Rapid quantification method and kit for lipidosome in-vivo sample based on FRET (Fluorescence Resonance Energy Transfer) technology

Abstract

The invention belongs to the technical field of detection, discloses a method and a kit for rapidly quantifying a sample in a liposome based on an FRET (Fluorescence Resonance Energy Transfer) technology, and particularly discloses an FRET liposome. The FRET lipidosome provided by the invention can be used for checking the existing lipidosome sample treatment method, if the integrity of the lipidosome is damaged in the treatment process, the change of the FRET efficiency is caused, and the influence of the treatment process on the integrity of the sample lipidosome can be judged by comparing the FRET efficiency of the lipidosome before and after treatment. Therefore, misjudgment of a final content determination result in a poor treatment process can be effectively avoided, and the accuracy of pharmacokinetic research is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a rapid quantification method and kit for in-vivo samples of liposomes based on FRET technology. Background Art

[0002] Among nanopharmaceuticals, liposomes are the most successful and widely applied nanotechnology. Currently, multiple liposome varieties have been approved for market use in anti-fungal, anti-cancer, and analgesic applications, including oxaliplatin, camptothecin, doxorubicin, amphotericin B, paclitaxel, irinotecan, and morphine sulfate. Carrier-based nanopharmaceuticals such as liposomes are formed by the interaction between drug molecules and carriers. After entering the body, as the drug is continuously released from the carrier, there are various forms such as drug-loaded particles, free drugs, carrier materials, and their metabolites. The "drug-loaded particle-free drug-carrier material" is in a dynamic change process. Therefore, accurately analyzing and quantifying the change process of each molecular form in the body is the key to the pharmacokinetic study of carrier-based drugs. According to the requirements of the "Technical Guidelines for Non-Clinical Pharmacokinetic Studies of Nanopharmaceuticals" promulgated by the CDE, the study of the in-vivo pharmacokinetics of liposomes should distinguish the released free drug and the drug encapsulated in the liposome to further study the effective concentration, safe dose, and ADME process of liposomes in the body. Therefore, rapidly and efficiently separating the free drug and the drug encapsulated in the liposome is currently a difficult point in the study.

[0003] In existing studies, there are mainly the following three problems: (1) The separation steps are complex, and sample loss or damage to liposomes may occur during the separation process, affecting the accurate quantification of free drugs; (2) There is a lack of direct indication for distinguishing free drugs and liposomes, and it must be determined through subsequent analysis processes; (3) No widely applicable and efficient separation and quantification method has been established.

[0004] Accurate analytical quantification is based on accurate separation. Common existing separation methods include solid-phase extraction (SPE), ultrafiltration, dialysis, ultracentrifugation, etc. However, these separation methods have complex separation steps, and sample loss may occur during the separation process, or the integrity of liposomes may be damaged, affecting the accurate quantification of free drugs. Moreover, they cannot indicate or distinguish between free drugs and liposomes, resulting in a complex subsequent analysis process. There is an urgent need to provide a method to solve the problems of cumbersome steps, high cost, and liposome damage during sample processing in existing separation methods, thereby improving the efficiency of liposome pharmacokinetic studies. Additionally, in traditional in vivo liposome sample processing, it is necessary to distinguish the amount of drug in liposomes and the amount of free drug. A certain proportion of organic solvents is used in liposome sample separation schemes such as solid-phase extraction and dialysis. The high-intensity mechanical force in ultrafiltration or ultracentrifugation schemes may also damage the integrity of liposomes, leading to drug leakage, thus affecting the determination of liposome and free drug concentrations in pharmacokinetic assays. Therefore, a liposome with a stable FRET effect is needed to verify whether the existing in vivo liposome sample analysis methods will affect the integrity of liposomes.

[0005] In summary, the study of in vivo liposome pharmacokinetics should distinguish between released free drugs and drugs encapsulated in liposomes to further study the effective concentration, safe dose, and ADME process of liposomes in vivo. Therefore, there is an urgent need for a method that can quickly detect and quantify, and confirm whether liposomes are damaged during the separation process. Summary of the Invention

[0006] The purpose of the first aspect of the present invention is to provide a FRET liposome.

[0007] The purpose of the second aspect of the present invention is to provide a kit.

[0008] The purpose of the third aspect of the present invention is to provide the application of the FRET liposome of the first aspect of the present invention or the kit of the second aspect of the present invention in detecting the content of liposome nano-drugs in in vivo samples.

[0009] The purpose of the fourth aspect of the present invention is to provide a rapid quantification method for in vivo samples of liposome nano-drugs based on FRET technology.

[0010] The purpose of the fifth aspect of the present invention is to provide the application of the quantification method of the fourth aspect of the present invention in the pharmacokinetic study of carrier drugs.

[0011] To achieve the above purposes, the technical solutions adopted by the present invention are:

[0012] In the first aspect of the present invention, there is provided a FRET liposome prepared by the following preparation method: Dissolve phospholipids and cholesterol in ethanol, add a fluorescent dye, and after mixing, dropwise add the mixture to water under stirring conditions, and then remove the ethanol to obtain the FRET liposome.

[0013] In some embodiments of the present invention, the mass ratio of the phospholipids, cholesterol to the fluorescent dye is 100 - 400:1.

[0014] In some preferred embodiments of the present invention, the mass ratio of the phospholipids, cholesterol to the fluorescent dye is 200 - 400:1.

[0015] In some embodiments of the present invention, the fluorescent dye includes a first fluorescent dye and a second fluorescent dye.

[0016] In some embodiments of the present invention, the first fluorescent dye includes any one of DiO, DiI, Cy3, FITC, Coumarin6, Alexa Fluor 488, Alexa Fluor 488, BODIPY-FL, and the second fluorescent dye includes any one of DiI, DiD, Cy5, DiI, Alexa Fluo, Rhodamine B, Cy5, BODIPY-TR; the first fluorescent dye is different from the second fluorescent dye, and the two can produce a FRET effect.

[0017] In some embodiments of the present invention, the fluorescent dye includes DiI and DiD, and the mass ratio of DiI to DiD is 1:1 - 5.

[0018] In some preferred embodiments of the present invention, the mass ratio of DiI to DiD is 1:1 - 4.

[0019] In some more preferred embodiments of the present invention, the mass ratio of DiI to DiD is 1:1 - 2.

[0020] The fluorescence labeling method with FRET effect is a method based on fluorescence intensity and molecular distance. In 1948, Theodor proposed an equation for quantifying the energy transfer efficiency between fluorophores with known distances, realizing the quantification of distance and energy transfer efficiency. The FRET effect is the non-radiative resonance transfer of the energy of electron excitation from the donor fluorophore in the excited state to the acceptor molecule in the ground state, and it is a near-field interaction. The occurrence of FRET requires partial overlap between the fluorescence emission spectrum of the donor molecule and the fluorescence excitation spectrum of the acceptor molecule. An overlap of more than 30% is the minimum condition for the effective and reliable detection of FRET. Secondly, energy transfer can occur when the distance between the donor molecule and the acceptor molecule is very close (1 - 10 nm), such as Figure 1 andFigure 2 As shown. The FRET group is a fluorophore with environmental responsiveness, characterized by high sensitivity, good responsiveness, no radioactivity, and non-invasiveness. It has been widely used in the research of the interaction between nanodrugs and the biological environment and the visualization in biological systems to explore the integrity and in vivo fate of particles after entering the systemic circulation.

[0021] In some embodiments of the present invention, the phospholipid includes soy lecithin.

[0022] In some embodiments of the present invention, the mass ratio of the phospholipid to cholesterol is 1-4:1.

[0023] In some preferred embodiments of the present invention, the mass ratio of the phospholipid to cholesterol is 2-4:1.

[0024] In some more preferred embodiments of the present invention, the mass ratio of the phospholipid to cholesterol is 2-2:1.

[0025] In some embodiments of the present invention, the water is water at 45-60 °C.

[0026] In some embodiments of the present invention, the conditions for stirring are 400-550 rpm.

[0027] In some embodiments of the present invention, ethanol is removed by rotary evaporation.

[0028] The second aspect of the present invention provides a kit, including the FRET liposome of the first aspect of the present invention.

[0029] The use method of this kit: Mix the liposome with FRET effect and the liposome nanodrug in a certain proportion and apply it to the study sample of blood drug concentration. Use the pretreatment method in the first aspect of the present invention to process the sample and monitor the change of FRET efficiency in the sample to verify the applicability of the method or analyze the accuracy of the results.

[0030] The effects of this kit:

[0031] The effect is intuitive. The integrity damage and drug leakage during the processing of the liposome sample are obtained through the intuitive result of the change in fluorescence efficiency of the FRET effect.

[0032] The operation is efficient. After introducing the FRET liposome, there is no need to repeatedly perform multiple comparative verifications on the cumbersome separation process, and the applicability of the existing sample processing process can be quickly optimized or guaranteed.

[0033] The result is reliable. During the separation and analysis of in vivo drug concentration samples, introducing the FRET liposome to verify the sample can be used for the assessment of the sample processing method and the verification of the data accuracy of the final analysis result.

[0034] In a third aspect of the present invention, there is provided the use of the FRET liposomes of the first aspect of the present invention or the kit of the second aspect of the present invention in detecting the content of liposomal nanodrugs in in vivo samples.

[0035] In a fourth aspect of the present invention, there is provided a rapid quantification method for in vivo samples of liposomal nanodrugs based on FRET technology, comprising the following steps:

[0036] Mix the sample to be tested with FRET liposomes to obtain a working solution;

[0037] Perform pre-treatment steps on the working solution and the FRET liposomes respectively, measure the FRET effect of the pre-treated working solution and the FRET liposomes using a FRET detector, establish the correlation between the FRET liposomes and the FRET effect, calculate the content of liposomal nanodrugs in the sample to be tested, and obtain the ratio of damaged liposomes to liposomal nanodrugs.

[0038] In some embodiments of the present invention, the pre-treatment is carried out by at least one method including solid-phase extraction, gel column separation, protein precipitation filtration, and centrifugation.

[0039] In some embodiments of the present invention, the pre-treatment is carried out by solid-phase extraction or gel column separation.

[0040] For the separation of free drugs and liposome particles, the solid-phase extraction method is selected based on the difference in the interaction forces of the two on the solid-phase extraction column, or the gel column is selected as the separation means based on the difference in their molecular weights and particle sizes. On the premise of ensuring the separation of free drugs and liposome particles, protein precipitation or ultracentrifugation is used to remove other interfering components in plasma as much as possible. Among them, the overall idea of the solid-phase extraction method is to first measure the concentration of free drugs, and then measure the total drug concentration in plasma. The difference between the two is the drug concentration encapsulated in liposomes; the overall idea of the gel column separation method is to first measure the drug concentration encapsulated in liposomes in plasma, and then measure the total drug concentration in plasma. The difference between the two is the concentration of free drugs. In order to ensure that the drugs encapsulated in liposome particles are not released or are not completely released during the entire separation process, FRET fluorescence is used to monitor liposome particles. The separated drug monomers are accurately quantified by HPLC or LC-MS / MS.

[0041] In some embodiments of the present invention, the quantification method further includes the step of detecting the content of free drugs in the sample to be tested using SPE, HPLC, or LC-MS / MS methods.

[0042] In the present invention, FRET fluorescent pairs will be used to label liposome particles. When the liposomes enter the systemic circulation, intact liposome particles exhibit the FRET effect, while particles that have lysed and released the drug do not exhibit the FRET fluorescence effect. The separation and quantification system will combine separation methods and fluorescence detection methods to detect fluorescence in real time during the process of separating free drug and liposomes in plasma, and use the presence or absence of the FRET effect as a criterion for determining whether the drug has been released to achieve rapid and accurate separation and quantification.

[0043] In a fifth aspect of the present invention, there is provided the use of the quantification method of the fourth aspect of the present invention in the pharmacokinetic study of carrier-based drugs.

[0044] The beneficial effects of the present invention are as follows:

[0045] In existing methods for processing in vivo samples of liposomes, there is a risk of destroying the integrity of liposomes, which in turn affects the determination results of the drug concentration encapsulated in liposomes and free drug. The FRET liposomes provided by the present invention can verify the existing methods for processing liposome samples. If the integrity of the liposomes is damaged during the processing, it will cause a change in the FRET efficiency. By comparing the FRET efficiencies of the liposomes before and after processing, the impact of the processing on the integrity of the sample liposomes can be determined. This will effectively avoid misjudgment of the final measured content results caused by poor processing procedures and effectively improve the accuracy of pharmacokinetic studies.

[0046] The present invention combines pretreatment methods (such as solid-phase extraction separation method and gel column separation method) with a fluorescence quantification method having the FRET effect, and encapsulates FRET fluorescent pairs in liposomes to real-time indicate the released free drug and the drug encapsulated in liposomes, so as to achieve rapid and accurate separation.

[0047] The present invention uses FRET fluorescent pairs to label liposome particles. When the liposomes enter the systemic circulation, intact liposome particles exhibit the FRET effect, while particles that have lysed and released the drug do not exhibit the FRET fluorescence effect. Fluorescence is detected in real time during the process of separating free drug and liposomes in plasma, and the presence or absence of the FRET effect is used as a criterion for determining whether the liposomes are damaged during the processing and to ensure the accuracy of the final result. It can also quickly verify the existing pretreatment separation and analysis methods without using multiple types of processing methods to verify each other.

[0048] The present invention uses an effective separation method for free drug and liposome particles. Combining the characteristics of FRET fluorescent pairs can visually judge and distinguish the two, greatly improving the efficiency of liposome pharmacokinetic studies in vivo. The FRET fluorescence labeling method does not require sample destruction and has wide applicability. Its synchronous operation with the separation and detection system can clearly and quickly identify free drug and liposome particles.

[0049] For the separation of free drugs and liposome particles, the solid-phase extraction method is selected by taking advantage of the differences in the interaction forces of the two on the solid-phase extraction column, or a gel column is selected as the separation means by taking advantage of the differences in their molecular weights and particle sizes. To ensure the effective separation of free drugs and liposome particles, protein precipitation or ultracentrifugation is used to remove other interfering components in the plasma as much as possible before subsequent separation. Among them, the overall idea of the solid-phase extraction method is to first measure the concentration of free drugs, and then measure the total drug concentration in the plasma. The difference between the two is the drug concentration encapsulated in the liposomes; the overall idea of the gel column separation method is to first measure the drug concentration encapsulated in the liposomes in the plasma, and then measure the total drug concentration in the plasma. The difference between the two is the concentration of free drugs. During the whole separation process, FRET fluorescent liposome particles are used for monitoring, and the released free drugs and intact liposome particles are indicated in real time through the change of FRET efficiency. The separated drug monomers are accurately quantified by HPLC or LC-MS / MS. Description of the Drawings

[0050] Figure 1 For the conditions and principles of FRET occurrence (Chen, T., et al, Advanced Drug Delivery Reviews, 143, 177-205).

[0051] Figure 2 For the relationship between FRET effect and fluorescence with respect to distance (Kaeokhamloed, N., Legeay, S., & Roger, E. (2022). Journal of Controlled Release, 349, 156-17).

[0052] Figure 3 Schematic diagram of the preparation of liposomes by the ethanol injection method.

[0053] Figure 4 For the FRET fluorophore pair located in the phospholipid bilayer of liposomes.

[0054] Figure 5 For the relationship between the FRET efficiency of FRET liposomes and the addition ratio of the fluorophore pair.

[0055] Figure 6 For the relationship between the FRET efficiency result of FRET liposomes and their integrity.

[0056] Figure 7 For the active loading of doxorubicin liposomes.

[0057] Figure 8 Schematic diagram of SPE separation.

[0058] Figure 9Schematic diagram of the process for judging liposome integrity by FRET (combined with SPE separation).

[0059] Figure 10 Schematic diagram of the process for preparing FRET liposomes and in vivo experiments.

[0060] Figure 11 In vitro verification results of doxorubicin liposomes.

[0061] Figure 12 Investigation on the stability of DiI and DiD FRET liposomes.

[0062] Figure 13 Effect of different ratios of DiD and DiR FRET liposomes on FRET efficiency.

[0063] Figure 14 Relationship between FRET efficiency and FRET liposome concentration. After diluting FRET liposomes (2 - 16 times), the total fluorescence intensity decreases but the FRET efficiency remains stable.

[0064] Figure 15 Color differences of the first, second, third, and fourth filtrates of non - drug - loaded FRET liposomes after solid - phase extraction by HLB column.

[0065] Figure 16 Color differences of the first, second, third, and fourth filtrates of DOX - loaded FRET liposomes after solid - phase extraction by HLB column.

[0066] Figure 17 Color differences of the first, second, third, and fourth filtrates of DOX - loaded FRET liposomes (containing a small amount of free drug) after solid - phase extraction by HLB column.

[0067] Figure 18 FRET efficiency of the first, second, third, and fourth filtrates of DOX - loaded FRET liposomes after solid - phase extraction by HLB column.

[0068] Figure 19 FRET efficiency of the first, second, third, and fourth filtrates of non - drug - loaded FRET liposomes after solid - phase extraction by HLB column.

[0069] Figure 20 Fluorescence efficiency of in - vivo samples of non - PEGylated FRET liposomes.

[0070] Figure 21 Fluorescence efficiency of in - vivo samples of PEGylated FRET liposomes.

[0071] Figure 22 Particle size distribution of blank liposomes prepared by the ethanol injection method.

[0072] Figure 23 Potential distribution of blank liposomes prepared by the ethanol injection method.

[0073] Figure 24 Particle size distribution of doxorubicin hydrochloride-loaded liposomes prepared by the ethanol injection method.

[0074] Figure 25 Potential distribution of doxorubicin hydrochloride-loaded liposomes prepared by the ethanol injection method.

[0075] Figure 26 Particle size distribution of blank liposomes prepared by the thin-film dispersion method.

[0076] Figure 27 Potential distribution of blank liposomes prepared by the thin-film dispersion method.

[0077] Figure 28 Particle size distribution of doxorubicin hydrochloride-loaded liposomes prepared by the thin-film dispersion method.

[0078] Figure 29 Potential distribution of doxorubicin hydrochloride-loaded liposomes prepared by the thin-film dispersion method. Detailed implementation manners

[0079] The content of the present invention will be further described in detail below through specific examples.

[0080] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0081] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0082] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0083] Example 1

[0084] In the traditional in vivo liposome sample treatment, it is necessary to distinguish the amount of drug in the liposome and the amount of free drug. In liposome sample separation schemes such as SPE, a certain proportion of organic solvents is used, and the high-intensity mechanical force in the ultrafiltration or ultracentrifugation scheme may also damage the integrity of the liposome, thereby causing drug leakage, which in turn affects the determination of liposome and free drug concentrations in pharmacokinetic assays.

[0085] The inventors labeled FRET fluorescent pairs for various marketed liposome products, including doxorubicin, amikacin, vincristine, oxaliplatin, mitoxantrone, and irinotecan. DiD and DiR were added to the above liposome products at a concentration of 50 μg / mL and incubated at room temperature for 0.5 h and 1 h respectively. Then, they were transferred to an ultrafiltration tube (MWCO 3500) and centrifuged at 4000 rpm for 30 min to remove free fluorescent molecules. The liposomes were resuspended and collected with PBS buffer. 200 μL of the above liposomes incubated with fluorescence were placed in a 96-well plate, and the fluorescence intensities of DiD and DiR were measured under an enzyme-linked immunosorbent assay (ELISA) reader. The results showed that the fluorescence intensities were low and no FRET effect was demonstrated. This may be due to the low fluorescence incubation efficiency and small amount of fluorescence, which could not excite the fluorescence of the acceptor fluorescent pair. Therefore, for the prepared marketed liposomes, the external standard incubation method cannot meet the requirements of FRET quantification, and an effective method for labeling marketed liposomes has not been found yet.

[0086] Aiming at the problems existing in the above sample treatment, the present invention designs and introduces an FRET liposome that can intuitively reflect the integrity of liposomes, which can effectively screen out the liposome breakage caused by poor treatment processes, thereby optimizing the treatment method.

[0087] This embodiment provides a preparation method of blank liposomes with FRET effect ( Figure 3 ) and the optimization of the preparation method.

[0088] A preparation method of FRET liposomes with FRET effect, including the following steps: Soybean phosphatidylcholine (SPC): cholesterol (Chol) are mixed and dissolved in ethanol according to a ratio of 57:23 (m / m). Subsequently, two fluorescent dyes, DiI and DiD, are added thereto. Finally, a 400 μL mixed solution containing 16 mg of total lipids and different masses of fluorescent dyes is obtained. The above mixed solution is added dropwise to 5 mL of pure water at 50 °C under stirring at 500 rpm, and ethanol is removed by subsequent rotary evaporation to obtain FRET liposomes (FRET liposomes are as Figure 4 ).

[0089] To investigate the effects of the corresponding two fluorescence ratios, the ratios of fluorescent dyes to lipids on the FRET efficiency, while keeping the lipid mass constant, experimental groups with different fluorescence addition amounts and two fluorescence ratios were designed. The FRET efficiency of the FRET liposomes prepared according to different volume ratios of DiI and DiD was tested and calculated by referring to the method of "Chen, T., He, B., Tao, J., He, Y., Deng, H., Wang, X., & Zheng, Y. (2019). Application of Forster Resonance Energy Transfer (FRET) technique to elucidate intracellular and InVivo biofate of nanomedicines. Adv Drug Deliv Rev, 143, 177 - 205. doi:10.1016 / j.addr.2019.04.009".

[0090] The results showed that when the addition ratio of the two fluorescent dyes was 1:1 (40 μg:40 μg), the efficiency of the FRET liposomes was 60%; when the addition ratio of the two fluorescent dyes was 1:2 (40 μg:80 μg), the efficiency of the FRET liposomes was high and stable (80%); when the addition ratio of the two fluorescent dyes was 1:3 (40 μg:120 μg), the efficiency of the FRET liposomes was higher than 80%; when the addition ratio of the two fluorescent dyes was 2:2 (80 μg:80 μg), the efficiency of the FRET liposomes did not increase( Figure 5 ). Therefore, the 1:2 ratio was selected as the optimal ratio. When the addition amount of the fluorescent pair was too much, it would affect the stability of the liposomes, while when the concentration of the fluorescent pair was lower than this concentration, it would seriously affect the FRET efficiency. The dye - lipid ratio (m / m) of 1:400 or 1:200 had better effects. The fluorescent dyes had poor water solubility and good affinity with lipids. After the above preparation method, the fluorescent pair would be distributed in the phospholipid layer. The adjusted concentration of the fluorescent pair made the two have a suitable distance in the phospholipid layer, so that a high - efficiency FRET effect could be achieved. The results of the stability investigation showed that the FRET liposomes prepared within 72 h had good FRET efficiency stability and could be applied to subsequent research( Figure 12 ).

[0091] To investigate the effects of different fluorescent pairs on the FRET efficiency, the fluorescent dyes were replaced with DiD and DiR, while keeping the total lipid mass constant (16 mg). The ratio and addition amount of the fluorescent dyes were investigated by the same preparation method. The results were as Figure 13. Under the condition of constant total DiD, increasing the concentration of DiR can improve the FRET efficiency. However, considering the influence of excessive dyes on the lipid bilayer structure of liposomes, the ratio of 1:2 can meet the subsequent usage requirements ( Figure 13 ).

[0092] During the preparation of liposomes, it is added, and the amount and ratio of fluorescent dyes added are adjusted to improve the spacing distance of dyes in the lipid bilayer to obtain liposomes with a stable FRET effect. Its FRET efficiency has a high correlation with the integrity of liposomes. Therefore, it can be used to verify whether the existing in-vivo sample analysis method of liposomes will affect the integrity of liposomes. Too high a total concentration of fluorescent dyes will affect the self-stability of liposomes (the surface potential and particle size distribution of liposomes).

[0093] In the previous experimental results, it was found that doxorubicin hydrochloride (DOX) in the drug-loaded liposomes and the FRET liposomes would have a cross-effect, thus affecting the judgment of the FRET effect on the encapsulation efficiency of liposomes. The FRET technique relies on the energy transfer in a pair of related fluorescent pairs. If a third fluorescent group is introduced, it will cause a large error in the fluorescence quantification. The absorption wavelength of doxorubicin hydrochloride applied in this study is in the range of 450nm - 550nm, and it has a significant impact on the fluorescence with an emission wavelength in the range of 550nm - 650nm. Therefore, the fluorescence spectra of DiI and DiD dyes have a cross with DOX, while DiD and DiR can avoid this influence to a certain extent.

[0094] Furthermore, the above-prepared FRET liposomes were applied, and Triton was used as an emulsifier breaker. Different concentrations of Triton were added to damage the liposomes to different degrees, and the changes in their FRET efficiency were measured. The results showed that as the added concentration increased, the proportion of damaged liposomes increased, and the FRET efficiency gradually decreased from the initial approximately 80% ( Figure 6 ). The above illustrates the correlation between the FRET efficiency of FRET liposomes and their integrity. The change in liposome integrity can be reflected by measuring the change in FRET efficiency, which has high sensitivity to the process of liposomes from complete to partially damaged. The above FRET changes can be used to indicate the complete information of liposomes. However, after the liposomes were completely damaged, the FRET ratio still remained at 0.2, indicating that the lipid fragments still retained a small amount of FRET ability.

[0095] To examine whether the concentration of FRET liposomes has an impact on the FRET efficiency, the above liposomes were serially diluted with PBS buffer solution and their fluorescence FRET efficiency was measured. From Figure 14As can be seen from the results, within the dilution range of 2 to 16 times, the total fluorescence intensity of the FRET liposomes decreases with the increase of the dilution factor. However, the calculated FRET efficiency does not change significantly. Therefore, the FRET efficiency can effectively indicate the integrity of liposomes within a certain concentration range, which provides a guarantee for separating free drugs from intact particles.

[0096] Example 2

[0097] This example provides two separation methods suitable for separating samples of liposome-containing drugs, as follows:

[0098] 1. Solid-phase extraction method (separation is achieved by taking advantage of the retention differences between liposomes and free drugs on the solid-phase extraction column, Figure 8 )

[0099] Determination of free drugs: Take 200 μL of plasma sample (plasma sample containing liposome drug with FRET effect) in an anti-adsorption EP tube, sequentially add 13CD3-DOX and 5% glucose solution, mix well, add the mixed solution to the HLB column. The HLB column is activated with methanol and water in sequence before use, and pretreated with blank plasma (i.e., plasma without liposome drug) and 5% glucose solution. After loading the sample, allow it to drip dry naturally and collect the first filtrate. Add 1 mL of inorganic reagent (water) or low-concentration organic reagent (methanol) for elution and then collect the second filtrate. Then add 200 μL of methanol for elution, allow it to drip dry naturally and squeeze dry, collect the eluate, dry it with nitrogen, dissolve it in the initial mobile phase (composed of acetonitrile - 5 mM acetic acid - ammonium formate in a volume ratio of 30:70:0.3), and perform HPLC or LC-MS / MS determination to obtain the content of free drugs.

[0100] Determination of total drug amount: Select the protein precipitation filtration method (PPT), solid-phase supported liquid-liquid extraction method (SLE), solid-phase extraction method or high-speed centrifugation method to separate the drug from the plasma. Taking PPT separation as an example, take plasma, add it to the PPT plate, collect the filtrate, dry it with nitrogen, dissolve it in the initial mobile phase, and perform HPLC or LC-MS / MS determination.

[0101] Calculation of the encapsulated drug in liposome particles: The difference between the total drug amount and the free drug amount is the content of the encapsulated drug in the liposome.

[0102] Calculate the content of the encapsulated drug A before treatment in 200 μL of plasma sample through the FRET fluorescence effect, and then compare it with the sum B of the content of the encapsulated drug in the first filtrate and the second filtrate. If B ≥ 95% A, it indicates that the separation method does not cause obvious damage to the liposomes.

[0103] Mix the FRET liposomes with in vivo samples. Referring to the SPE treatment method, take the separated samples for FRET efficiency test in fluorescence. If the FRET efficiency still maintains more than 95% of the original sample ratio, it is considered that the existing separation method does not affect the integrity of the liposomes in the sample. If the FRET ratio drops below 95% of the original sample ratio, it indicates that the treatment process will affect the integrity of the liposomes, and the existing separation treatment method needs to be further optimized and improved.

[0104] Although solid-phase extraction (SPE) may cause damage to liposomes in the sample due to the use of organic solvents and the overall operation is cumbersome, SPE is also used for subsequent quantification in this example. Using FRET liposomes can effectively avoid the risk of damage therein.

[0105] 2. Gel column separation method (separate the two by taking advantage of the different retention abilities of liposomes and free drugs on the gel column due to the difference in molecular weights)

[0106] Determination of the concentration of the drug encapsulated in liposomes: Drop 0.5 mL of plasma sample (plasma sample containing liposome drug with FRET effect) onto the swollen gel column (sephadex G50), elute with deionized water at a flow rate of 0.5 mL / min, collect one fraction of the effluent every 2 mL, monitor the liposome situation in the effluent with FRET fluorescence effect, and combine the solutions containing liposomes as filtrate B. Destroy the liposome membrane by means such as adding organic solvents, adjusting the pH value, heating, sonication, and mechanical means to release the encapsulated drug. Taking the addition of organic solvents as an example, take 200 μL of plasma sample, add 600 μL of methanol, vortex and mix well, ultracentrifuge, and take the supernatant for HPLC or LC-MS / MS determination.

[0107] Determination of the total drug concentration: Take 200 μL of plasma sample, destroy the liposome membrane by means such as heating, sonication, and mechanical means, ultracentrifuge, take the supernatant for HPLC or LC-MS / MS determination, and monitor the destruction of the liposome membrane with FRET fluorescence effect;

[0108] Calculation of the free drug concentration: The difference between the total drug concentration determination and the concentration of the encapsulated drug is the free drug concentration in plasma

[0109] Calculate the content A of the drug encapsulated before treatment in 0.5 mL of plasma sample through the FRET fluorescence effect, and then compare it with the content of the encapsulated drug in filtrate B. If B≥95%A, it indicates that the pre-separation method will not cause obvious damage to the liposomes.

[0110] For the separation of the above-mentioned free drug and liposome particles, solid-phase extraction is selected by taking advantage of the difference in the interaction forces of the two on the solid-phase extraction column, or a gel column is selected as the separation means by taking advantage of the differences in their molecular weights and particle sizes. On the premise of ensuring the separation of the free drug and liposome particles, protein precipitation or ultracentrifugation is used to remove other interfering components in the plasma as much as possible. Among them, the overall idea of the solid-phase extraction method is to first measure the concentration of the free drug, and then measure the total drug concentration in the plasma. The difference between the two is the drug concentration encapsulated in the liposome; the overall idea of the gel column separation method is to first measure the drug concentration encapsulated in the liposome in the plasma, and then measure the total drug concentration in the plasma. The difference between the two is the free drug concentration. In order to ensure that the drug encapsulated in the liposome particles is not released or is incompletely released during the entire separation process, FRET fluorescence is used to monitor the liposome particles. The separated drug monomer is accurately quantified by HPLC or LC-MS / MS.

[0111] Example 3

[0112] This example provides a rapid quantification method for in vitro samples of liposomes based on FRET technology. The method includes the following steps:

[0113] Mix the FRET liposome (prepared in Example 1) and the liposome nanodrug in a certain proportion to obtain a mixed solution as the sample to be measured. At the same time, use FRET liposomes with different concentrations as standard reference substances; perform pretreatment on the mixed solution or standard reference substance as follows: Place the mixed solution or standard reference substance in a 3K NMWL ultrafiltration tube and centrifuge at 4000 g at 4 °C for 30 minutes. The formed coprecipitate is washed and resuspended with an equal volume (equal to the volume of the filtrate) of PBS buffer. After repeating 3 times, centrifuge to separate the free drug not encapsulated in the liposome and the formed liposome. After dilution, the FRET efficiency in the solution before and after pretreatment is detected using a FRET detector. At the same time, after dilution, the particle size, particle size distribution and zeta potential of the liposome particles are measured using a Malvern particle size analyzer. The determination of the encapsulation efficiency and drug loading is accurately quantified by high performance liquid chromatography (HPLC). The correlation (such as a standard curve) between the concentration of the FRET liposome and the FRET efficiency is established using the FRET efficiency of the standard reference substance, and the concentration of the intact liposome in the liposome nanodrug is quantified through this correlation.

[0114] Example 4

[0115] In this example, the applicability and robustness of the method in Example 3 are further verified by constructing doxorubicin liposomes.

[0116] When preparing liposome particles, drugs with different solubilities were selected as model drugs for research to detect the adaptability and robustness of the separation and quantification methods. Doxorubicin and amikacin are both approved liposome products on the market. The solubilities of the two drugs differ greatly, representing the release and metabolic behaviors of liposomes prepared from different water-soluble drugs. Therefore, doxorubicin and amikacin were selected as model drugs to prepare liposomes in this example.

[0117] Doxorubicin is the hydrochloride salt of an anthracycline antibiotic, a drug that acts on DNA and can be embedded in DNA for the chemotherapy of various cancers. Its prototype, adriamycin, has a low solubility in aqueous solution, approximately 1.18 mg / mL. Currently, there is a marketed liposomal injection of doxorubicin for the treatment of malignant tumors such as AIDS-related Kaposi's sarcoma, ovarian cancer, and multiple myeloma, and it is a first-line chemotherapy drug.

[0118] According to the physicochemical characteristics of doxorubicin, an active loading method based on the pH gradient was used for the preparation of doxorubicin liposomes, as Figure 7 shown. Hydrogenated phosphatidylcholine (HSPC), DSPE-PEG2000, and cholesterol were dissolved in 2 mL of ethanol according to a mass ratio of 3:1:1 (32 mg in total). The organic solvent was removed by rotary evaporation and placed in a vacuum drying oven to completely volatilize the solvent to form a lipid film. 5 mL of citrate buffer was added and hydrated at 45 °C for 2 hours to obtain liposomes, and the pH inside the liposomes was maintained at 4 at this time. Sodium carbonate was added to the liposome suspension to adjust the external solution environment of the liposomes to pH 7.8 neutral. 4 mg of doxorubicin was added to the solution, and the pH difference inside and outside the liposome vesicles drove the drug to be encapsulated inside the liposomes. Doxorubicin forms a complex with citrate anions inside the liposomes, so the loading efficiency is much higher than that of the conventional passive drug loading method (Li, X.; Hirsh, D. J.; Cabral-Lilly, D.; Zirkel, A.; Gruner, S. M.; Janoff, A. S.; Perkins, W. R. Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient. Biochim Biophys Acta 1998, 1415(1), 23 - 40. DOI: 10.1016 / s0005 - 2736(98)00175 - 8 From NLM Medline).

[0119] The above-prepared doxorubicin liposomes and the FRET liposomes (DiI and DiD in a mass ratio of 1:2) prepared in Example 1 were mixed and detected using the method of Example 3.

[0120] The results showed that the FRET liposomes in the mixed liposomes could maintain stability and FRET efficiency. And after adding Triton as a liposome disruptor, there was a correlation between the encapsulation efficiency (EE) and FRET efficiency of liposomes at different degrees of disruption ( Figure 11 ), that is, as the concentration of Triton added increased, the FRET ratio decreased, and the encapsulation efficiency of doxorubicin hydrochloride liposomes also decreased. This indicates that the integrity of existing liposomes can be indicated by adding FRET liposomes.

[0121] Using the FRET liposomes prepared above and combining with the existing solid-phase extraction separation conditions, the applicability of the solid-phase extraction conditions (the same solid-phase extraction method as in Example 2) to FRET liposomes was verified, and the integrity of drug-free and DOX-loaded liposomes was investigated in turn. From Figures 15 - 17 the results, it can be directly seen that after the liposome sample was loaded, both drug-loaded and drug-free liposomes could quickly pass through the HLB solid-phase extraction column and had lower residual concentrations in the subsequent two rounds of aqueous elution, but there was still a small amount of fluorescent color remaining in the final organic eluent (the fourth filtrate). Among them, the drug-loaded liposomes showed a dark brown color due to the co-presence of the drug and the fluorescence, and were mainly concentrated in the first eluent during the elution process, with less color distribution in the subsequent elution. Similarly, there was a small amount of residual sample color in the final eluent, and there was no color separation in the first, second, and third eluents. The yellow DOX in the liposomes was not separated from the liposomes. However, in the FRET liposome sample containing free drug, it could be seen that only the free drug color appeared in the fourth filtrate and there was less residual fluorescent probe.

[0122] By reading the plate with an enzyme-linked immunosorbent assay reader (Ex 640 nm, EM 670 nm - 770 nm), the fluorescence efficiency of DOX-loaded and drug-free FRET liposomes was analyzed. From the results ( Figure 18 and Figure 19 ), the first filtrate in the drug-loaded and drug-free liposomes had FRET efficiency and fluorescence intensity (the concentration of liposomes in the sample) similar to those of the original sample. The second and third filtrates were diluted by water filtration, and the overall fluorescence concentration decreased, but they still maintained FRET efficiency close to that of the original sample. However, a significant decrease in FRET efficiency occurred in the fourth filtrate, indicating that the liposomes in the fourth filtrate had been disrupted. Based on the above results, with the help of the FRET liposome tool, the separation of free drugs and intact liposomes by the SPE separation method can be more accurately indicated, the separation method can be optimized, and the separation efficiency can be improved.

[0123] Example 5

[0124] This embodiment provides a rapid quantification method for in vivo samples of liposomes based on FRET technology. The method comprises the following steps:

[0125] (1) Pretreat the sample to be tested by using the separation method in Example 2 to obtain a sample solution to be tested. The sample to be tested is a plasma sample containing liposomes with known concentration of FRET effect and liposome nano-drugs (i.e., without FRET effect); that is, it is a mixture of liposomes with known concentration of FRET effect and the plasma of a subject treated with liposome nano-drugs.

[0126] (2) Use a FRET detector to measure the sample solution to be tested obtained after pretreatment. According to the FRET effect result, determine the integrity of the liposome drug. Use FRET liposomes to represent the overall liposomes, and establish the correlation between FRET liposomes and the FRET effect. In this way, the ratio of free and intact liposomes (the ratio of damaged liposomes) in the in vivo sample process can be quickly obtained without LC-MS analysis, and then the concentration of liposome nano-drugs in vivo can be obtained. Further, use the method mentioned in the pretreatment separation method to accurately quantify the content of free drug and total drug in the sample to be tested, and calculate the content of encapsulated drug in liposome particles.

[0127] The principle of the above method is as follows: Label liposome particles with FRET fluorescence pairs. When the liposomes enter the systemic circulation, the intact liposome particles have FRET effect, while the particles that have dissolved and released drugs do not have FRET fluorescence effect. The separation and quantification system combines the separation method and the fluorescence detection method, and detects fluorescence in real time during the process of separating free drugs and liposomes in plasma, and uses whether there is FRET effect as the standard for judging whether the drug is released to achieve rapid and accurate separation and quantification. Since the FRET efficiency will decrease after the integrity of the liposomes is damaged, the damage situation of the liposomes can be intuitively obtained. Further combining SPE and LC-MS methods can accurately quantify the drug concentration in the sample (for example, using SPE separation and subsequent supporting fluorescence detection device, directly judging liposome damage through FRET, and the process schematic diagram is as Figure 9 ).

[0128] Example 6

[0129] Inject the doxorubicin liposomes prepared in Example 4 into mice, and collect 2 mL of blood samples by orbital blood collection at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after administration ( Figure 10 ). Put it into a centrifuge at 4000 r for 4 min to separate plasma, mix the plasma with FRET liposomes with known concentration, and use it as the sample to be tested, and perform quantitative detection by using the method of Example 5.

[0130] The results showed that the method of Example 5 could quickly obtain the proportions of free and intact liposomes (the proportion of damaged liposomes) during the process of in vivo samples.

[0131] Example 7

[0132] To simulate whether the FRET technology in the present invention can achieve good indication effects for the mainstream liposomes (non-PEG and PEGylated FRET liposomes) on the market and verify the feasibility of applying FRET liposomes to the study of the distribution law of liposomes in mice, non-PEG and PEGylated FRET liposomes (both commercially available liposomes) (DiD-DiR is the FRET fluorescence pair) were injected into mice, and the mice were euthanized at fixed time points, mouse serum was collected, and the serum samples were placed in black microplates to measure the FRET efficiency of liposomes at each time point, and the pharmacokinetic distribution of liposomes in mice was investigated.

[0133] For non-PEG FRET liposomes, the clearance in vivo was relatively fast, and the fluorescence intensity decreased by about half (the number of FRET liposomes) every 0.5 h, while the FRET efficiency measured in the whole serum remained relatively stable (0 h: 76.6%, 0.5 h: 79.6%, 1 h: 76.4%) ( Figure 20 ). Therefore, it can be speculated that non-PEGylated liposomes are more likely to be cleared as a whole in circulation.

[0134] For PEGylated FRET liposomes, their circulation time in vivo was longer, and the FRET efficiency decreased slowly with the extension of time within 8 h, but the overall fluorescence intensity decreased more significantly, indicating that the number of liposomes in circulation in vivo was also continuously decreasing, and the proportion of intact liposomes was gradually decreasing ( Figure 21 ).

[0135] Based on the above results, FRET liposomes can be used as a rapid tool for in vivo analysis, and through the changes in FRET efficiency and the total fluorescence intensity, the changes in the circulating concentration and integrity of liposomes in mice can be quickly analyzed and judged. Subsequently, through further optimization, rapid analysis results can be achieved without going through a cumbersome in vivo sample separation and treatment process. This method has high application potential.

[0136] Example 8

[0137] In this example, liposomes were prepared by the ethanol injection method and the thin film dispersion method, and the effects of the two on liposomes were investigated. Specifically as follows:

[0138] (1) Preparation of liposomes by the ethanol injection method

[0139] The injection method for preparing liposomes is a convenient and rapid means of liposome preparation. Especially in the small-scale process research in the laboratory, liposomes with smaller particle sizes can be quickly prepared. The basic method is to dissolve phospholipid materials including phospholipids and cholesterol in a suitable organic phase. Common organic solvents include ethanol, methanol, and ether, etc. Then, the above-mentioned mixed solution is dropped or injected into the stirred aqueous phase. When necessary, the temperature of the aqueous phase can also be controlled. The lipid concentration in the organic phase, the relative ratio of the organic phase to the aqueous phase, etc. are all optimization parameters in the preparation process.

[0140] A method for preparing blank liposomes includes the following steps: First, dissolve soybean phosphatidylcholine (SPC): cholesterol (Chol) in ethanol according to a ratio of 57:23 (m / m) to finally obtain a 400 μL mixed solution containing 16 mg of total lipids. Drop the above-mentioned mixed solution drop by drop into 5 mL of water at 50 °C under stirring at 500 rpm, and then use the rotary evaporation method to remove ethanol in the subsequent process to obtain blank liposomes.

[0141] Use DLS to measure the particle size and zeta potential of the blank liposomes. The results show that the particle size is 122.90 ± 2.95 nm ( Figure 22 ), and the zeta potential is 0.351 ± 0.27 mV ( Figure 23 , Table 1), which is comparable to the properties of commercially available mainstream liposomes.

[0142] Prepare blank liposomes by a similar method, and load doxorubicin hydrochloride using the pH gradient. First, adjust the pH of the aqueous phase to 4 during the preparation of liposomes, and then adjust the external aqueous phase to pH 8, add doxorubicin hydrochloride, incubate at 50 °C for 30 min, and remove free doxorubicin by dialysis to obtain doxorubicin hydrochloride liposomes. Use DLS to measure the particle size and zeta potential of the blank liposomes. The results show that the doxorubicin hydrochloride liposomes exhibit a similar particle size and zeta potential distribution to the blank liposomes ( Figure 24 and Figure 25 ).

[0143] (2) Preparation of liposomes by the thin-film dispersion method

[0144] The thin film dispersion method is the most classical method for preparing liposomes. Its process mainly includes first dissolving lipids in a suitable organic solvent, removing the organic solvent using a rotary evaporator to form a uniformly dispersed thin film, then adding a suitable hydration medium, and adjusting the hydration temperature and time according to different phospholipid types and process requirements to obtain large unilamellar liposomes. Subsequently, the above liposomes are passed through polycarbonate membranes with different pore sizes in sequence to gradually reduce the particle size, and they can be extruded multiple times to finally obtain liposomes with appropriate particle size and uniform distribution. If the active drug loading method is used, it is necessary to construct a change in the internal and external ion concentration or pH gradient, and then add and adjust the external aqueous phase according to the drug properties, incubate and use dialysis and other means to remove the free drug to obtain the drug-loaded liposomes.

[0145] A method for preparing blank liposomes includes the following steps: Dissolve HSPC (19.16 mg), cholesterol (6.38 mg), and DSPE-PEG2000 (6.38 mg) in 1 mL of dichloromethane. Then evaporate the mixture under vacuum to form a thin film layer. Then add 3 mL of ammonium sulfate (final concentration 200 mM) and incubate in a 60 °C water bath for 30 minutes. Subsequently, use a liposome extrusion device (AVESTIN LiposoFast-Basic, Ottawa, Canada) to reduce the particle size of the liposomes using 400, 200, and 100 nm filters more than 16 times. If the blank liposomes reach a suitable size and good polydispersity index (PDI), the ammonium sulfate in the outer phase will be removed by dialysis in PBS (pH 7.2).

[0146] The average particle size, PDI, and zeta potential of the liposomes will be analyzed by Zetasizer Nano. The results show that the particle size is 121.51 ± 2.212 nm, and the potential is -23.60 ± 0.306 mv ( Figure 26 and Figure 27 , Table 1), which is comparable to the properties of commercially available mainstream liposomes. For the drug-loaded liposomes, after preparing the blank liposomes, doxorubicin hydrochloride is added, and with the help of the ammonium sulfate gradient, doxorubicin hydrochloride is actively loaded into the liposomes, and the free drug is removed by dialysis to obtain the drug-loaded liposomes, whose particle size and potential distribution are similar to those of the blank liposomes ( Figure 28 and Figure 29 ).

[0147] Table 1 Particle size and potential distribution of liposomes prepared by different methods (n = 3)

[0148]

[0149] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A FRET liposome is prepared by the following preparation method: Dissolve phospholipids and cholesterol in ethanol, add a fluorescent dye, and after mixing, dropwise add the mixture to water under stirring conditions, and remove ethanol to obtain the FRET liposome.

2. The FRET liposome according to claim 1, wherein The mass ratio of the phospholipids, cholesterol to the fluorescent dye is 100-400:

1.

3. The FRET liposome according to claim 2, wherein The fluorescent dye includes a first fluorescent dye and a second fluorescent dye.

4. The FRET liposome according to claim 3, wherein The first fluorescent dye includes any one of DiO, DiI, Cy3, FITC, Coumarin 6, Alexa Fluor 488, Alexa Fluor 488, BODIPY-FL, and the second fluorescent dye includes any one of DiI, DiD, Cy5, DiI, Alexa Fluo, Rhodamine B, Cy5, BODIPY-TR; the first fluorescent dye is different from the second fluorescent dye, and the two can produce a FRET effect.

5. The FRET liposome according to claim 4, characterized in that, The fluorescent dye includes DiI and DiD, and the mass ratio of DiI to DiD is 1:1-5.

6. A kit includes the FRET liposome according to any one of claims 1-5.

7. The application of the FRET liposome according to any one of claims 1-5 or the kit according to claim 6 in the detection of the content of liposome nano-drugs in in vivo samples.

8. A rapid quantitative method for in vivo samples of liposome nanoparticles based on FRET technology, comprising the following steps: Mix the sample to be tested with the FRET liposome according to any one of claims 1-5 to obtain a working solution; Perform a pretreatment step on the working solution and the FRET liposome respectively, measure the FRET effect of the pretreated working solution and the FRET liposome using a FRET detector, establish the correlation between the FRET liposome and the FRET effect, and calculate the content of liposome nano-drugs in the sample to be tested and the ratio of damaged liposomes to liposome nano-drugs.

9. The quantification method according to claim 8, wherein The pretreatment is carried out by at least one method including solid-phase extraction, gel column separation, protein precipitation filtration, and centrifugation.

10. The application of the quantification method according to claim 8 or 9 in the pharmacokinetic study of carrier drugs.

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