阅读说明：本技术 一种负载抗癌药物喜树碱的纳米药物载体的荧光免疫定量检测方法 (Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier ) 是由 张明翠 连中宇 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种负载抗癌药物喜树碱的纳米药物载体荧光免疫定量检测方法,将CPT@PSI<Sub>OAm</Sub>包被抗原包被于酶标板中,封闭、加入不同浓度的CPT@PSI<Sub>OAm</Sub>标准品和FITC标记的抗PSI<Sub>OAm</Sub>抗体,建立直接竞争荧光免疫分析法定量检测CPT@PSI<Sub>OAm</Sub>；通过检测CPT@PSI<Sub>OAm</Sub>包被抗原、FITC标记抗体复合物荧光信号达到定量检测CPT@PSI<Sub>OAm</Sub>的目的,基于抗原抗体特异性,与荧光标记物相结合,使免疫反应信号进一步提高,可进行高通量测定,此检测方法的线性范围为0.12-1638.7ng/mL,检出限为0.04ng/mL,本发明为负载抗癌药物的纳米药物载体提供了更加灵敏的检测方案。(The invention discloses a nano-drug carrier fluorescence immunoassay quantitative detection method for loading anticancer drug camptothecin, which comprises the following steps of carrying out quantitative detection on CPT @ PSI OAm Coating the coated antigen in an enzyme label plate, sealing, and adding CPT @ PSI with different concentrations OAm Standard and FITC-labeled anti-PSI OAm Antibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI OAm (ii) a By detecting CPT @ PSI OAm quantitative detection CPT @ PSI achieved by fluorescent signals of coating antigen and FITC labeled antibody complex OAm The detection method aims at further improving immunoreaction signals and carrying out high-throughput determination by combining with a fluorescent marker based on antigen-antibody specificity, the linear range of the detection method is 0.12-1638.7ng/mL, the detection limit is 0.04ng/mL, and the invention provides a more sensitive detection scheme for the nano-drug carrier loaded with the anti-cancer drug.)

1. A fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin is characterized by comprising the following steps:

(1) preparation of CPT @ PSI_OAm(camptothecin-loaded oleylamine-grafted polysuccinimide) coating antigen;

(2) Preparation of FITC-labeled anti-PSI_OAmAn antibody;

(3) Will CPT @ PSI_OAmDiluting the coating antigen with a coating solution, coating the diluted coating antigen in an enzyme label plate, sealing, and adding CPT @ PSI with different concentrations_OAmStandard and FITC-labeled anti-PSI_OAmAntibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI_OAm；

(4) In CPT @ PSI_OAmThe logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, a standard curve is drawn to obtain a linear equation, and the CPT @ PSI is quantitatively detected_OAmthe concentration of (c).

2. the detection method according to claim 1, wherein the step (1) specifically comprises the steps of:

(1-1) mixing PSI_OAmDissolving (oleylamine graft polysuccinimide), Camptothecin (CPT) and polyethylene-b-polyethylene glycol in trichloromethane, adding into sodium hydroxide solution, performing ultrasonic reaction, evaporating to remove trichloromethane, centrifuging, and dispersing the precipitate in PBS buffer solution to obtain hydrolyzed CPT @ PSI_OAm-COO^-A hapten solution;

(1-2) To CPT @ PSI_OAm-COO^-Adding a PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride into the hapten solution for reaction for 10-20 min, then adding egg white albumin, incubating, centrifuging, and dispersing the obtained precipitate into the PBS buffer solution to obtain the CPT @ PSI_OAmThe envelope antigen of (1).

3. the detection method according to claim 2, wherein in step (1-1), the PSI_OAmThe dosage ratio of the camptothecin to the polyethylene-b-polyethylene glycol to the trichloromethane to the sodium hydroxide is (10-60) mg: (0.5-2) mg: (0.5-2) mL: (2-16) mL; the ultrasonic reaction condition is 200-400W ultrasonic for 5-10 min.

4. the detection method according to claim 2, wherein the step (1-2) comprises the steps of: to 1mL CPT @ PSI_OAm-COO^-Adding 1mL of PBS buffer solution containing 0.1-1 mg of N-hydroxysuccinimide and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the hapten solution, reacting for 10-20 min, adding 1-10 mg of chicken egg albumin, incubating for 2-4 h at 25 ℃, centrifuging for 10-15 min, taking the precipitate, dispersing into 3mL of neutral PBS buffer solution, and thus obtaining the CPT @ PSI_OAmThe envelope antigen of (1).

5. The detection method according to claim 1, wherein the step (2) specifically comprises the steps of: general PSI_OAmMixing the antibody solution with fluorescein isothiocyanate solution, stirring in the dark for reaction for 3-6 h, and purifying by dialysis and gel filtration after the reaction is finished to obtain FITC-labeled anti-PSI_OAman antibody.

6. The detection method according to claim 5, wherein the PSI_OAmThe antibody solution is PSI_OAmThe antibody is dissolved in 0.01M PBS buffer solution with pH7.1 to obtain the antibody, and the protein concentration of the antibody is 10-20 mg/mL; the fluorescein isothiocyanate solution is isothioureafluorescein cyanate was dissolved in carbonate buffer at pH 9.6 and the concentration was 2 mg/mL.

7. Detection method according to claim 5 or 6, characterized in that said PSI_OAmThe volume ratio of the antibody solution to the fluorescein isothiocyanate solution is 10: 1; the eluent purified by gel filtration is 0.01M PBS buffer solution with pH7.1, and the flow rate is 0.1-0.5 mL/min.

8. the detection method according to claim 1, wherein the step (3) comprises the following steps:

(3-1) coating: use of coating buffer to coat CPT @ PSI_OAmdiluting the coating antigen by 80 times, coating a 96-hole enzyme label plate with 100 mu L of each hole, and keeping the temperature in a refrigerator at 4 ℃ overnight;

(3-2) sealing: washing the 96-well enzyme label plate for 3 times by using a PBST solution, wherein each time is 3-5 min, then adding 1 wt% casein for sealing, keeping the volume of each well at 200 mu L, and incubating for 1-2 h at 37 ℃; the purpose of the PBST solution wash is to wash away unbound CPT @ PSI_OAmCoating antigen;

(3-3) sample addition Competition: washing the 96-well enzyme label plate for 3 times by using PBST solution, wherein each time is 3-5 min, and then washing 50 mu L of CPT @ PSI with different concentrations_OAmStandard solution and 50. mu.L of FITC-labeled PSI_OAmmixing the antibodies in advance, adding the mixture into each hole in gradient, incubating for 1-2 h at 37 ℃, washing for 2-4 times by using a washing solution, and drying for 3-5 minutes each time;

And (3-4) measuring the fluorescence intensity of each hole on a microplate reader when the excitation wavelength is 485nm and the emission wavelength is 525 nm.

9. The detection method according to claim 1, wherein in the step (4), the linear equation of the standard curve is F ═ 8116.6-311.7lgC, F is fluorescence intensity, and C is CPT @ PSI_OAmThe concentration of (c); coefficient of correlation R²Linear range 0.12-1638.7ng/mL, detection limit 0.04ng/mL ═ 0.991.

Technical Field

The invention relates to a quantitative detection method of a drug-loaded nano-drug carrier, in particular to a fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin.

Background

Now, cancer has become one of the biggest killers of human health and is a major challenge difficult to solve in world medicine, and the worldwide incidence rate of cancer has risen by 33% in the past decade, and only 2018, 1810 thousands of people have been diagnosed as cancerAnd 980 thousands of people died. The anticancer drugs approved to be on the market at present are only hundreds, however, the development of cancer treatment is influenced by low water solubility, poor biocompatibility, low blood clearance rate, poor tumor targeting, serious toxic and side effects on normal tissues and the like of many anticancer drugs, the nano-drug carrier can greatly improve the water solubility and stability of the drugs, prolong the blood circulation time, enhance the permeability and retention (EPR) effect of passive accumulation of tumors, and the macromolecular nano-drug carrier is oleamide grafted polysuccinimide PSI_OAmthe carrier is an emerging drug carrier in recent years, can load anti-cancer drugs, has a wide application prospect in the aspect of cancer treatment, and provides a more feasible experimental scheme for the research on the kinetic processes of quantitative detection, delivery, tracing, drug release and the like of the anti-cancer drugs.

Although many liposome drug carriers are in clinical use, the high molecular drug carriers are used as nano materials, and the biological effect generated by the complex chemical structure of the high molecular drug carriers and the quantitative analysis method of drug carrier-loaded drugs have no unified evaluation standard and evaluation method internationally. At present, researchers have provided many challenges faced by nano-drug carriers, and meanwhile, the real-time dynamic monitoring and quantitative analysis research of processes of the nano-drug carriers loaded with drugs, such as circulation in blood after entering a human body, transportation to the surface of cancer cells for enrichment, permeation into the cancer cells, drug release of the drug carriers and the like, are few, so that the establishment of high-sensitivity quantitative detection of the nano-drug carriers loaded with anticancer drugs camptothecin is urgent.

At present, the detection of the anticancer drugs mainly takes HPLC and ultraviolet detection as main parts, and if an immunoassay method is established, the quantitative detection of the anticancer drug loaded nano-drug carrier has more advantages of more sensitively quantifying the anticancer drugs.

Disclosure of Invention

the invention provides a fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin, which is used for quantitatively detecting the drug loaded on the nano-drug carrier by adopting a direct competition fluorescence immunoassay method, is simple and quick, does not need a complex sample pretreatment process, and is combined with high specificity and high sensitivity of an antigen and an antibody.

The technical scheme adopted by the invention is as follows:

A fluorescence immunoassay quantitative detection method of a nano-drug carrier loaded with an anticancer drug camptothecin comprises the following steps:

(1) Preparation of CPT @ PSI_OAm(camptothecin-loaded oleylamine-grafted polysuccinimide) coating antigen;

(2) Preparation of FITC-labeled anti-PSI_OAmAn antibody;

(3) will CPT @ PSI_OAmDiluting the coating antigen with a coating solution, coating the diluted coating antigen in an enzyme label plate, sealing, and adding CPT @ PSI with different concentrations_OAmstandard and FITC-labeled anti-PSI_OAmAntibody, establishing direct competition fluoroimmunoassay method for quantitatively detecting CPT @ PSI_OAm；

(4) In CPT @ PSI_OAmthe logarithm of the concentration of the standard substance is an abscissa, the fluorescence intensity value is an ordinate, a standard curve is drawn to obtain a linear equation, and the CPT @ PSI is quantitatively detected_OAmThe concentration of (c).

Further, the step (1) specifically includes the steps of:

(1-1) mixing PSI_OAmDissolving (oleylamine graft polysuccinimide), Camptothecin (CPT) and polyethylene-b-polyethylene glycol in trichloromethane, adding into sodium hydroxide solution, performing ultrasonic reaction, evaporating to remove trichloromethane, centrifuging, and dispersing the precipitate in PBS buffer solution to obtain hydrolyzed CPT @ PSI_OAm-COO^-A hapten solution;

(1-2) to CPT @ PSI_OAm-COO^-Adding a PBS buffer solution containing N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride into the hapten solution for reaction for 10-20 min, then adding egg white albumin, incubating, centrifuging, and dispersing the obtained precipitate into the PBS buffer solution to obtain the CPT @ PSI_OAmThe envelope antigen of (1).

In step (1-1), the PSI_OAmThe dosage ratio of the camptothecin to the polyethylene-b-polyethylene glycol (PE-b-PEG) to the trichloromethane to the sodium hydroxide is (10-60) mg:(0.5-2) mg: (0.5-2) mL: (2-16) mL; the ultrasonic reaction condition is 200-400W ultrasonic for 5-10 min. The PSI_OAmhas an average particle diameter of 200 nm.

The step (1-2) specifically comprises the following steps: to 1mL CPT @ PSI_OAm-COO^-adding 1mL of PBS buffer solution containing 0.1-1 mg of N-hydroxysuccinimide and 0.1-1 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the hapten solution, reacting for 10-20 min, adding 1-10 mg of chicken egg albumin, incubating for 2-4 h at 25 ℃, centrifuging for 10-15 min, taking the precipitate, dispersing into 3mL of neutral PBS buffer solution, and thus obtaining the CPT @ PSI_OAmThe envelope antigen of (1).

The step (2) specifically comprises the following steps: general PSI_OAmMixing the antibody solution with fluorescein isothiocyanate solution, stirring in the dark for reaction for 3-6 h, and purifying by dialysis and gel filtration after the reaction is finished to obtain FITC-labeled anti-PSI_OAmAn antibody.

The PSI_OAmthe antibody solution is PSI_OAmthe antibody is dissolved in 0.01M PBS buffer solution with pH7.1 to obtain the antibody, and the protein concentration of the antibody is 10-20 mg/mL; the fluorescein isothiocyanate solution is obtained by dissolving fluorescein isothiocyanate in a carbonate buffer solution with the pH value of 9.6, and the concentration of the fluorescein isothiocyanate solution is 2 mg/mL.

the PSI_OAmThe volume ratio of the antibody solution to the fluorescein isothiocyanate solution is 10: 1; the eluent purified by gel filtration is 0.01M PBS buffer solution with pH7.1, and the flow rate is 0.1-0.5 mL/min.

the step (3) specifically comprises the following steps:

(3-1) coating: use of coating buffer to coat CPT @ PSI_OAmDiluting the coating antigen by 80 times, coating a 96-hole enzyme label plate with 100 mu L of each hole, and keeping the temperature in a refrigerator at 4 ℃ overnight;

(3-2) sealing: washing the 96-well enzyme label plate for 3 times by using a PBST solution, wherein each time is 3-5 min, then adding 1 wt% casein for sealing, keeping the volume of each well at 200 mu L, and incubating for 1-2 h at 37 ℃; the purpose of the PBST solution wash is to wash away unbound CPT @ PSI_OAmCoating antigen;

(3-3) sample addition Competition: PBST solution washes 96-well enzyme label plate 3 times, each time3-5 min, then adding 50 mu L of CPT @ PSI with different concentrations_OAmStandard solution and 50. mu.L of FITC-labeled PSI_OAmMixing the antibodies in advance, adding the mixture into each hole in gradient, incubating for 1-2 h at 37 ℃, washing for 2-4 times by using a washing solution, and drying for 3-5 minutes each time;

And (3-4) measuring the fluorescence intensity of each hole on a microplate reader when the excitation wavelength is 485nm and the emission wavelength is 525 nm.

In the step (4), the linear equation of the standard curve is F ═ 8116.6-311.7lgC, F is fluorescence intensity, and C is CPT @ PSI_OAmThe concentration of (c); coefficient of correlation R²linear range 0.12-1638.7ng/mL, detection limit 0.04ng/mL ═ 0.991.

In the preparation method provided by the invention, PSI with the particle size of 200nm is used_OAmHigh-titer PSI obtained by immunizing white rabbits with nanoparticles as immunogen_OAman antibody. By combining CPT and PSI_OamThe polymer and polyethylene-b-polyethylene glycol are dissolved in chloroform, and then added into sodium hydroxide solution for ultrasonic reaction to realize drug loading and PSI_OAmThe hydrolysis is completed in one step to obtain CPT @ PSI_OAm-COO^-Hapten and further preparing CPT @ PSI_OAmThe envelope antigen of (1). The polyethylene-b-polyethylene glycol as an amphiphilic block polymer can play a role in modification and protection, can prolong the circulation time of the nano-drug carrier in vivo and promote accumulation of target tissues. In addition, the polyethanol block part can provide good hydrophilicity, and the hydrophobic polyethylene block part can promote the encapsulation of hydrophobic drugs, so that a uniform and stable nano-drug carrier is formed.

The oleylamine grafted polysuccinimide is a pH response nano-drug carrier, the inner core of the carrier has hydrophobicity, and the anticancer drug Camptothecin (CPT) can be mixed with PSI_OAmThe hydrophobic part of the nano-drug carrier forms hydrogen bonds, and the hydrophobic drug is successfully wrapped in the hydrophobic cavity of the nano-drug carrier; meanwhile, the shell has hydrophilicity, can provide good biocompatibility and can convey the anti-cancer drug to a target position. Camptothecin (CPT) and PSI due to acidic pH of cancer cell environment_OAmThe hydrogen bonds are easy to break, so that the medicine is released to achieve the purpose of treating the cancer.

General PSI_OAmCPT @ PSI after loading the anticancer drug camptothecin_OAmThe nanoparticles have a specific PSI_OAmThe smaller size of the nanoparticles (-100 nm) indicates that loading of the drug gives the nanoparticles a more compact appearance. Meanwhile, experimental results prove that the nano-drug carrier PSI_OAmNot only with anti-PSI_OAmThe antibody has high specificity combination, and the nano-drug carrier and the anti-PSI after the antibody loads the anti-cancer drug camptothecin_OAmAntibodies also bind with high specificity. Based on the method, a nano-drug carrier (CPT @ PSI) for loading the anticancer drug camptothecin is established_OAm) Detection, which provides a better quantitative detection scheme and wider practical application value for the nano-drug carrier loaded with the anticancer drug.

The invention establishes a nano-drug carrier (CPT @ PSI) for loading an anticancer drug camptothecin_OAm) Compared with the prior art, the fluorescence immunoassay quantitative detection method has the following advantages:

(1) the invention realizes the quantitative detection of the nano-drug carrier loaded with the anticancer drug camptothecin by using a direct competitive fluorescence immunoassay method for the first time, and provides a more effective detection means for the future anticancer drug detection.

(2) The invention uses a nano-drug carrier (CPT @ PSI) for loading an anticancer drug camptothecin_OAm) As coating antigen, and whether the coated medicine can be combined with anti-PSI_OAmAntibody specific binding and further validation of PSI using fluorescence immunoassay_OAmwrapping with PSI_OAmthe antibody still has specificity and meets the requirement of quantitative detection of CPT @ PSI_OAmThe purpose is.

(3) at present, the detection of the anticancer drugs mainly takes HPLC and ultraviolet detection as main materials, and the quantitative detection of the nano-drug carrier loaded with the anticancer drug camptothecin is more advantageous for the high-sensitivity quantification of the anticancer drugs by establishing an immunoassay method, so that a more sensitive detection scheme is provided for the detection of the nano-drug carrier and the drugs.

(4) The method establishes direct competition fluoroimmunoassay quantitative detection CPT @ PSI_OAmby detecting CPT @ PSI_OAmenvelope antigen, FITC-labeled PSI_OAmquantitative detection CPT @ PSI achieved by antibody complex fluorescent signal_OAmthe object of (1) is to further improve an immunoreaction signal by binding to a fluorescent label based on the antigen-antibody specificity, and to enable high-throughput measurement.

(5) nano medicine carrier PSI_OAmThe antibody can be further applied to wrapping other anti-cancer drugs, can specifically identify the nano-drug carrier loaded by other anti-cancer drugs by utilizing one antibody, has wide application range and has practical application value.

Drawings

in FIG. 1, (a) is PSI_OAmTEM image of hapten, (b) is CPT @ PSI_OAmA TEM image of the hapten;

CPT @ PSI in FIG. 2_OAm-COO^-The hapten is (a) an infrared characteristic pattern and (b) an XRD characteristic pattern;

FIG. 3 shows the CPT @ PSI_OAmThe logarithm of the concentration of the standard substance is the abscissa, and the fluorescence intensity value at the wavelength of 525nm is the ordinate, thereby establishing a standard curve.

Detailed Description

The present invention will be described in detail with reference to examples.

The raw materials used in the invention are as follows:

Polysuccinimide (Mw ═ 6000) was purchased from shijiazhuang dessai chemical ltd;

Camptothecin was purchased from Shanghai Mecline Biotechnology, Inc.;

FITC was purchased from Aladdin;

Polyethylene-b-polyethylene glycol (PE-b-PEG, Mn 1400) was purchased from ALDRICH;

The chicken egg white albumin was purchased from bio-engineering (Shanghai) Co., Ltd;

Other reagents are commercially available from commercial vendors.

High molecular polymer PSI_OAmThe preparation method comprises the following steps: heating 32mL of N, N-dimethylformamide to 90 ℃, adding 1.6g of polysuccinimide and 2.17mL of oleylamine, keeping the temperature at 100 ℃, heating for reacting for 5 hours, finally adding 360mL of methanol for precipitating, centrifuging,taking the precipitate and weighing to obtain the high molecular polymer PSI_OAm。

anti-PSI_OAmThe preparation method of the antibody refers to the method disclosed in CN201810459487.8, and the anti-PSI is prepared_OAmThe titer of the antibody was 1: 64000.

The preparation method of each solution used in the invention comprises the following steps:

PBS buffer: weighing 8.0g of NaCl, 0.1g of KCl and NaH₂PO₄·2H₂O 0.29g、Na₂HPO₄·12H₂Dissolving O2.96 g in distilled water, and fixing the volume to 1000mL to obtain PBS buffer solution with the concentration of 0.01 mol/LpH-7.4;

PBST solution: adding 500 mu L of Tween-20 into 1000mL of PBS, and uniformly mixing;

Coating buffer CB: weighing Na₂CO₃ 1.59g、NaHCO₃2.94g of the extract is dissolved in distilled water and the volume is up to 1000 mL; thus obtaining 0.05mol/L coating buffer solution with pH value of 9.6;

1 wt% casein: 0.3g of casein was weighed out and dissolved in 30mL of 0.01mol/L PBS buffer solution with pH 7.4, and the mixture was stirred well.