CN112294764A - Preparation method and application range of 3-bromopyruvate lipid cubic liquid crystal - Google Patents
Preparation method and application range of 3-bromopyruvate lipid cubic liquid crystal Download PDFInfo
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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Abstract
The invention discloses a preparation method and an application range of 3-bromopyruvate lipid cubic liquid crystal, belonging to the technical field of biology and comprising the following steps: weighing GMO0.889g and F1270.111g, placing the mixture in a beaker, and carrying out constant-temperature water bath at 60 ℃; magnetically stirring until the solution is completely melted, adding 3-BP26.67mg, and slowly adding 25ml of pre-preheated pure water into the oil phase to serve as a precursor solution; the precursor solution was homogenized under high pressure at 14900psi for 9 cycles to give milky white 3-BP-LCNP. According to the invention, the influence of auxiliary materials, a stabilizing agent, the dosage of a dispersion phase and the dosage of a medicament on the crystal form of the cubic liquid crystal of the lipid is preliminarily screened, the preparation prescription process is optimized by adopting orthogonal design, the particle size and the encapsulation rate meet the requirements, the screened prescription and the preparation process are simple and feasible, and the prepared cubic liquid crystal nanoparticles have good stability.
Description
Technical Field
The invention relates to prescription screening and an application range, in particular to a preparation method and an application range of 3-bromopyruvate lipid cubic liquid crystal, and belongs to the technical field of biology.
Background
3-bromopyruvate, 3-BP, the molecular weight is 166.96, the key rate-limiting enzyme acting on glycolysis is hexokinase II HK II, as a powerful glycolysis inhibitor, because the structure is similar to the structure of in vivo cell metabolite lactic acid and pyruvic acid, the key rate-limiting enzyme can be transported into tumor cells by virtue of proteins for transporting lactic acid and pyruvic acid, such as monocarboxylic acid transport protein MCT1, the generation of ATP in the tumor cells can be effectively reduced, the energy supply of the tumor cells is cut off, the purpose of killing the tumor cells is achieved by starving the tumor cells, but because of simple and undefined side effects of the structure, the long-term use safety of the 3-BP still needs to be considered, the clinical use is still limited, so the anti-tumor research is carried out on the structural modification of the 3-BP, such as the formation of drug copolymers, or the use of novel drug delivery systems, such as gold liposome, nano-particles, microencapsulati, has great significance.
LCNPs are closed lipid bilayer honeycomb structures containing a bicontinuous water region and a lipid region, which are formed by dispersing amphipathic lipids with a certain concentration in an aqueous solution spontaneously, the amphipathic lipids spontaneously form a thermodynamically stable lipid bilayer generally, and then are recombined into liquid crystal systems with various shapes and structures, such as lamellar liquid crystal lamellar, cubic liquid crystal cubosomes and hexagonal liquid crystal hexagonal, cubic liquid crystal has a unique double-channel structure, can entrap drugs with different polarities and particle sizes, so that the drugs can be less influenced by organisms and immune systems, the cubic liquid crystal is a self-stabilizing system, the stability is superior to that of the traditional nanoparticles, and the carrier material is safe, nontoxic and good in fluidity, can be used for intravenous injection and the like, and can be used for preparing the 3-bromopyruvate lipid cubic liquid crystal nano preparation aiming at the problems of 3-bromopyruvate.
Disclosure of Invention
The invention mainly aims to provide a preparation method and an application range of 3-bromopyruvate lipid cubic liquid crystal, which can be used for quickly coding commodities and conveniently using codes.
The purpose of the invention can be achieved by adopting the following technical scheme:
a preparation method of 3-bromopyruvate lipid cubic liquid crystal comprises the following steps:
step 1: weighing GMO0.889g and F1270.111g, placing the mixture in a beaker, and carrying out constant-temperature water bath at 60 ℃;
step 2: magnetically stirring until the solution is completely melted, adding 3-BP26.67mg, and slowly adding 25ml of pre-preheated pure water into the oil phase to serve as a precursor solution;
and step 3: the precursor solution was homogenized under high pressure at 14900psi for 9 cycles to give milky white 3-BP-LCNP.
Preferably, the method comprises the following steps: chromatographic conditions, preparation of a solution, establishment of a standard curve and investigation of sample quality;
the chromatographic conditions are as follows: a chromatographic column: ZOBARXSB-AQ 4.6X 150mm, 5 m; mobile phase: 0.1% trifluoroacetic acid-water: 0.1% trifluoroacetic acid-acetonitrile 90: 10, V/V; the flow rate is 0.8 mL/min; the ultraviolet detection wavelength is 204 nm; the sample volume is 20 mul; column temperature: room temperature;
preparation of the solution:
precisely weighing a proper amount of a3-BP reference substance into a 10mL brown measuring flask, adding a proper amount of mobile phase for dissolving, diluting and fixing the volume to a scale, and preparing a reference substance stock solution with the concentration of 1 mg/mL;
precisely measuring 1ml of blank LCNP solution, placing the blank LCNP solution in a 10ml brown volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 10min, and carrying out constant volume to reach scales to obtain a blank lipid cubic liquid crystal solution;
precisely measuring 1ml of 3-BP-LCNP solution, placing in a 10ml brown volumetric flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 10min, and fixing the volume to a scale to obtain a test solution;
establishment of a standard curve: diluting the control stock solution into control solutions of 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 90. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL and 750. mu.g/mL, sampling 20. mu.l of the sample, recording the chromatogram, and performing linear regression on the mass concentration (C,. mu.g/mL) by using the peak area (A);
examination of sample quality: and inspecting and screening the specificity, precision, stability, recovery rate, encapsulation rate and dialysis time of the sample to obtain the 3-bromopyruvate lipid cubic liquid crystal.
Preferably, during the preparation of the solution, the solution is filtered through a 0.45 μm microporous membrane, and the filtrate is injected.
Preferably, the special investigation is to take a reference substance in the establishment of a standard curve, a prepared test substance and a blank lipid cubic liquid crystal solution, sample injection and record a chromatogram, and observe whether the auxiliary material interferes with the determination of the content of 3-BP.
Preferably, the precision is measured by respectively preparing the 3-BP reference substance into solutions with different concentrations of 10 mug/ml, 90 mug/ml and 250 mug/ml, and repeatedly injecting the sample 6 times in a day to calculate the precision in the day; the measurement was continued for 3 days, the day-to-day precision was calculated, and the relative standard deviation was calculated.
Preferably, the stability test is to take the same 3-BP test sample solution, respectively place for 0h, 2h, 4h, 6h, 8h, 12h and 24h at room temperature, sample and record peak areas; precisely measuring 3-BP-LCNP1ml into a dialysis bag, dialyzing for 4h at room temperature by using pure water as a dialysis medium, performing ultrasonic demulsification on a sample or sampling free 3-BP continuous filtrate in dialysate, measuring the peak area of the 3-BP, and calculating the encapsulation rate; the drug loading calculation formula is as follows: the drug loading% (% of drug contained in the lipid cubic liquid crystal)/total weight of the lipid cubic liquid crystal × 100%.
Preferably, the recovery rate is examined by adding a3-BP control into a blank lipid cubic liquid crystal, preparing solutions with different concentrations of low, medium and high of 25.3 mug/ml, 101.2 mug/ml and 253 mug/ml respectively, injecting each sample in parallel for 3 times, and recording the peak area of 3-BP.
Preferably, the dialysis time is considered as precisely measuring 3-BP-LCNP1ml into a pretreated dialysis bag, pure water is used as a dialysis medium at room temperature, and magnetic stirring is carried out; sampling at 1h, 2h, 3h, 4h, 5h, 6h and 7h respectively, supplementing the corresponding volume, taking 20 mu l of the filtrate for injection, and recording the peak area of 3-BP.
Preferably, the recovery rate investigation is divided into a method recovery rate and a sample adding recovery rate, the method recovery rate precisely measures 3-BP reference substance solution, and the 3-BP reference substance solution is prepared into 3 solutions with different concentrations, namely low, medium and high; taking 1ml into a pretreated dialysis bag, and dialyzing for 4 hours at room temperature by using 200ml of pure water as a dialysis medium; sampling 20 μ l of the filtrate, and recording the peak area of 3-BP; measuring a proper amount of 3-BP reference substance solution at the sample adding recovery rate, metering the volume to 10ml by using blank LCNP, preparing into low, medium and high concentrations of 3, placing 1ml into a pretreated dialysis bag, dialyzing for 4h at room temperature by using 200ml of pure water as a dialysis medium, taking 20 mu l of subsequent filtrate for sample injection, and recording the peak area of 3-BP.
The preparation method of the 3-bromopyruvate lipid cubic liquid crystal is applied in the range of antitumor research.
The invention has the beneficial technical effects that: according to the preparation method and the application range of the 3-bromopyruvate lipid cubic liquid crystal, the influence of auxiliary materials, stabilizing agents, the dosage of dispersed phases and the dosage of medicaments on the crystal form of the lipid cubic liquid crystal is preliminarily screened, and an orthogonal design optimization preparation prescription process is adopted, so that the cubic liquid crystal prepared by an injection method has larger grain diameter, is unstable and inhomogeneous, and therefore, nanoparticles prepared by certain external energy such as homogenization, ultrasound, high-speed stirring and the like can not only increase the dispersion degree of the nanoparticles in water, but also improve the medicament encapsulation rate; meanwhile, the influence of the homogenization pressure and the homogenization frequency on the particle size and the encapsulation rate of the 3-BP-LCNP is respectively inspected through a single factor, an optimal preparation process is selected, the particle size of the 3-bromopyruvate lipid cubic liquid crystal nanoparticle obtained by homogenizing for 9 times is stabilized to be about 192.7nm under 14900psi, the encapsulation rate reaches more than 70%, the particle size and the encapsulation rate meet the requirements, the screening formula and the preparation process are simple and feasible, and the prepared cubic liquid crystal nanoparticle is good in stability.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a preferred embodiment of a3-BP standard curve graph according to the present invention;
FIG. 2 is a schematic diagram showing the overall structure of a preferred embodiment of a high performance liquid chromatogram of-BP (A)3-BP-LCNP (B) LCNP (C) according to the present invention;
FIG. 3 is a schematic diagram of the overall structure of a preferred embodiment of a cubic liquid crystal polarization microscope view according to the present invention;
FIG. 4 is a schematic diagram of the overall structure of a preferred embodiment of particle size distribution under different homogenization pressures in accordance with the present invention;
fig. 5 is a schematic view showing the overall structure of a preferred embodiment of the particle size distribution at different homogenization times according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail with reference to the following examples, but the embodiments of the present invention are not limited thereto.
Example 1:
the preparation method of the 3-bromopyruvate lipid cubic liquid crystal provided by the embodiment comprises the following steps:
step 1: weighing GMO0.889g and F1270.111g, placing the mixture in a beaker, and carrying out constant-temperature water bath at 60 ℃;
step 2: magnetically stirring until the solution is completely melted, adding 3-BP26.67mg, and slowly adding 25ml of pre-preheated pure water into the oil phase to serve as a precursor solution;
and step 3: the precursor solution was homogenized under high pressure at 14900psi for 9 cycles to give milky white 3-BP-LCNP.
The method comprises the following steps: chromatographic conditions, preparation of a solution, establishment of a standard curve and investigation of sample quality; the chromatographic conditions are as follows: a chromatographic column: ZOBARXSB-AQ 4.6X 150mm, 5 m; mobile phase: 0.1% trifluoroacetic acid-water: 0.1% trifluoroacetic acid-acetonitrile 90: 10, V/V; the flow rate is 0.8 mL/min; the ultraviolet detection wavelength is 204 nm; the sample volume is 20 mul; column temperature: room temperature; preparation of the solution: precisely weighing a proper amount of a3-BP reference substance into a 10mL brown measuring flask, adding a proper amount of mobile phase for dissolving, diluting and fixing the volume to a scale, and preparing a reference substance stock solution with the concentration of 1 mg/mL; precisely measuring 1ml of blank LCNP solution, placing the blank LCNP solution in a 10ml brown volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 10min, and carrying out constant volume to reach scales to obtain a blank lipid cubic liquid crystal solution; precisely measuring 1ml of 3-BP-LCNP solution, placing in a 10ml brown volumetric flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 10min, and fixing the volume to a scale to obtain a test solution; establishment of a standard curve: diluting the control stock solution into control solutions of 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 90. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL and 750. mu.g/mL, sampling 20. mu.l of the sample, recording the chromatogram, and performing linear regression on the mass concentration (C,. mu.g/mL) by using the peak area (A); examination of sample quality: and inspecting and screening the specificity, precision, stability, recovery rate, encapsulation rate and dialysis time of the sample to obtain the 3-bromopyruvate lipid cubic liquid crystal. In the preparation process of the solution, the solution is filtered by a 0.45 mu m microporous filter membrane, and the filtrate is taken for sample injection.
The special investigation is to take a reference substance in the establishment of a standard curve, a prepared test substance and a blank lipid cubic liquid crystal solution, sample introduction and chromatogram recording, and observe whether the auxiliary material interferes with the determination of the content of 3-BP. The precision investigation is that the 3-BP reference substance is respectively prepared into three solutions with different concentrations, namely 10 mug/ml, 90 mug/ml and 250 mug/ml, which are low, medium and high, and the sample introduction is repeated for 6 times in one day, and the precision in the day is calculated; the measurement was continued for 3 days, the day-to-day precision was calculated, and the relative standard deviation was calculated. Taking the same 3-BP test sample solution, respectively standing for 0h, 2h, 4h, 6h, 8h, 12h and 24h at room temperature, injecting a sample and recording peak areas; precisely measuring 3-BP-LCNP1ml into a dialysis bag, dialyzing for 4h at room temperature by using pure water as a dialysis medium, performing ultrasonic demulsification on a sample or sampling free 3-BP continuous filtrate in dialysate, measuring the peak area of the 3-BP, and calculating the encapsulation rate; the drug loading calculation formula is as follows: the drug loading% (% of drug contained in the lipid cubic liquid crystal)/total weight of the lipid cubic liquid crystal × 100%.
The recovery rate is examined by adding a3-BP reference substance into blank lipid cubic liquid crystal, preparing three solutions with different concentrations of low, medium and high, namely 25.3 mug/ml, 101.2 mug/ml and 253 mug/ml respectively, carrying out parallel sample injection for 3 times for each sample, and recording the peak area of 3-BP. The dialysis time is considered as that 3-BP-LCNP1ml is precisely measured and put into a pretreated dialysis bag, pure water is used as a dialysis medium at room temperature, and magnetic stirring is carried out; sampling at 1h, 2h, 3h, 4h, 5h, 6h and 7h respectively, supplementing the corresponding volume, taking 20 mu l of the filtrate for injection, and recording the peak area of 3-BP. The recovery rate investigation is divided into a method recovery rate and a sample application recovery rate, the method recovery rate precisely measures 3-BP reference substance solution, and the 3-BP reference substance solution is respectively prepared into low, medium and high 3 solutions with different concentrations; taking 1ml into a pretreated dialysis bag, and dialyzing for 4 hours at room temperature by using 200ml of pure water as a dialysis medium; sampling 20 μ l of the filtrate, and recording the peak area of 3-BP; measuring a proper amount of 3-BP reference substance solution at the sample adding recovery rate, metering the volume to 10ml by using blank LCNP, preparing into low, medium and high concentrations of 3, placing 1ml into a pretreated dialysis bag, dialyzing for 4h at room temperature by using 200ml of pure water as a dialysis medium, taking 20 mu l of subsequent filtrate for sample injection, and recording the peak area of 3-BP. The preparation method of the 3-bromopyruvate lipid cubic liquid crystal is applied in the range of antitumor research.
Prescription GMO/F127 with different proportions is adopted, the proportion is 9: 1. 8: 1. 7: 1. 6: 1. 5: 1. 4: 1. 3: 1. 2: 1. 1: 1, wt% pure water 20, 70, 80 and 90 wt%, and a preliminary screening of cubic liquid crystal prescriptions was performed using a polarization microscope. The optimal prescription of the 3-BP-LCNP is screened by adopting an orthogonal design L9 and taking the encapsulation rate as an evaluation index and taking four factors of the proportion of 3-BP in GMO, the proportion of GMO/F127, the dosage of a disperse phase and the stirring time.
TABLE 13-BP-LCNP prescription screening orthogonal design factor Table
Homogenizing 3-BP-LCNP at 7450, 14900, and 22500psi for 9 times, collecting homogenized 3-BP-LCNP, and determining particle size and encapsulation efficiency.
Homogenizing for 3-BP-LCNP at fixed pressure of 14900psi for 3, 6, and 9 times, collecting homogenized 3-BP-LCNP, and determining particle size and encapsulation efficiency.
The 3-BP methodology results in a standard curve: the linear relation of 3-BP in 5-750 mu g/mL meets the test requirements. Standard curve equation: a is 0.0464C-0.0719, and R2 is 0.9999.
The retention time of the 3-BP chromatogram (A) is consistent with that of the 3-BP-LCNP chromatogram (B), and the blank LCNP chromatogram (C) has no interference peak in the retention time, so that the auxiliary material has no interference on the determination of the 3-BP content and has good specificity.
The daily precision RSD of the 3-BP with low, medium and high concentrations is respectively 1.3, 3 and 2.17 percent, the daily precision RSD is respectively 0.8, 1.63 and 2.12 percent and is less than 4 percent, and the measurement requirement is met. The RSD of the peak area of the 3-BP in 24h of the sample is less than 2 percent, which indicates that the stability of the test solution in 24h is good. The average recovery rates of the 3-BP with low, medium and high concentrations are respectively 96.15%, 97.96% and 92.86%, the recovery rates are all more than 90%, and the test requirements are met
TABLE 23 BP recovery test results
The examination of dialysis time was conducted by hourly sampling, and it was found that the content of free 3-BP in the dialysate was 1.566, 1.67, 1.727, 1.751, 1.753, 1.754, and 1.754. mu.g/mL, and the amount of free drug increased with the increase of dialysis time, but the content of the dialysate did not increase substantially after 5 hours of dialysis, indicating that the dialysate reached an equilibrium state after 5 hours of dialysis, and thus the dialysis time was set to 5 hours.
The average recovery rates of 3-BP with low, medium and high concentrations are 83.16%, 90.24% and 98.2% respectively, the recovery rates are all more than 80%, and 3-BP can freely permeate a dialysis membrane, which indicates that the dialysis method can be used for determining the encapsulation efficiency of 3-BP-LCNP.
TABLE 33-BP dialysis methodology method recovery test results
The average recovery rates of the three concentrations of low, medium and high sample recovery rate of 3-BP are respectively 92.0%, 90.03% and 91.89%, and the recovery rates are all more than 90%, which indicates that the blank LCNP has small influence on the encapsulation efficiency of the drug determined by the dialysis method.
TABLE 43-sample recovery test results of BP dialysis methodology
The prescription screening result is that under a polarizing microscope, the lamellar liquid crystal has optical birefringence and is in a cross pattern or an oily pattern; since cubic liquid crystals do not have optical birefringence, they exhibit a dark field under a polarizing microscope. The results show that when the water content is 20%, most of the liquid crystal is not cubic, and a small part of the liquid crystal is lamellar; when the water content exceeds 70%, cubic liquid crystal is formed, but when the water content is 40% -60%, the cubic liquid crystal is sticky and jelly-like, which is not favorable for intravenous administration, so that it is selected to add excessive water in the preparation process.
TABLE 5 lipid cubic liquid Crystal prescription screening
C, cubic liquid crystal; l is a lamellar liquid crystal; n-unformed liquid Crystal
The results of orthogonal experimental design show (see table 6), the influence factors are that the dosage of C dispersed phase is larger than BGMO/F127 ratio, A3-BP accounts for GMO ratio and stirring time is larger than D from large to small, and the influence strength of each level in different factors on the results is as follows: a1> A3> A2, B1> B3> B2, C2> C3> C1, D3> D1> D2. According to the analysis result of the orthogonal test variance, the proportion of GMO/F127, the dosage of a disperse phase and the stirring time have obvious influence on the prescription encapsulation rate, and the optimal prescription obtained through the orthogonal test is A1B1C2D3, namely the prescription is as follows: 3-BP26.67mg, GMO0.889g, F1270.111g and 25ml of preheated pure water to obtain a preparation prescription with the encapsulation rate of 72.53 percent.
TABLE 63-BP-LCNP prescription optimized orthogonal design L9(34) results Table
TABLE 7L 9(34) results of analysis of variance in orthogonal test
F0.05(2,2)＝19.00
The 3-BP-LCNP application range and the homogenization pressure result show that the encapsulation efficiency and the particle size of the 3-BP-LCNP are gradually reduced when the homogenization pressure is gradually increased. As can be seen, 3-BP-LCNP dispersity is less correlated to the homogenization pressure, and considering that too high a homogenization pressure may destroy the structure of the cubic liquid crystal, 14900psi was selected as the final homogenization pressure in this experiment.
Table 8 homogeneous pressure investigation
The homogenization times result shows that the encapsulation efficiency of the 3-BP is less related to the homogenization times, but the influence on the average particle size of the 3-bromopyruvic acid is larger, and when the homogenization times are gradually increased, the particle size of the cubic liquid crystal nanoparticles is gradually reduced. When the homogenization times exceed 6 times, the particle size reduction amplitude is small; when the number of homogenization times exceeds 9, the particle size distribution is concentrated. Therefore, the cycle number of 9 times is finally selected as the optimal preparation process condition.
TABLE 9 examination of number of homogenizers
In summary, in this embodiment, according to the preparation method and the application range of the 3-bromopyruvate lipid cubic liquid crystal in this embodiment and the preparation method and the application range of the 3-bromopyruvate lipid cubic liquid crystal in this embodiment, in this experiment, the influence of the auxiliary material, the stabilizer, the usage amount of the dispersion phase and the usage amount of the drug on the crystal form of the lipid cubic liquid crystal is preliminarily screened, and the preparation formula process is optimized by adopting an orthogonal design. Because the cubic liquid crystal prepared by the injection method has larger grain diameter, is unstable and inhomogeneous, the nanoparticles prepared by certain external energy such as homogenization, ultrasound, high-speed stirring and the like can not only increase the dispersion degree of the nanoparticles in water, but also improve the drug encapsulation rate.
The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.
Claims (10)
1. A preparation method of 3-bromopyruvate lipid cubic liquid crystal is characterized by comprising the following steps:
step 1: weighing GMO0.889g and F1270.111g, placing the mixture in a beaker, and carrying out constant-temperature water bath at 60 ℃;
step 2: magnetically stirring until the solution is completely melted, adding 3-BP26.67mg, and slowly adding 25ml of pre-preheated pure water into the oil phase to serve as a precursor solution;
and step 3: the precursor solution was homogenized under high pressure at 14900psi for 9 cycles to give milky white 3-BP-LCNP.
2. The method for preparing 3-bromopyruvate lipid cubic liquid crystal according to claim 1, comprising: chromatographic conditions, preparation of a solution, establishment of a standard curve and investigation of sample quality;
the chromatographic conditions are as follows: a chromatographic column: ZOBARXSB-AQ 4.6X 150mm, 5 m; mobile phase: 0.1% trifluoroacetic acid-water: 0.1% trifluoroacetic acid-acetonitrile 90: 10, V/V; the flow rate is 0.8 mL/min; the ultraviolet detection wavelength is 204 nm; the sample volume is 20 mul; column temperature: room temperature;
preparation of the solution:
precisely weighing a proper amount of a3-BP reference substance into a 10mL brown measuring flask, adding a proper amount of mobile phase for dissolving, diluting and fixing the volume to a scale, and preparing a reference substance stock solution with the concentration of 1 mg/mL;
precisely measuring 1ml of blank LCNP solution, placing the blank LCNP solution in a 10ml brown volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 10min, and carrying out constant volume to reach scales to obtain a blank lipid cubic liquid crystal solution;
precisely measuring 1ml of 3-BP-LCNP solution, placing in a 10ml brown volumetric flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 10min, and fixing the volume to a scale to obtain a test solution;
establishment of a standard curve: diluting the control stock solution into control solutions of 5. mu.g/mL, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 90. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL and 750. mu.g/mL, sampling 20. mu.l of the sample, recording the chromatogram, and performing linear regression on the mass concentration (C,. mu.g/mL) by using the peak area (A);
examination of sample quality: and inspecting and screening the specificity, precision, stability, recovery rate, encapsulation rate and dialysis time of the sample to obtain the 3-bromopyruvate lipid cubic liquid crystal.
3. The method for preparing 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the solutions are filtered by 0.45 μm microporous membrane during the preparation process of the solutions, and the filtrate is injected.
4. The method for preparing 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the special investigation is to take a reference substance in the establishment of a standard curve, a prepared test substance and a blank lipid cubic liquid crystal solution, sample injection and record chromatogram, and observe whether the auxiliary material interferes with the determination of the 3-BP content.
5. The preparation method of the 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the precision is measured by preparing the 3-BP reference substance into three solutions with different concentrations of 10 μ g/ml, 90 μ g/ml and 250 μ g/ml, repeating the sample injection for 6 times in a day, and calculating the precision in the day; the measurement was continued for 3 days, the day-to-day precision was calculated, and the relative standard deviation was calculated.
6. The preparation method of the 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the stability test comprises taking the same 3-BP test solution, respectively standing for 0h, 2h, 4h, 6h, 8h, 12h and 24h at room temperature, injecting sample and recording peak area; precisely measuring 3-BP-LCNP1ml into a dialysis bag, dialyzing for 4h at room temperature by using pure water as a dialysis medium, performing ultrasonic demulsification on a sample or sampling free 3-BP continuous filtrate in dialysate, measuring the peak area of the 3-BP, and calculating the encapsulation rate; the drug loading calculation formula is as follows: the drug loading% (% of drug contained in the lipid cubic liquid crystal)/total weight of the lipid cubic liquid crystal × 100%.
7. The preparation method of the 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the recovery rate is measured by adding a3-BP reference substance into a blank lipid cubic liquid crystal to prepare three solutions with different concentrations, namely low, medium and high, of 25.3 μ g/ml, 101.2 μ g/ml and 253 μ g/ml respectively, and each sample is subjected to parallel sample injection for 3 times, and the 3-BP peak area is recorded.
8. The preparation method of 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 2, wherein the dialysis time is measured by precisely measuring 3-BP-LCNP1ml into a pretreated dialysis bag, pure water at room temperature is used as a dialysis medium, and magnetic stirring is carried out; sampling at 1h, 2h, 3h, 4h, 5h, 6h and 7h respectively, supplementing the corresponding volume, taking 20 mu l of the filtrate for injection, and recording the peak area of 3-BP.
9. The method for preparing 3-bromopyruvate lipid cubic liquid crystal as claimed in claim 7, wherein the recovery rate test is divided into a method recovery rate and a sample application recovery rate, the method recovery rate precisely measures 3-BP reference solution, and the 3-BP reference solution is prepared into 3 solutions with different concentrations, namely low, medium and high; taking 1ml into a pretreated dialysis bag, and dialyzing for 4 hours at room temperature by using 200ml of pure water as a dialysis medium; sampling 20 μ l of the filtrate, and recording the peak area of 3-BP; measuring a proper amount of 3-BP reference substance solution at the sample adding recovery rate, metering the volume to 10ml by using blank LCNP, preparing into low, medium and high concentrations of 3, placing 1ml into a pretreated dialysis bag, dialyzing for 4h at room temperature by using 200ml of pure water as a dialysis medium, taking 20 mu l of subsequent filtrate for sample injection, and recording the peak area of 3-BP.
10. The preparation method of 3-bromopyruvate lipid cubic liquid crystal according to any one of claims 1 to 9, which is applied in the field of antitumor research.
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