invention

[0004] In order to overcome the bottleneck problem that "living cells will metabolize or excrete intracellular substances at any time, resulting in the inability to correctly quantify intracellular drugs" and achieve an effective evaluation of the "intracellular drug release performance" of the carrier-drug conjugate, during the search for possible solutions, the present invention noticed that drug entry into cells is mediated by a carrier and the carrier is usually more stable than the linker. Therefore, the present invention proposes a solution strategy: using the intracellular carrier as a dynamic reference object to correct the total amount of drug entering the cell. Because according to the law of conservation of matter, if the total amount of drug entering the cell is known, the present invention only needs to detect the measurable part in the cell, including the prototype drug and the drug still conjugated to the carrier, which is also the most interesting part of the present invention, that is, the task of monitoring intracellular release is completed, and there is no need to consider the cumbersome detection of the unknown part.

[0005] In order to overcome the shortcomings and deficiencies of the existing methods for detecting the intracellular drug release rate in the above-mentioned prior art, the primary object of the present invention is to provide a general method for evaluating the intracellular drug release performance of a carrier-drug conjugate. The most important feature of this method is to use the delivery carrier as a dynamic reference object to correct the total amount of drug entering the cell, and complete the quantitative monitoring of the prototype drugs in different forms in the cell. Then, based on the law of conservation of matter, the effective evaluation of the intracellular drug release rate of the CDC is finally achieved. In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: The general method for evaluating the intracellular drug release performance of a carrier-drug conjugate at least includes a release experiment step and a think tank research and judgment step; The release experiment step: The formal release evaluation experiment includes the following parts: (1) Conduct a cytotoxicity experiment of the carrier-drug conjugate to determine the appropriate cell administration concentration of the carrier-drug conjugate; (2) According to the labeling strategy of the carrier, select a matching detection method to conduct a cell uptake experiment of the carrier-drug conjugate to determine the appropriate time range for drug release research; (3) Based on the optimized conditions obtained from the above two experimental conclusions, conduct a formal drug release study. Among them, the carrier uptake amount represents the total amount of drug entering the cell, and the traditional quantitative detection method quantitatively monitors the free prototype drug in the cell and the prototype drug still conjugated to the delivery carrier. The obtained data is converted with a single cell as the smallest measurement unit for think tank research and judgment; The think tank research and judgment step: In order to be able to analyze the obtained data better and more accurately, the present invention combines reference documents and conducts a logical analysis from the perspectives of cell uptake, intracellular substance transport, extracellular excreted substances, etc., and establishes a conversion parameter Figure 2 of the intracellular carrier content and the total amount of drug entering the cell as shown of the research and judgment flow chart; before conducting the research and judgment, the present invention also needs to calculate the obtained data according to where Representing the calculated values at each time point Potential approximations Is the measurable carrier content in the cell Is the content of the carrier-conjugated form of the drug measurable in the cell Is the content of the free drug measurable in the cell, and then enters the flowchart for judgment. According to the judgment result, appropriate auxiliary judgment experiments are added; the process includes the following steps: First, judge whether all the calculated Values meet Condition 1, where Condition 1: Any The difference from the minimum value among them ( ) is less than Times the minimum value ( ), The value range of is between 0.01 and 0.1; In addition, (1) if the obtained Value meets Condition 1, that is, enter the next operation, increase the concentration of the incubation liquid medicine, carry out a new round of intracellular drug release research, and calculate the new According to ; At this time, it is necessary to compare whether there is a difference in the Values of the two experiments before and after; If the difference between the Values at the corresponding time points in the two experiments before and after is less than Times the minimum value ( ), The value range of is between 0.01 and 0.1, which means that the conditions in the first experiment meet The value can be approximated as The constraint condition, that is, the minimum value among the Values calculated in the first experiment is the Required by the present invention, and the judgment work on Is completed; If the difference between the Values of the two experiments before and after is large, significantly greater than Times the minimum value ( ), it means that the conditions in the first experiment do not meet The value can be approximated as The constraint condition, then it is necessary to further increase the concentration of the incubation liquid medicine or replace other control cells to carry out a new release experiment until the obtained Value drops to meet Condition 1. At the same time, for the uptake results obtained from the experiment, it is also necessary to ensure that the cell uptake of the drug meets the prerequisite condition of uptake acceleration ≥ 0; at this time, the minimum value among the Values calculated in the latest experiment is the target , and the judgment on Research work; (2) If only some of the obtained values meet Condition 1, it is necessary to further evaluate whether the obtained values meet Condition 2. Condition 2: A. Among the obtained series of values, the minimum value appears at the first detection time point, and the value at the last time point is the maximum value; B. Including the first time point, there are at least two consecutive time points where the value has a difference from the minimum value less than times the minimum value ( ), where the value range of is between 0.01 and 0.1; C. The difference between the maximum value of the value and the minimum value of the value is greater than times the minimum value of the value ( ), where the value range of is between 0.01 and 0.1, and the value range of is between 2 and 10; If the obtained value meets Condition 2, a judgment can be made that the minimum value among the obtained values in this experiment is the target , and the research work on is completed; values do not meet Condition 1, or some values meet Condition 1 but the values do not meet Condition 2, then it is necessary to further increase the concentration of the incubation liquid or replace other control cells to conduct a new release experiment until the obtained value drops to meet Condition 1 or Condition 2; at the same time, for the uptake results obtained from the experiment, it is necessary to ensure that the cell uptake of the drug meets the prerequisite that its uptake acceleration is greater than or equal to 0; at this time, the minimum value among the values calculated from the latest experiment is the target , and the research work on is completed; Finally, according to the determined Values are calculated for cell uptake, drug release, and drug elimination at each time point within the cell. Then, using time as the independent variable, polynomial curve fitting is performed. Starting from a linear equation, the degree of the fitting curve is incremented successively until the r2 of the fitting curve is greater than 0.98, at which point the process stops. The degree is selected for curve fitting, and the concentration curve fitting equation is obtained. Subsequently, the concentration-time curve equation is differentiated with respect to time to obtain the velocity-time equation, and software is used to convert the velocity-time equation into an image for intuitive analysis of subsequent release rates.

[0006] Preferably, before the release experiment step, there is also a carrier labeling step or a cell medium selection step; The carrier labeling step is as follows: Based on the physicochemical property characteristics of the delivery carrier itself, a suitable labeling method is selected, and a probe-labeled delivery carrier is prepared. Subsequently, an evaluation is carried out on the labeling stability and whether the labeling method affects the drug delivery performance of the carrier to determine the most suitable carrier labeling method; The cell medium selection step includes: First, potential target cell lines are selected according to the treatment requirements. Second, according to the basic chemical bond properties of the linker used in the carrier-drug conjugate, an evaluation method for the intracellular drug release response mechanism to be investigated is selected. Finally, immunofluorescence staining, addition of fluorescent probes, or Western Blot method is used to evaluate the content of active substances capable of triggering drug release in the target cell line and other cell lines in the in-house cell bank. Eventually, in addition to the target cells, control cell lines with high and low expression of active substances are screened out. Through statistical analysis based on the above semi-quantitative results, a suitable cell line is finally selected for subsequent investigation of the intracellular drug release process.

[0007] More preferably, before the release experiment step, there are also a carrier labeling step and a cell medium selection step.

[0008] The implementation process of the method including the carrier labeling step, cell medium selection step, release experiment step, and think tank research step is as Figure 1 shown.

[0009] More preferably, in the carrier labeling step, if the carrier is a polypeptide substance containing free amino groups, or a protein, polylactic acid, polylysine, or polyglutamic acid polymer, fluorescence labeling, biotin labeling, or stable metal isotope labeling can be used for labeling; Fluorescence labeling and biotin labeling can be directly coupled and labeled based on the amidation reaction of active esters with amino groups; The stable metal isotope labeling method requires connection modification with a bifunctional chelating agent such as isothiocyanatobenzyl diethylenetriaminepentaacetic acid (DTPA-SCN), isothiocyanatobenzyl ethylenediaminetetraacetic acid (EDTA-SCN), or isothiocyanatobenzyliminodisuccinic acid (IDHA-SCN); If the carrier forms a stable nanopreparation through hydrophobic interaction, the labeling can be carried out by encapsulating a hydrophobic probe; If the carrier is a commonly used protein carrier, it can also be directly used for subsequent detection without probe labeling.

[0010] Further preferably, for different carrier probe labeling methods, a matching labeling stability evaluation method is selected: For the fluorescence labeling method, a microplate reader and a flow cytometer are used as the stability evaluation methods; For the biotin labeling method, in combination with HRP-labeled streptomycin, the ELISA method is used for stability evaluation; For the stable isotope labeling method, an inductively coupled plasma mass spectrometer or an inductively coupled plasma atomic emission spectrometer is used for stability evaluation; For protein carriers without probe modification, in combination with the corresponding detection antibody pair, the ELISA method is used for stability evaluation.

[0011] Further preferably, in the carrier labeling step, regarding the evaluation of whether the labeling method will affect the drug delivery performance of the carrier, a combined scheme of in vivo pharmacokinetic investigation, cell-level pharmacodynamic evaluation, and evaluation of intracellular drug uptake and metabolism based on liquid chromatography-mass spectrometry (LC-MS) is adopted, or a combined scheme of cell-level pharmacodynamic evaluation and evaluation of intracellular drug uptake and metabolism based on LC-MS is carried out.

[0012] Further preferably, in the cell medium selection step: Immunofluorescence staining and western blot are used for semi-quantitative characterization of various proteases and glucosidases in cells. The proteases include but are not limited to cathepsin A, cathepsin B, cathepsin L, matrix metalloproteinase-2, matrix metalloproteinase-7, and the glucosidase is β-glucosidase or β-galactosidase; The fluorescence probe method is used for semi-quantitative characterization of reactive oxygen species, GSH, and protons in cells.

[0013] Preferably, in the cell toxicity evaluation in the release experiment step (1), dose-dependent cell proliferation inhibition is selected for investigation. The investigation time range is 24 - 96 h. The cell viability is detected using CCK-8, and then the IC50 of the carrier-drug conjugate is obtained through statistical analysis. After that, the drug concentration near the IC50 is selected as the dosing concentration for the first experiment.

[0014] Preferably, in the cell uptake experiment of the release experiment step (2): For the fluorescently labeled carrier, quantitative detection is performed by flow cytometry; For the biotin-labeled carrier, intracellular fluorescence staining is performed by combining with fluorescently labeled streptomycin, and quantitative detection is performed by flow cytometry; For the carrier labeled with stable isotopes, quantitative detection is performed by an inductively coupled plasma mass spectrometer or an inductively coupled plasma atomic emission spectrometer combined with a flow cytometer; For the protein carrier without probe modification, intracellular immunofluorescence staining is performed by combining with the corresponding detection antibody pair, and quantitative detection is performed by flow cytometry; Taking time as the abscissa and the carrier uptake amount as the ordinate, an uptake amount-time correlation graph is established. Subsequently, the inflection point of the change in the carrier uptake rate can be visually observed from the graph, and the time corresponding to the inflection point is judged. The time range from the 0 moment to this time point is the appropriate release research time range.

[0015] Preferably, in the release experiment step (3), within the appropriate release research time range determined in the above step (2), a formal intracellular drug release study is carried out at the first administration concentration determined in step (1), including the following steps: 1) The target cells and control cells are respectively amplified and cultured in a 6 - 10 cm culture dish in a 37°C carbon dioxide incubator until the number of culture dishes sufficient for the experiment is reached; 2) The culture dish is removed, and a working solution of the carrier-drug conjugate is prepared with a complete medium containing 10% FBS. During the formal experiment, 1 - 3 mL of the working solution is added to each culture dish, and at the same time, a control is set by adding an equal volume of 10% FBS complete medium without the carrier-drug conjugate; 3) Take 15 - 30 minutes after the start of drug administration incubation as the first detection time point, and then set another 5 - 10 detection time points at the same time interval, for a total of 6 - 11 time points with a time interval between 15 - 30 minutes. Collect at least 3 culture dishes at each time point. At each time point, wash the cells 3 times with cold PBS, then digest the cells on ice with 0.5 - 1 mL of 0.25% trypsin for 1 - 5 minutes, then add 1 mL of cold PBS for rinsing. After combining the cell samples, place them in a centrifuge at 4°C and centrifuge at 1500 rpm for 5 minutes. After aspirating the supernatant, resuspend the cells by adding 1 mL of cold PBS and centrifuge again for cell washing, for a total of 3 washes. When adding 1 mL of cold PBS for the last resuspension, take out 50 μL of the cell suspension and place it on ice for flow cytometry to detect the cell count. Centrifuge the remaining 950 μL again, discard the supernatant, and immediately freeze it in an -80°C refrigerator for storage. Wait until all samples are collected and then process them together. After all samples are collected, add cold PBS to the cell suspension placed on ice to make up to 250 μL, and then count the cell number with a flow cytometer. Set the flow rate to 50 - 200 μL / minute and the collection time to 1 - 4 minutes. For the cell samples stored at -80°C, thaw them and add 10 μL of an internal standard aqueous solution containing 0.5 - 5 μg / mL. The internal standard is determined according to the properties of the substance to be measured. Then, add 200 μL of methanol for protein precipitation. The methods for protein precipitation include vortexing for 5 - 20 minutes or ultrasonic treatment in an ultrasonic cleaner for 5 - 20 minutes. Then, centrifuge at 13000 rpm for 5 - 10 minutes at 4°C. Finally, gently aspirate 50 μL of the centrifuged supernatant and transfer it to a liquid phase vial with a liner tube, make a mark, and store it in a -20°C refrigerator for further testing. For the remaining sample containing the precipitate, according to the characteristics of the linker used, add 10 μL of a suitable chemical bond-breaking reagent, usually a strong acid, strong base, or enzyme, and use vortexing or ultrasonic method to make the drug fully fall off the carrier. Finally, centrifuge at 13000 rpm for 5 - 10 minutes at 4°C. Finally, gently aspirate 50 μL of the centrifuged supernatant and transfer it to a liquid phase vial with a liner tube, make a mark, and store it in a -20°C refrigerator for further testing. If an enzyme is used, after fully removing the methanol, resuspend the centrifuged precipitate with an enzyme digestion buffer solution, then add the enzyme and incubate for 24 hours, then centrifuge at 13000 rpm for 5 - 10 minutes. Finally, gently aspirate 50 μL of the centrifuged supernatant and transfer it to a liquid phase vial with a liner tube, make a good mark, and store it in a -20°C refrigerator for further testing. Finally, according to the type of drug, select LC-MS, inductively coupled plasma mass spectrometry, or inductively coupled plasma atomic emission spectrometry for quantitative detection.

[0016] Further preferably, if it is a fluorescently labeled vector in step 3), the fluorescence signal of the label is simultaneously acquired as the content of the intracellular entry vector; If it is a biotin-labeled vector, fluorescence-labeled streptomycin is combined for intracellular fluorescence staining, and the fluorescence signal used is collected as the content of the intracellular entry vector; If it is a vector labeled with stable isotopes, quantitative detection is carried out by an inductively coupled plasma mass spectrometer or an inductively coupled plasma atomic emission spectrometer combined with a flow cytometer as the content of the intracellular entry vector; If it is a protein vector without probe modification, the corresponding detection antibody pair is combined for intracellular immunofluorescence staining, and the fluorescence signal used is collected as the content of the intracellular entry vector.

[0017] Based on the basic understanding that drug entry into cells is mediated by a vector and usually the stability of the vector is better than that of the linker, the present invention proposes an innovative strategy of using the intracellular vector as a dynamic reference object to correct the total amount of intracellular drug in real time. By combining common substance quantitative detection methods, the intracellular drug release process of the conjugate of the vector and the drug is monitored "from top to bottom", and the key parameters for coordinate transformation of the reference system established based on the previous theoretical analysis The judgment logic flow chart is used to judge whether it can truly reflect the intracellular drug release performance of the substance. For the experimental results that do not meet the conditions, improvement measures are proposed to finally meet the research requirements. The application of this technology will help to break through the bottleneck in the research and development of conjugates of the vector and the drug at the present stage and achieve the original innovation of the technology.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) So far, there has been no relevant technical report at home and abroad. This technology is an innovative strategy based on the understanding of the underlying logic of the intracellular drug release process, which can effectively solve the problem of "incorrect quantification of intracellular drugs caused by the metabolism or efflux of intracellular drugs by living cells", and belongs to an innovative technology.

[0019] (2) Based on the "from top to bottom" innovative strategy of the present invention, by using common substance quantitative techniques, the effective monitoring of different forms of drugs in cells can be achieved, avoiding the cumbersome quantification process of potential metabolites and extracellular efflux drugs in traditional methods, greatly improving the experimental efficiency and accuracy, and easily realizing the monitoring of the intracellular drug release process, which will help the high-throughput screening of the key linker of the conjugate of the vector and the drug.

[0020] (3) The judgment think tank in the present invention combines the basic understanding of multiple processes such as cell uptake, intracellular substance transport, and intracellular substance metabolism. It can not only be used as a guiding opinion for drug release research, but also be used to guide other basic research related to drug uptake and release, and has good universality.

[0021] (4) The implementation of the method of the present invention will help people to more clearly understand the functional role of the linker in the two contradictory processes of drug protective delivery and target cell-specific release, and enable the design of carrier-drug conjugates that are logical, meet practical needs, and maximize the "efficacy-to-toxicity ratio" from the underlying logic, having broad application prospects in the research and development of anti-tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flowchart of the method of the present invention; Figure 2 is a flowchart of the think tank research and judgment step of the present invention; Figure 3 is a morphology diagram of the drug-loaded polymer after fluorescence labeling in Example 1 of the present invention; Figure 4 is a comparison diagram of in vivo pk results of fluorescence-labeled and unlabeled drug-loaded nanoparticles in Example 1 of the present invention; Figure 5 is a cell-level function evaluation diagram of the fluorescence-labeled carrier-drug conjugate in Example 1 of the present invention; Figure 6 is an in vitro cathepsin B release diagram of the drug-loaded nanoparticles in Example 1 of the present invention; Figure 7 is an immunofluorescence diagram of 4 types of cell cathepsin B in Example 1 of the present invention; Figure 8 is a western blot result diagram of 4 types of cell cathepsin B in Example 1 of the present invention; Figure 9 is a cytotoxicity experiment diagram in Example 1 of the present invention; Figure 10 is a screening diagram of cell uptake in Example 1 of the present invention; Figure 11 is an extracellular vector stability diagram in Example 1 of the present invention; Figure 12 is a formal intracellular free drug and carrier-conjugated drug concentration diagram in Example 1 of the present invention; Figure 13 is a diagram of k(t) changing with time in Example 1 of the present invention; Figure 14 is a diagram of the intracellular drug content changing with time in Example 1 of the present invention; Figure 15 is a diagram of the intracellular drug release rate changing with time in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] The following listed examples help those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0024] For the preparation processes described in the following examples, all chemical reagents used are of analytical purity unless otherwise specified.

[0025] Example 1 A general method for evaluating the intracellular drug release performance of a carrier-drug conjugate includes the following steps: (1) Fluorescent labeling of the carrier Synthesis of the fluorescently labeled carrier-drug conjugate and determination of the drug loading: Weigh 100 mg of the carrier-drug conjugate (PEG-polyamino acid carrier-gemcitabine conjugate), and react it with water-soluble Cy5-NHS in PBS with a molar ratio of (1:1) by stirring for 2 hours. Then transfer the carrier-drug conjugate to a dialysis bag with a molecular weight cut-off of 2000 Da and dialyze it in PBS for 48 hours to completely dialyze out the remaining unreacted Cy5. After that, add a freeze-drying protectant and then freeze-dry to obtain the Cy5-labeled carrier-drug conjugate. The carrier-drug conjugate successfully labeled with the Cy5 probe is blue as Figure 3 shown.

[0026] Reconstitute the freeze-dried product with an injection vehicle. After completely releasing the drug by adding 0.1 N sodium hydroxide aqueous solution for lysis, detect the drug loading by LC-MS.

[0027] Comparison of in vivo PK between the fluorescently labeled and unlabeled carrier-drug conjugates: After that, use the unlabeled carrier-drug conjugate with the same concentration as a control to conduct a PK study in normal mice. The dosage is 10 mg / kg and the administration volume is 200 μL. At 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 12 hours after administration, collect 100 μL of blood from the tail vein, and obtain plasma after centrifuging at 2000 rpm for 5 minutes. Take 5 μL of plasma, add 95 μL of PBS, place it in a 96-well plate, and detect the fluorescence emission signal excited at 633 nm / emitted at 675 nm by an enzyme-linked immunosorbent assay (ELISA) reader. After adding methanol containing 500 ng / mL internal standard to precipitate proteins, centrifuge at 13000 rpm for 5 minutes, take a part of the supernatant and place it in a liquid phase vial with a liner tube as the test sample for free drug; then add 0.1 N NaOH aqueous solution to the remaining supernatant and protein precipitate, vortex for 30 minutes to fully lyse the sample, centrifuge at 13000 rpm for 5 minutes, and take the supernatant as the test sample for all drugs. Finally, quantitatively determine the drug content in the sample by LC-MS, and the results are as Figure 4As shown, by comparing the in vivo PK of the fluorescently labeled and unlabeled conjugate of the carrier drug, it is evaluated whether the fluorescent labeling will affect the behavior of the conjugate of the carrier drug. The results are shown in the figure. Generally, the labeling with hydrophilic fluorescent probes does not affect the function and activity of the original conjugate of the carrier drug, indicating good stability.

[0028] Investigation of the DNA damage function of the fluorescently labeled conjugate of the carrier drug at the cellular level: KPC cells were seeded in a 6-well plate at a density of 20,000 cells / well. After the cells were completely adherent, the supernatant was discarded. Fluorescently labeled and unlabeled conjugates of the carrier drug at a concentration of 10 μg / mL were added to two different wells respectively. After treatment for 12 hours, the supernatant was removed, washed 3 times with PBS, fixed with 4% paraformaldehyde for 5 minutes, then washed 3 times with PBS, incubated overnight with rabbit anti-mouse γ-H2AX, then washed 3 times with PBS, and then immunofluorescently stained with FITC-labeled goat anti-rabbit secondary antibody. Fluorescent images were obtained using a fluorescence microscope. The results are as Figure 5 shown. Compared with the control group, the fluorescent probe labeling does not affect the DNA damage efficacy of the drug loaded in the conjugate of the carrier drug.

[0029] Investigation of the release of the fluorescently labeled conjugate of the carrier drug in response to cathepsin B in vitro: The fluorescently labeled conjugate of the carrier drug was placed in an enzymatic digestion buffer solution, and cathepsin B was added. After shaking reaction at 37 °C and a rotational speed of 150 rpm in a shaker for 24 hours, it was taken out. After protein precipitation with methanol, it was quantified by LC-MS. At the same time, a control without adding cathepsin B was set. The results are as Figure 6 shown. After adding cathepsin B, more free drug in the release medium was observed, indicating that cathepsin B is a mechanism for triggering drug release.

[0030] (2) Selection of cell medium Investigation of the distribution of cathepsin B in cells by immunofluorescent staining: 4T1 cells, KPC cells, NIH-3T3 cells and HEK293 cells were seeded on a confocal dish at a density of 5000 cells / well. After the cells were adherent, the supernatant was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde, then washed 3 times with PBS, and then incubated overnight with rabbit anti-mouse cathepsin B primary antibody. After that, it was washed 3 times with PBS, immunofluorescently stained with FITC-labeled goat anti-rabbit secondary antibody at 4 °C, and the expression of cathepsin B in each cell was observed under a laser confocal microscope. The results are as Figure 7As shown, cathepsin B in KPC cells and 4T1 cells is distributed in large vesicles within the cells, while in NIH-3T3 cells and HEK-293 cells, cathepsin B is distributed in relatively uniform small vesicles.

[0031] The expression of cathepsin B in cells was examined by western blot: 4T1 cells, KPC cells, NIH-3T3 cells and HEK293 cells were seeded in 6-well plates at a density of 20,000 cells / well. After the cells adhered, the supernatant was discarded, and the cells were washed 3 times with PBS. Then, 150 μL of cell lysis buffer was added. After the cells were fully lysed, intracellular proteins were separated by western blot. After overnight incubation with anti-cathepsin B primary antibody, HRP-labeled secondary antibody was added for bioluminescence color development, and finally images were obtained using a bioluminescence imager. The results are as Figure 8 shown. The expression level of cathepsin B in NIH-3T3 cells is the highest. Based on the comprehensive Figure 7 and Figure 8 results, the present invention determined that NIH-3T3 cells and KPC cells are used for subsequent release studies.

[0032] (3) Conduct of release experiments The appropriate drug administration concentration was determined through a cytotoxicity experiment: KPC cells and NIH-3T3 cells were seeded in 96-well plates at a density of 5000 cells per well. After the cells adhered and grew for 12 hours, the medium was removed. A series of concentration stock solutions of carrier-drug conjugates were prepared by diluting with complete medium at an initial concentration of 1000 μg / mL in multiples of 3, and added in descending order of concentration. After further incubation in a 37 °C carbon dioxide incubator for 24 hours, the cell supernatant was removed, 100 μL of CCK-8 working solution was added, and the cells were further cultured in the cell incubator for 2 hours. Then, the cell viability was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the results were curve-fitted to calculate the IC50. The results are as Figure 9 shown. In both types of cells, the lowest IC50 value is 31 ng / mL, so the present invention selected this concentration as the initial working concentration.

[0033] The appropriate time range for investigation was determined through a cell uptake experiment: KPC cells and NIH-3T3 cells were seeded in 6-well plates at a density of 20,000 cells per well. After the cells adhered and grew for 12 hours, complete cell culture medium containing the carrier-drug conjugate at a concentration of 5 μg / mL was added. At 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours, the cells were digested with trypsin on ice, and then fluorescence detection was performed using a flow cytometer. The fluorescence intensity in the APC channel was used as the uptake amount of each group of cells, and an uptake-time graph was made with time as the independent variable. As Figure 10 shown, between the 4th and 8th hours after drug administration, an inflection point with a decreasing uptake rate appeared. Therefore, the time range within 4 hours was selected as the investigation time range for the drug release experiment.

[0034] Flow cytometer to obtain cell carrier uptake and cell counting: KPC cells and NIH-3T3 cells were seeded in 6-cm dishes at a density of 200,000 cells per dish and cultured in a 37 °C carbon dioxide incubator for expansion until there were enough dishes for the experiment. The working solution of the carrier-drug conjugate at a concentration of 20 μg / mL prepared with complete medium containing 10% FBS was removed. At 0.5 hour, 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours, the cells were washed 3 times with cold PBS, then digested with 0.5 mL of trypsin on ice for 3 minutes, followed by rinsing with 1 mL of cold PBS. After combining the cell samples, they were centrifuged at 1500 rpm for 5 minutes in a 4 °C centrifuge. After aspirating the supernatant, the cells were resuspended by adding 1 mL of cold PBS and centrifuged again for cell washing, with a total of 3 washes. When resuspending with 1 mL of cold PBS for the last time, 50 μL of the cell suspension was taken and placed on ice for flow cytometry to detect the cell count, and the remaining 950 μL was centrifuged again, the supernatant was discarded, and it was immediately frozen and stored in an -80 °C refrigerator. After all samples were collected, they were processed together. After all samples were collected, cold PBS was added to the cell suspension placed on ice to make up to 250 μL, and then fluorescence detection and cell number counting were performed on the APC channel using a flow cytometer. The flow rate was set at 150 μL / minute, and the collection time was 1 minute. The fluorescence intensity in the APC channel was used as the uptake amount of each group of cells, and an uptake-time graph was made with time as the independent variable. As Figure 10 shown, within 3 hours after drug administration, both types of cells showed relatively linear cell uptake behavior, meeting the requirements of the present invention for cell uptake.

[0035] LC-MS to obtain the content of free drugs inside and outside the cells and the drug content in the form of carrier conjugation: For cell samples placed at -80 °C, after thawing, add 10 μL of an aqueous internal standard solution containing 2 μg / mL. The selected internal standard is cytidine ribonucleoside. Then, add 200 μL of methanol for protein precipitation and vortex for 10 minutes. After that, centrifuge at 13,000 rpm for 10 minutes at 4 °C. Finally, gently aspirate 50 μL of the centrifuged supernatant and transfer it to a liquid phase vial containing a liner tube, and label it as free drug, then place it in a -20 °C refrigerator for further testing; for the remaining sample containing the precipitate, add 10 μL of sodium hydroxide aqueous solution (0.1 N), vortex for 20 minutes, and finally centrifuge at 13,000 rpm for 10 minutes at 4 °C. Finally, gently aspirate 50 μL of the centrifuged supernatant and transfer it to a liquid phase vial containing a liner tube, and label it as the sum of free drug and drug in the form of conjugate with the carrier, then place it in a -20 °C refrigerator for further testing; finally, perform LC-MS injection for quantitative detection of the concentration. The results are as Figure 11 shown. Almost no free drug was detected extracellularly, and most of the drug existed in the form of conjugate with the carrier, indicating that the drug entry into the cell is indeed mediated by the carrier; in addition, as Figure 14 shown, the difference in the total detectable drug content in the two types of cells was consistent with the cell uptake results. NIH-3T3 cells showed significantly stronger uptake, which also proved that the method of the present invention is scientific and has good feasibility. The measured experimental data can be used to reflect the real situation.

[0036] (4) Think tank research and judgment, and draw the intracellular drug release curve According to the formula calculate value, and then draw a time-related graph. As Figure 13 shown, in this example, the average values of the two types of cells at each time point were calculated as shown in Table 1 below. Then, compared with the research and judgment flow chart of the present invention, in this example, it was observed that the result of the current NIH-3T3 cell group was the smallest in the of the present invention's research and judgment, was the largest, and from the first time point, there were 3 time points where the SD was less than 0.02, was greater; meeting the research and judgment criteria. Therefore, in this example, it was determined that the of the NIH-3T3 cell group was the approximate value of the in this experiment; and based on this, the change situation of 3 forms of substances in the cell related to time was drawn ( ), one is the total amount of uptake into the cell Figure 14 , another is the release amount , and the other is the undetectable elimination amount ​ , polynomial fitting was performed, and the equation with the least number of times when the fitting curve r2 reached 0.98 was selected as the fitting curve. Subsequently, the derivative was taken with respect to time t to obtain the velocity equation, and the velocity curve was presented as an image through software as Figure 15 . As shown in the figure, in KPC cells, there is an absolute release rate superior to that of NIH-3T3 cells , as well as a greater relative release rate . This release result was mutually verified with the previous cytotoxicity results, demonstrating that the difference in release indeed played a certain role in the cytotoxicity results.

[0037] Table 1 The average value

Claims

1. A general method for evaluating the intracellular drug release performance of a carrier-drug conjugate, characterized in that: At least include release experiment steps and think tank research and judgment steps; The release experiment steps: The release evaluation experiment includes the following parts: (1) Conducting cytotoxicity experiments on carrier-drug conjugates to determine the appropriate cellular administration concentration of carrier-drug conjugates; (2) Selecting a matching detection method based on the carrier labeling strategy to conduct a cellular uptake experiment on the carrier-drug conjugate to determine the appropriate time range for drug release research; (3) Based on the optimized conditions obtained from the conclusions of the above two experiments, conducting a formal drug release study, in which the carrier uptake amount represents the total amount of drug entering the cell, and the traditional quantitative detection method quantitatively monitors the free prototype drug in the cell and the prototype drug still coupled to the carrier. The obtained data is converted to a single cell as the minimum measurement unit for use in think tank research and judgment; The think tank's judgment steps are as follows: Establishing conversion parameters between intracellular carrier content and total amount of drug entering the cell Before making a judgment, you need to The obtained data are calculated, where Represents the calculated value at each time point Potential approximation, is the intracellular carrier content that can be detected, is the amount of carrier-coupled drug that can be detected in the cell, The content of free drugs that can be measured in the cell is then entered into the flow chart for judgment, and appropriate auxiliary judgment experiments are added according to the judgment results; the process includes the following steps: First, determine all the calculated Whether the value satisfies condition 1, condition 1: any With the minimum value ( ) is less than times the minimum value ( ), The value range of is between 0.01 and 0.1; In addition, (1) if the If the value meets condition 1, the next step is to increase the concentration of the incubation solution, conduct a new round of intracellular drug release research, and Calculate new ; At this point, we need to compare the results of the two experiments. Whether the values ​​are different; If the corresponding time points in the two experiments The difference between the values ​​is less than times the minimum value ( ), The value range of is between 0.01 and 0.1, which means that the conditions in the first experiment are met. The value can be approximated as The constraints of The minimum value among the values ​​is the target , complete the pair Research and judgment work; If the two experiments The difference between the values ​​is large and significantly greater than times the minimum value ( ), it means that the conditions in the first experiment are not met The value can be approximated as If the constraint condition is not met, it is necessary to further increase the concentration of the incubation solution or replace other control cells to conduct a new release experiment until the obtained The value drops to meet the condition 1. At the same time, for the uptake results obtained in the experiment, it is also necessary to ensure that the cell's uptake of the drug meets the prerequisite of uptake acceleration ≥ 0; at this time, the latest experimental calculation The minimum value among the values ​​is the target , complete the pair Research and judgment work; (2) If the If only part of the data in the value meets condition 1, further evaluation is required. Whether the value satisfies condition 2, condition 2: A. The obtained series The minimum value appears at the first detection time point and the minimum value appears at the last detection time point. The value is the maximum value; B. Including the first time point, there are at least two consecutive time points Value and the minimum value among them The difference is less than times the minimum value ( ), The value range is between 0.01 and 0.1; C. The maximum value and The difference between the minimum and maximum values ​​is greater than times Minimum value ( ), The value range is between 0.01 and 0.

1. The value range of is between 2 and 10; If the obtained If the value meets condition 2, we can make a judgment that the The minimum value among the values ​​is the target , complete the pair Research and judgment work; (3) If the The value does not meet condition 1, or part of Value data meets condition 1 but If the value does not meet condition 2, it is necessary to further increase the concentration of the incubation solution, or replace other control cells to conduct a new release experiment until the obtained The value drops to meet condition 1 or condition 2; at the same time, for the uptake results obtained in the experiment, it is necessary to ensure that the cell's uptake of the drug meets the prerequisite that its uptake acceleration is greater than or equal to 0; at this time, the latest experimental calculation The minimum value among the values ​​is the target , complete the pair Research and judgment work; Finally, according to the determined eligible The values ​​of cellular uptake, drug release, and drug elimination at each time in the cell were calculated, and then polynomial curve fitting was performed with time as the independent variable. Starting from the first-order equation, the power of the fitting curve was gradually increased until the r2 of the fitting curve was greater than 0.98, that is, the curve fitting was stopped, and the power was selected for curve fitting to obtain the concentration curve fitting equation; subsequently, the concentration-time curve equation was differentiated with respect to time to obtain the speed-time equation, and the speed-time equation was converted into an image using software for intuitive analysis of subsequent release rates.

2. A general method for evaluating the intracellular drug release performance of a carrier-drug conjugate according to claim 1, characterized in that: The step of labeling a vector or selecting a cell medium is also included before the release experiment step; The carrier marking steps are as follows: According to the physical and chemical properties of the delivery vector, select the appropriate labeling method and prepare the probe-labeled delivery vector, and then conduct an evaluation of the labeling stability and whether the labeling method will affect the drug delivery performance of the vector to determine the most appropriate vector labeling method; The cell medium selection steps are as follows: First, potential target cell lines are selected according to treatment needs. Second, based on the basic chemical bond properties of the linker used in the carrier-drug conjugate, an evaluation method for the intracellular drug release response mechanism to be investigated is selected. Finally, immunofluorescence staining, the addition of fluorescent probes, or Western Blot is used to evaluate the content of active substances that trigger drug release in the target cell line and other cell lines in the own cell bank. Finally, control cell lines with high and low expression of active substances, respectively, are screened out in addition to the target cells. After statistical analysis based on the above semi-quantitative results, a suitable cell line is finally selected for subsequent investigation of the intracellular drug release process.

3. A general method for evaluating the intracellular drug release performance of a carrier-drug conjugate according to claim 2, characterized in that: The release experiment step also includes a vector labeling step and a cell medium selection step.

4. A general method for evaluating the intracellular drug release performance of a carrier-drug conjugate according to claim 2, characterized in that: In the carrier labeling step, if the carrier is a polypeptide substance or protein, polylactic acid, polylysine, or polyglutamic acid polymer containing a free amino group, it can be labeled using a fluorescent labeling method, a biotin labeling method, or a stable metal isotope labeling method; Fluorescent labeling and biotin labeling can be directly coupled based on the amidation reaction between active esters and amino groups; The stable metal isotope labeling method requires the use of a bifunctional chelating agent for connection modification, wherein the bifunctional chelating agent is isothiocyanatobenzyldiethylenetriaminepentaacetic acid or isothiocyanatobenzylethylenediaminetetraacetic acid or isothiocyanatobenzyliminodisuccinic acid; If the carrier forms a stable nanoformulation through hydrophobic interaction, it can be labeled by encapsulating the hydrophobic probe; If the carrier is a commonly used protein carrier, it can be directly used for subsequent detection without probe labeling.

5. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 4, characterized in that: For different vector probe labeling methods, select matching labeling stability assessment methods: For the fluorescent labeling method, microplate reader and cell flow cytometry were used as stability assessment methods; For the biotin labeling method, stability assessment was performed using ELISA in combination with HRP-labeled streptavidin; For the stable isotope labeling method, stability assessment was performed using inductively coupled plasma mass spectrometry or inductively coupled plasma atomic emission spectrometry; For protein carriers that are not modified with probes, the stability is evaluated by ELISA in combination with corresponding detection antibody pairs.

6. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 4, characterized in that: In the carrier labeling step, whether the labeling method affects the drug delivery performance of the carrier is evaluated by adopting a combination of three methods: in vivo pharmacokinetic investigation, cellular level pharmacodynamic evaluation, and intracellular loaded drug uptake and metabolism evaluation based on liquid chromatography-mass spectrometry, or a combination of cellular level pharmacodynamic evaluation and LC-MS-based intracellular loaded drug uptake and metabolism evaluation.

7. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 2, characterized in that: In the cell medium selection step: immunofluorescence staining and western blot are used for semi-quantitative characterization of various proteases and glucosidases in cells, wherein the proteases include but are not limited to cathepsin A, cathepsin B, cathepsin L, matrix metalloproteinase-2, and matrix metalloproteinase-7, and the glucosidase is β-glucosidase or β-galactosidase; Fluorescence probe method is used for semi-quantitative characterization of intracellular reactive oxygen species, GSH, and protons.

8. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 1, characterized in that: The cytotoxicity evaluation in the release experiment step (1) is a dose-dependent cell proliferation inhibition study, the study time range is 24-96 hours, CCK-8 is used to detect cell viability, and then the IC50 of the carrier drug conjugate is obtained by statistical analysis. After that, the drug concentration near the IC50 is selected as the drug concentration for the first experiment.

9. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 1, characterized in that: In the release experiment step (2) of the cell uptake experiment: For fluorescently labeled vectors, quantitative detection was performed by flow cytometry; For biotin-labeled vectors, fluorescent-labeled streptomycin was used for intracellular fluorescence staining, and quantitative detection was performed by flow cytometry; For stable isotope-labeled vectors, quantitative detection is performed by an inductively coupled plasma mass spectrometer or an inductively coupled plasma atomic emission spectrometer coupled to a flow cytometer; For protein carriers that are not modified with probes, intracellular immunofluorescence staining is performed in combination with corresponding detection antibodies, and quantitative detection is performed by flow cytometry; With time as the horizontal axis and the carrier uptake as the vertical axis, an uptake-time correlation graph is established. Subsequently, the inflection point of the change in the carrier uptake rate can be visually observed from the graph, and the time corresponding to the inflection point can be judged. From time 0 to this time point is the appropriate time range for release research.

10. A general method for evaluating intracellular drug release performance of carrier-drug conjugates according to claim 1, characterized in that: The release experiment step (3) is to conduct a formal intracellular drug release study at the first administration concentration determined in step (1) within the appropriate release study time range determined in step (2), and includes the following steps: 1) The target cells and control cells were cultured in 6-10 cm culture dishes in a 37°C carbon dioxide incubator for expansion and culture until the number of culture dishes was sufficient for the experiment; 2) Remove the culture dish and prepare the carrier-drug conjugate working solution with complete culture medium containing 10% FBS. In the formal experiment, add 1-3 mL of working solution to each culture dish, and add an equal volume of 10% FBS complete culture medium without carrier-drug conjugate as a control; 3) The first detection time point is 15-30 minutes after the start of drug administration and incubation, and then 5-10 detection time points are set at the same time interval, for a total of 6-11 time points, with a time interval of 15-30 minutes, and at least 3 culture dishes are collected at each time point; at each time point, after washing the cells 3 times with cold PBS, use 0.5-1 mL of 0.25% trypsin to digest the cells on ice for 1-5 minutes, and then add 1 mL of cold PBS for rinsing. After the cell samples are combined, they are placed in a centrifuge at 4 degrees and centrifuged at 1500 rpm for 5 minutes. After aspirating the supernatant, the cells are resuspended by adding 1 mL of cold PBS and then centrifuged again for washing the cells, for a total of 3 washes; when adding 1 mL of cold PBS for the last resuspending treatment, take out 50 μL of the cell suspension and place it on ice, wait for the flow cytometry to detect its cell number, centrifuge the remaining 950 μL again, discard the supernatant, and immediately place it in a -80℃ refrigerator for freezing and storage, and wait for all samples to be collected and then centrally processed; After all samples are collected, add cold PBS to the cell suspension on ice to make up to 250 μL, and then count the cells by flow cytometer; set the flow rate to 50-200 μL / min, and the collection time is 1-4 minutes. For cell samples placed at -80 degrees, thaw them and add 10 μL of an aqueous solution containing 0.5-5 μg / mL of an internal standard. The internal standard is determined according to the properties of the substance to be tested; then, add 200 μL of methanol for protein precipitation. The protein precipitation method includes vortexing for 5-20 minutes, or ultrasonic treatment in an ultrasonic cleaner for 5-20 minutes, and then centrifuge at 13,000 rpm for 5-10 minutes at 4 degrees. Finally, gently aspirate 50 μL of the supernatant after centrifugation and transfer it to a liquid phase vial containing an inner liner tube, mark it, and place it in a -20 degree refrigerator for testing; for the remaining samples containing precipitation, add 10 μL of a suitable chemical bond breaking reagent, usually a strong acid, a strong base or an enzyme, is used to fully remove the drug from the carrier by vortexing or ultrasonication, and then centrifuged at 13,000 rpm for 5-10 minutes at 4 degrees. Finally, 50 μL of the supernatant after centrifugation is gently transferred to a liquid phase vial containing an inner liner tube, marked, and placed in a -20 degree refrigerator for testing; if an enzyme is used, the methanol needs to be fully removed, and the centrifugal precipitate is resuspended with an enzyme digestion buffer solution, and then the enzyme is treated for 24 hours, and then centrifuged at 13,000 rpm for 5-10 minutes. Finally, 50 μL of the supernatant after centrifugation is gently transferred to a liquid phase vial containing an inner liner tube, marked, and placed in a -20 degree refrigerator for testing; Finally, depending on the type of drug, LC-MS or inductively coupled plasma mass spectrometry or inductively coupled plasma atomic emission spectrometry is selected for quantitative detection; Preferably, in step 3), if the vector is a fluorescently labeled vector, the labeled fluorescent signal is simultaneously obtained as the intracellular vector content; If it is a biotin-labeled vector, it is combined with fluorescent-labeled streptomycin for intracellular fluorescence staining, and the used fluorescence signal is collected as the intracellular vector content; If it is a stable isotope-labeled vector, it is quantitatively detected by an inductively coupled plasma mass spectrometer or an inductively coupled plasma atomic emission spectrometer combined with a flow cytometer as the intracellular vector content; If the protein carrier is not modified with a probe, then the corresponding detection antibody pair is used for intracellular immunofluorescence staining, and the fluorescent signal used is collected as the intracellular carrier content.

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