Method for detecting albumin-bound paclitaxel nanoparticles

Abstract

The present application relates to the detection method of albumin-bound paclitaxel nanoparticles, and belongs to the technical field of quality control. The present application provides a detection method of albumin-bound paclitaxel nanoparticles, comprising the following steps: preparing a test solution, preparing a dialysis medium, dialysis, detecting paclitaxel concentration and detecting fluorescence intensity. The method mainly adopts the dialysis method, and through optimizing the test solution concentration, the dialysis medium concentration, the dialysis time and the temperature condition of detecting fluorescence intensity, the fluorescence quenching type of nanoparticles, the binding site number, the binding constant and the force type of paclitaxel and albumin can be accurately detected. The instrument and equipment used in the detection method of the present application are not complex, the operation is simple and fast, the specificity is strong, the reproducibility is good, the required sample amount is small, the data fitting linearity is good, the binding properties of albumin and paclitaxel can be quickly and accurately judged, and the present application has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a method for detecting albumin-bound paclitaxel nanoparticles, belonging to the field of quality control technology. Background Technology

[0002] Paclitaxel for injection (albumin-bound) is a lyophilized powder for injection, prepared using nanotechnology, consisting of paclitaxel and human serum albumin. The advantage of conjugating paclitaxel to human serum albumin lies in its ability to improve efficacy while reducing toxicity. The properties of the interaction between albumin and paclitaxel, including the fluorescence quenching type, the number of binding sites, binding constant, and type of interaction force, are crucial for process control and product quality characterization. Therefore, it is necessary to develop an efficient detection method to assess the properties of the albumin-paclitaxel interaction in albumin-bound paclitaxel nanoparticles. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in the prior art. Therefore, the object of this invention is to provide a method for detecting albumin-bound paclitaxel nanoparticles, particularly a method for detecting the interaction properties between albumin and paclitaxel (including fluorescence quenching type, number of binding sites between paclitaxel and albumin, binding constant, and interaction force type).

[0004] This invention provides a method for detecting albumin-bound paclitaxel nanoparticles, comprising the following steps:

[0005] Preparation of test solution: Take albumin-bound paclitaxel nanoparticles or the test sample containing them, add sodium chloride aqueous solution to the albumin concentration of 0.7-1.1 mg / ml and the paclitaxel concentration of 0.095-0.108 mg / ml, disperse evenly, and set aside.

[0006] Preparation of dialysis medium: Take albumin, add sodium chloride aqueous solution to the albumin concentration to 0.08-0.12% g / ml, disperse evenly, and set aside;

[0007] Dialysis: Dialyze the test solution in dialysis medium. Take out 4 to 5 portions of the retention solution within 15 to 90 minutes of dialysis, with an interval of more than 15 minutes between adjacent retention solution collections, and set them aside for later use.

[0008] Detection of paclitaxel concentration: Detect the concentration of paclitaxel in the test solution and 4-5 portions of the retained solution.

[0009] Fluorescence intensity detection: The fluorescence intensity of the test solution and 4-5 retained solutions were detected at 28℃ and 37±3℃ respectively.

[0010] In the above detection method, dialysis is used to release a portion of paclitaxel from the test solution into the dialysis medium, thereby changing the paclitaxel concentration in the test solution while keeping the albumin concentration unchanged. Furthermore, by controlling the dialysis time, a model is created where different concentrations of paclitaxel bind to the same concentration of albumin. The type of fluorescence quenching is determined by detecting the quenching of the intrinsic fluorescence of albumin at two different temperatures. The number of binding sites and the binding constant of paclitaxel to albumin are quantitatively calculated, and the type of binding force between paclitaxel and albumin is determined.

[0011] The above detection method is based on the inventor's following findings:

[0012] Experimental results showed that an albumin concentration of 0.7–1.1 mg / ml and a paclitaxel concentration of 0.095–0.108 mg / ml in the test solution ensured accurate detection results. When the concentration of the test solution was too low or too high, the instrument could not accurately detect changes in albumin fluorescence intensity.

[0013] Accurate sampling is only possible when the albumin concentration in the dialysis medium is 0.08–0.12% g / ml. If the concentration of the dialysis medium is too low, paclitaxel will precipitate out of the dialysis medium, resulting in a low concentration of paclitaxel outside the dialysis bag. This accelerates the process of paclitaxel moving from inside the dialysis bag to the outside, making sampling more difficult.

[0014] Collect 4-5 samples of the retention solution within 15-90 minutes of dialysis, with an interval of more than 15 minutes between adjacent samples. This ensures accurate detection results. If the dialysis time is too long, the paclitaxel concentration in the dialysis bag will be too low, resulting in insufficient quenching of the intrinsic fluorescence of albumin, which will prevent the instrument from accurately detecting changes in fluorescence intensity. Conversely, if the interval between dialysis sampling points is too short, fewer paclitaxel molecules will pass through, resulting in minimal changes in the paclitaxel concentration in the dialysis bag. Similarly, the instrument will not be able to accurately detect changes in albumin fluorescence intensity, leading to poor linear fitting.

[0015] Detecting fluorescence intensity at 28℃ and 37±3℃ respectively ensures the accuracy of the test results. The temperature affects the fluorescence intensity; the higher the temperature, the lower the fluorescence intensity. When the temperature is too high, the fluorescence intensity is too low, and instrument error has a significant impact on the experimental results. When the temperature is too low, it is difficult to perform the test at room temperature, and the test results will also be inaccurate.

[0016] Furthermore, in the step of preparing the test solution, sodium chloride aqueous solution is added until the albumin concentration is 0.8-0.9 mg / ml and the paclitaxel concentration is 0.100 mg / ml.

[0017] Furthermore, in the step of preparing the dialysis medium, sodium chloride aqueous solution is added until the albumin concentration is 0.1%, g / ml.

[0018] Furthermore, in the steps of preparing the test solution and preparing the dialysis medium, the concentration of the sodium chloride aqueous solution is 0.8–1.0%, g / ml.

[0019] Preferably, in the steps of preparing the test solution and preparing the dialysis medium, the concentration of the sodium chloride aqueous solution is 0.9%, g / ml.

[0020] Furthermore, during the dialysis process, the retention solution was collected at four time points: 15 min, 30 min, 45 min, and 60 min of dialysis.

[0021] Furthermore, in the dialysis step, the test solution is placed in a dialysis bag for dialysis. Here, the retention solution refers to the sample solution that remains in the dialysis bag and has not been dialyzed out.

[0022] Furthermore, the dialysis medium is pretreated as follows before dialysis: the container containing the dialysis medium is placed in a constant temperature incubator at 37°C and a rotation speed of 150 rpm for 30 min.

[0023] Furthermore, in the fluorescence intensity detection step, the fluorescence intensity was detected at 28℃ and 37℃ respectively.

[0024] Furthermore, the excitation wavelength for detecting fluorescence intensity was 280 nm; the fluorescence intensity at a wavelength of 348 nm was also detected.

[0025] Furthermore, the albumin is human serum albumin.

[0026] Furthermore, the concentration of paclitaxel was determined using liquid chromatography.

[0027] Preferably, the chromatographic column is Fluorosep-RP Phenyl 5μ 60A 250*4.0mm.

[0028] Preferably, the mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 9:11.

[0029] Preferably, the detection wavelength is 227 nm.

[0030] Furthermore, the detection method further includes the following steps: constructing a Stem-Volmer curve based on the paclitaxel concentration and fluorescence intensity detection results to obtain at least one of the quenching constant and diffusion constant. Specifically, according to the equation... The fluorescence intensity of the albumin solution was denoted as F0, where F represents the fluorescence intensity of the fluorescent sample at each time point, and C... Q The concentrations of paclitaxel at each time point are plotted as F0 / F versus paclitaxel concentration. sv K represents the quenching constant. qThis represents the diffusion constant.

[0031] Furthermore, the detection method further includes the following steps: constructing a double logarithmic curve based on the paclitaxel concentration and fluorescence intensity detection results to obtain at least one of the following: number of binding sites, binding constant, ΔG, ΔH, and ΔS. Specifically, according to the equation lg[(F0-F) / F]=lgK+nlg[D], the fluorescence intensity of the albumin solution is denoted as F0, where F is the fluorescence intensity of the fluorescent sample at each time point, and D is the concentration of paclitaxel at each time point. A graph is plotted between lg((F0-F) / F) and lg(D). n represents the number of binding sites between paclitaxel and albumin, and K represents the binding constant. The thermodynamic parameters ΔG, ΔH, and ΔS of the interaction between paclitaxel and albumin are further calculated based on the binding constant K.

[0032] This invention provides a method for detecting albumin-bound paclitaxel nanoparticles. The method primarily employs dialysis, and by optimizing the concentration of the sample solution, the concentration of the dialysis medium, the dialysis time, and the temperature conditions for detecting fluorescence intensity, it can accurately detect the fluorescence quenching type of the nanoparticles, as well as the number of binding sites, binding constant, and interaction type between paclitaxel and albumin. The detection method of this invention uses simple and rapid equipment, exhibits high specificity and reproducibility, requires a small sample volume, and demonstrates good linearity in data fitting. It can quickly and accurately determine the binding properties of albumin and paclitaxel, and has broad prospects for widespread application. Attached Figure Description

[0033] Figure 1 This is a Stern-Volmer curve of parallel sample 1A050C-1 in the example;

[0034] Figure 2 This is a Stern-Volmer curve of parallel sample 1A050C-2 in the example;

[0035] Figure 3 This is a double logarithmic curve of parallel sample 1A050C-1 in the example;

[0036] Figure 4 This is a double logarithmic curve of parallel sample 1A050C-2 in the example. Detailed Implementation

[0037] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0038] Example

[0039] 1) Test solution: Take one vial of paclitaxel for injection (albumin-bound) (Celgene, trade name) The analysis revealed that the ratio of paclitaxel to albumin was approximately 100 mg to 900 mg (batch number 1A050C). 20 ml of 0.9% g / ml sodium chloride injection was added, and the mixture was gently shaken to disperse evenly, yielding a reconstituted solution. 1 ml of this reconstituted solution was placed in a 50 ml volumetric flask, dissolved and diluted to the mark with 0.9% g / ml sodium chloride injection, and shaken well. This solution was then poured into a clean glass bottle for later use (this solution is the 0 min dialysis solution).

[0040] 2) Dialysis bag pretreatment: Cut eight dialysis bags with a length of about 12cm, put them in purified water and heat them. After boiling for 20 minutes, turn off the heat and let them cool to room temperature. Then rinse them with pure water and put them in purified water for later use.

[0041] 3) Preparation of dialysis medium: Weigh 22.516g of sodium chloride and place it in a 5L beaker. Add 2500ml of water to prepare a 0.9% g / ml sodium chloride aqueous solution. Add 10ml of human serum albumin (BPL Company, concentration 25g / 100ml, batch number AAD20089), shake well, and you will get a dialysis medium with a human serum albumin concentration of 0.1% g / ml.

[0042] 4) Dialysis medium pretreatment: Measure 150 ml of dialysis medium into a 250 ml beaker and place it in a constant temperature incubator at 37℃ and 150 rpm for 30 min (prepare 8 parallel portions).

[0043] 5) Dialysis: Take 5 ml of the test solution and place it in a dialysis bag. Prepare 8 parallel samples and place them in preheated dialysis medium. At each time point of 15 min, 30 min, 45 min and 1 h, use a pipette to aspirate the samples from the two dialysis bags into clean glass bottles, shake well and set aside.

[0044] 6) Paclitaxel detection: Take 1 ml of sample from clean glass vials at 0 min, 15 min, 30 min, 45 min, and 1 h respectively, and place them into separate 10 ml volumetric flasks. Dilute to the mark with acetonitrile, sonicate for 5 min, shake well, and filter. Prepare two parallel aliquots (filter head manufacturer: Cobetter, pore size: 0.22 μm). Inject 10 μl each of the reference solution and the paclitaxel detection solution into the liquid chromatograph. Specific detection conditions are as follows:

[0045] Column: Fluorosep-RP Phenyl 5μ60A250*4.0mm

[0046] Mobile phase: Acetonitrile: Water = 9:11, v / v

[0047] Flow rate: 1.5 ml / min

[0048] Detection wavelength: 227nm

[0049] Column temperature: 25℃

[0050] Injector temperature: 4℃

[0051] Injection volume: 10 μl

[0052] Reference solution: Take an appropriate amount of paclitaxel reference standard (Southern Pharmaceutical Company, purity 99.70%, batch number RS-902-2007401), and dilute it with acetonitrile to a concentration of 20 μg of paclitaxel per 1 ml.

[0053] The test results are shown in Table 1.

[0054] 7) Fluorescent Sample Preparation: 200 μl of sample from clean glass vials at 0 min, 15 min, 30 min, 45 min, and 1 h were placed into 96-well microplates (two wells per sample). The fluorescence intensity at 348 nm under an excitation wavelength of 280 nm was measured at 28℃ and 37℃. Separately, 9 ml of dialysis medium was transferred to a 10 ml volumetric flask and diluted to the mark with 0.9% g / ml sodium chloride aqueous solution. This concentration was approximately equal to the albumin concentration in the dialysis bag (0.9 mg / ml). The fluorescence intensity at 348 nm under an excitation wavelength of 280 nm was measured at 28℃ and 37℃. The results are shown in Table 2.

[0055] Table 1. Results of Paclitaxel Concentration Detection

[0056]

[0057] Table 2 Fluorescence intensity detection results

[0058]

[0059] 8) Examine the quenching type: based on the equation The fluorescence intensity of the albumin solution was denoted as F0, where F represents the fluorescence intensity of the fluorescent sample at each time point, and C... Q The values ​​represent the concentrations of paclitaxel at different time points. In this experiment, the relationship between F0 / F and paclitaxel concentration was plotted at different temperatures (28℃, 37℃), see [see figure]. Figure 1 , Figure 2 The linear slope represents the quenching constant K. sv The fitting calculation results are shown in Table 3; diffusion constant K q The fitting calculation results are shown in Table 4.

[0060] Table 3 Linear Slope

[0061]

[0062] Table 4 Diffusion constant K q (L / mol / s)

[0063]

[0064] Experimental results show that the Stern-Volmer curve exhibits a good linear relationship, and the slope of the curve decreases with increasing temperature, while the diffusion constant K... q >2*10 10 This indicates that the quenching mode is static quenching.

[0065] 9) Investigating the number of binding sites, binding constant, and type of binding force: According to the equation lg[(F0-F) / F]=lg K+nlg[D], the fluorescence intensity of the albumin solution is denoted as F0, where F is the fluorescence intensity of the fluorescent sample at each time point, and D represents the concentration of paclitaxel at each time point. In this experiment, at different temperatures (28℃, 37℃), a graph was plotted between 1g((F0-F) / F) and 1g(D), see [see figure]. Figure 3 , Figure 4 The number of binding sites n, binding constant K, and thermodynamic parameters of the interaction between paclitaxel and albumin at different temperatures were obtained by linear regression. The calculation results are shown in Tables 5 to 7.

[0066] Table 5 Number of binding sites

[0067] Parallel samples Linear slope at 28℃ (binding site) Linear slope at 37℃ (binding site) 1A050C-1 1.9882 2.0806 1A050C-2 2.1057 2.1962

[0068] Table 6. Binding constant K

[0069] Parallel samples Temperature 28℃ (lgK) Temperature 28℃ (K binding constant) Temperature 37℃ (lgK) Temperature 37℃ (K binding constant) 1A050C-1 7.3661 23232716.9 7.7156 51951728.3 1A050C-2 7.8349 68375418.9 8.1750 149623565.6

[0070] Table 7. Thermodynamic parameters of the interaction between paclitaxel and albumin

[0071]

[0072] It can be seen that the detection method of the present invention has a good linear relationship and good parallelism between the two samples. It can accurately detect the fluorescence quenching type of albumin-bound paclitaxel nanoparticles, as well as the number of binding sites, binding constant, and interaction type of paclitaxel and albumin.

[0073] Comparative Example 1

[0074] A self-prepared sample of paclitaxel for injection (albumin-bound) was used for testing. This experiment was conducted according to the method described in the above examples, with the difference being the concentration of the test solution and the dialysis time. Specifically, the reconstituted solution was diluted 100 times, at which point the albumin concentration in the test solution was approximately 0.45 mg / ml. Samples were taken from the retention solution at five time points: 20 min, 40 min, 60 min, 1.5 h, and 2 h of dialysis. The results of fluorescence intensity and paclitaxel concentration detection are shown in Table 8.

[0075] Table 8. Detection results of fluorescence intensity and paclitaxel concentration

[0076] dialysis time Fluorescence intensity at 28℃ -1 Fluorescence intensity at 28℃ -2 Paclitaxel concentration (mg / ml) 0h 347.914 347.376 0.0251 20min 324.485 314.293 0.0195 40min 347.656 341.467 0.0172 60min 342.618 340.084 0.0139 1.5h 362.199 363.853 0.0102 2h 366.628 366.641 0.00617

[0077] It can be seen that when the albumin concentration in the test solution is about 0.45 mg / ml, the paclitaxel concentration in the dialysis bag decreases with increasing dialysis time. At this time, the fluorescence intensity of albumin in the dialysis bag should have increased accordingly, but the experimental results did not show this, indicating that the change in fluorescence intensity under this condition cannot be accurately detected.

[0078] Comparative Example 2

[0079] A self-prepared sample of injectable paclitaxel (albumin-bound) was used for testing. This experiment was conducted according to the method described in the above examples, with the main difference being the dialysis time. Specifically, samples were taken from the retention solution at five time points: 20 min, 40 min, 1 h, 1.5 h, and 2 h after dialysis. The fluorescence intensity results are shown in Table 9.

[0080] Table 9 Results of fluorescence intensity detection

[0081]

[0082] It can be seen that the fluorescence intensity at 1.5h and 2h of dialysis is basically the same, indicating that the concentration of paclitaxel in the dialysis bag is too low at this time, and the degree of quenching of the intrinsic fluorescence of albumin is small, resulting in the instrument being unable to accurately detect the change in fluorescence intensity.

[0083] Comparative Example 3

[0084] A self-prepared sample of injectable paclitaxel (albumin-bound) was used for testing. This experiment was conducted according to the method described in the above examples, with the main difference being the sampling time intervals during dialysis. Specifically, samples were taken from the retention solution at six time points: 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min of dialysis. The fluorescence intensity results are shown in Table 10.

[0085] Table 10 Results of fluorescence intensity detection

[0086]

[0087] It can be seen that the instrument fluctuation value (the difference in fluorescence detection between two parallel samples of the same sample) is about 5. When the dialysis sampling time interval is less than 15 minutes, the fluorescence intensity change value may also be about 5, which is close to the instrument fluctuation value. At this time, the error is large and the detection result is inaccurate.

[0088] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in one or more embodiments. Furthermore, those skilled in the art can combine and integrate the different embodiments described in this specification and the features of those embodiments without contradiction.

Claims

1. A method for detecting albumin-bound paclitaxel nanoparticles, characterized by: Includes the following steps: Preparation of test solution: Take albumin-bound paclitaxel nanoparticles or the test sample containing them, add sodium chloride aqueous solution to the albumin concentration of 0.8~0.9 mg / ml and the paclitaxel concentration of 0.100 mg / ml, disperse evenly, and set aside; Preparation of dialysis medium: Take albumin, add sodium chloride aqueous solution to the albumin concentration to 0.1% g / ml, disperse evenly, and set aside; Dialysis: Dialyze the test solution in dialysis medium, and collect the retained solution at four time points: 15 min, 30 min, 45 min, and 60 min for later use. Detection of paclitaxel concentration: The concentration of paclitaxel in the test solution and the four retained solutions were determined respectively; Fluorescence intensity detection: The fluorescence intensity of the test solution and the four retained solutions were detected at 28℃ and 37±3℃ respectively. Stern-Volmer curves were prepared based on the results of paclitaxel concentration and fluorescence intensity detection to obtain at least one of the quenching constant and diffusion constant. Based on the results of paclitaxel concentration and fluorescence intensity detection, a double logarithmic curve was constructed to obtain at least one of the following: number of binding sites, binding constant, ΔG, ΔH, and ΔS.

2. The detection method according to claim 1, characterized in that: In the steps of preparing the test solution and preparing the dialysis medium, the concentration of the sodium chloride aqueous solution is 0.8~1.0%, g / ml.

3. The detection method according to claim 2, characterized in that: In the steps of preparing the test solution and preparing the dialysis medium, the concentration of the sodium chloride aqueous solution is 0.9% g / ml.

4. The detection method according to claim 1, characterized in that: In the fluorescence intensity detection step, the fluorescence intensity was measured at 28℃ and 37℃ respectively.

5. The detection method according to claim 1, characterized in that: The excitation wavelength for detecting fluorescence intensity was 280 nm; the fluorescence intensity was detected at a wavelength of 348 nm.

6. The detection method according to claim 1, characterized in that: The albumin in question is human serum albumin.

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