CN111298138A - Ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3/PTX@Anti-HE4 mAb - Google Patents

Ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3/PTX@Anti-HE4 mAb Download PDF

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CN111298138A
CN111298138A CN202010295841.5A CN202010295841A CN111298138A CN 111298138 A CN111298138 A CN 111298138A CN 202010295841 A CN202010295841 A CN 202010295841A CN 111298138 A CN111298138 A CN 111298138A
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ptx
bsa
ovarian cancer
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he4mab
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CN111298138B (en
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李菁菁
董丽娜
侯平甫
徐凯
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Xuzhou Medical University
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Abstract

The invention relates to an ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3A.weighing BSA, Gd (NO) for the/PTX @ Anti-HE4mAb3)3·6H2O and NaOH, and then mixing and stirring at constant temperature; b. pouring the mixture obtained in the step a into a dialysis bag, and dialyzing the mixture in ultrapure water to obtain BSA-Gd2O3Nanoparticles; c. concentrating the solution obtained in step b by 10 times, filtering with a filter, and adding NaOH to the solution after filteringAdjusting pH to 9, slowly adding PTX, performing ultrasonic action for 1.5 hr, ultrafiltering to remove unreacted PTX to obtain BSA-Gd2O3PTX solution; d. c, connecting the filtrate obtained in the step c with a monoclonal antibody HE4 antibody through an amide reaction, and finally performing ultrafiltration on the solution to remove unreacted impurities to obtain a nano probe BSA-Gd2O3the/PTX @ Anti-HE4 mAb. The invention has simple preparation steps, high loading rate and entrapment rate of the chemotherapeutic drug, good water solubility and stability and high T1 relaxation efficiency. The compound has good targeting for ovarian cancer cells and ovarian cancer tumor-bearing nude mice, successfully realizes the targeting magnetic resonance imaging and targeting anticancer effects, and also has the advantages of good biocompatibility, low toxicity and the like.

Description

Ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3/PTX@Anti-HE4 mAb
Technical Field
The invention relates to an ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3the/PTX @ Anti-HE4mAb belongs to the technical field of medicine.
Background
Ovarian cancer (oc) is currently the most fatal gynaecological malignancy in women worldwide and is characterized by high morbidity and mortality. Ovarian cancer, due to lack of specific clinical symptoms and biological indicators, usually has been diagnosed already to the advanced stage of the disease, and this stage is often followed by malignant metastasis to the pelvis and peritoneum. Another reason for the high mortality of ovarian cancer is limited and complex therapeutic measures. The current first-line treatment for ovarian cancer is surgical removal of the primary lesion followed by chemotherapy with Paclitaxel (PTX) and platinum drugs in combination. PTX is a taxane chemotherapeutic drug whose poor water solubility and pharmacokinetics limit its utility. Therefore, researchers have developed various nanocarriers for the delivery of PTX. However, it is noteworthy that most nanoparticles remain in the experimental phase due to uncertain biocompatibility in vivo. Researchers should therefore be forced to develop functional nanoprobes with greater clinical transformation potential.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3the/PTX @ Anti-HE4mAb has simple preparation process and less material consumption, and can realize targeted magnetic resonance enhanced imaging and targeted therapy of ovarian cancer.
In order to achieve the purpose, the invention provides the following technical scheme: ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3A/PTX @ Anti-HE4mAb comprising the steps of:
a. BSA, Gd (NO) were weighed3)3·6H2O and NaOH, then mixing and stirring at constant temperature; BSA is bovine serum albumin, Gd (NO)3)3·6H2O is hydrated gadolinium nitrate, and NaOH is sodium hydroxide;
b. pouring the mixture obtained in the step a into a dialysis bag, and dialyzing the mixture in ultrapure water to obtain BSA-Gd2O3Nanoparticles;
c. concentrating the solution obtained in the step b by 10 times, filtering by using a filter, adjusting the pH of the solution to 9 by using NaOH after filtering, slowly adding PTX, performing ultrasonic action for 1.5 hours, and performing ultrafiltration after ultrasonic action to remove unreacted PTX to obtain BSA-Gd2O3PTX solution; PTX is paclitaxel;
d. c, connecting the filtrate obtained in the step c with a monoclonal antibody HE4 antibody through an amide reaction, and finally performing ultrafiltration on the solution to remove unreacted impurities to obtain a nano probe BSA-Gd2O3(ii)/PTX @ Anti-HE4 mAb; HE4 is human epididymis protein 4.
Further, in the step a, the BSA is 250mg and the Gd (NO)3)3·6H2O was 1mL of 50mM and NaOH was 1mL of 2M.
Further, the total dialysis time in the step b is 48h, water is changed every 6h, and the water is changed for 8 times.
Further, in said step c, the size of the ultrafilter is 0.45 μ M, the NaOH concentration is 0.2M, and the PTX concentration and amount are 13mg/mL and 10 μ L, respectively.
Further, in the step d, 100. mu.L EDC (10mg/mL) was added to 20. mu.L monoclonal anti-HE4 antibody (1mg/mL), activated in an incubator at 37 ℃ for 15min, and then 100. mu.L NHS (10mg/mL), 500. mu.L BSA-Gd2O3PTX solution, and finally adding activated monoclonal anti-HE4 antibody into the mixture, and reacting at 37 ℃ for 2 h.
Compared with the prior art, the preparation method has the characteristics of simple preparation steps, high loading rate and encapsulation rate of the chemotherapeutic drug, good water solubility, good stability and high T1 relaxation efficiency. The probe has good targeting property for ovarian cancer cells and ovarian cancer tumor-bearing nude mice, successfully realizes the targeting magnetic resonance imaging and targeting anticancer effects, has the advantages of good biocompatibility, low toxicity and the like, and has good application prospect when being used as an ovarian cancer targeting diagnosis and treatment probe.
Drawings
FIGS. 1A-C are BSA-Gd preparations of the present invention2O3、BSA-Gd2O3PTX and BSA-Gd2O3(ii) electron micrograph of/PTX @ Anti-HE4 mAb;
FIGS. 1D-F are BSA-Gd of the present invention2O3、BSA-Gd2O3PTX and BSA-Gd2O3Particle size frequency distribution plot of/PTX @ Anti-HE4 mAb;
FIG. 2 shows BSA-Gd of the present invention2O3Zeta potential monitoring chart of the assembly process of the/PTX @ Anti-HE4mAb nanoprobe;
FIG. 3 shows BSA-Gd of the present invention2O3PTX in vitro drug release profile;
FIG. 4A shows different concentrations of BSA-Gd according to the present invention2O3And BSA-Gd2O3T1WI plot for/PTX;
FIG. 4B is a BSA-Gd preparation of the present invention2O3And BSA-Gd2O3A T1 relaxation rate fit plot for/PTX;
FIG. 5A is a graph showing BSA-Gd-concentration under 4 ℃ storage conditions according to the present invention2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb hydrated particle size time curve;
FIG. 5B is a graph showing BSA-Gd-concentration under 4 ℃ storage conditions in accordance with the present invention2O3PTX and BSA-Gd2O3Plot of the signal of the/PTX @ Anti-HE4mAb T1 as a function of time (the embedded images are the corresponding T1WI plots);
FIG. 6 shows BSA-Gd of the present invention2O3Toxicity test on HO-8910 ovarian cancer cells and EA.hy926 cells;
FIG. 7 EA at different concentrations of PTX.In vitro cytotoxicity assay of hy926 cells;
FIG. 8 is an in vitro cytotoxicity assay of HO-8910 ovarian cancer cells at different concentrations of PTX;
FIG. 9A is BSA-Gd of the present invention2O3PTX and BSA-Gd2O3Magnetic field after incubation of/PTX @ Anti-HE4mAb and HO-8910 ovarian cancer cellsT1WI for resonance imaging;
FIG. 9B shows BSA-Gd of the present invention2O3PTX and BSA-Gd2O3The magnetic resonance signal corresponds to the result after the/PTX @ Anti-HE4mAb is incubated with the cells;
FIG. 10A is HO-8910 tumor-bearing nude mouse tumor non-targeted enhanced MRI images;
FIG. 10B is HO-8910 tumor-bearing nude mouse tumor targeted enhanced MRI images;
FIG. 10C shows the change in signal intensity of HO-8910 tumor-bearing nude mouse tumor T1 WI;
FIG. 10D is an MRI image of HO-8910 tumor-bearing nude mouse liver after enhancement;
FIG. 11A is an image of each group of nude mice after treatment is over (a, b, and c represent PBS, BSA-Gd, respectively)2O3PTX and BSA-Gd2O3/PTX@Anti-HE4 mAb)；
FIG. 11B shows weekly MRI images of different groups of nude mice (a, B and c represent PBS, BSA-Gd, respectively)2O3PTX and BSA-Gd2O3(ii)/PTX @ Anti-HE4 mAb; from left to right for 0, 1 week, 2 weeks, 3 weeks in sequence);
figure 11C is an anatomical image of each group of nude mouse tumors after treatment is completed;
FIG. 11D is a tumor growth curve in nude mice of different treatment groups;
FIG. 12 is a graph showing the weight change of nude mice during the treatment of each group;
FIG. 13 shows the comparison of the tissue structures of the heart, liver, spleen, lung and kidney organs of nude mice after the treatment (HE staining, magnification × 200).
Detailed Description
The invention will be further explained with reference to the drawings.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention providesOvarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3Per PTX @ Anti-HE4mAb, 250mg BSA was weighed, dissolved in 9mL ultrapure water, stirred in a 37 ℃ water bath, and then 1mL 50mM Gd (NO) was added slowly3)3·6H2O, after 5min, adding 1mL of 2M NaOH, and reacting in a constant-temperature water bath at 37 ℃ for 12 h; after the reaction is finished, pouring the solution into a dialysis bag, dialyzing in ultrapure water for 48h, changing water every 6h for 8 times, and obtaining BSA-Gd after the dialysis is finished2O3A nanoparticle solution; BSA-Gd2O3Concentrating the solution by 10 times, and collecting the concentrated BSA-Gd2O3100 μ L of the solution was filtered using a 0.45 μ M size microporous membrane filter, the pH was adjusted to 9 with 0.2M NaOH, PTX was dissolved in absolute ethanol at a concentration of 13mg/mL in BSA-Gd2O3Adding 10 μ L PTX slowly into the solution, performing ultrasonic action for 1.5 hr, ultrafiltering to remove unreacted PTX to obtain BSA-Gd2O3PTX solution; mu.L EDC (10mg/mL) was added to 20. mu.L monoclonal anti-HE4 antibody (1mg/mL), activated in an incubator at 37 ℃ for 15min, followed by 100. mu.L NHS (10mg/mL), 500. mu.L BSA-Gd2O3PTX solution, adding activated monoclonal antibody HE4 antibody into the mixture, reacting at 37 deg.C for 2h, ultrafiltering the solution after reaction, and removing unreacted impurities to obtain nanometer probe BSA-Gd2O3PTX @ Anti-HE4 mAb. Finally, the prepared solution was kept in a refrigerator at 4 ℃ for further use.
As shown in Table 1, the drug loading rate and the encapsulation rate of paclitaxel were calculated for different reaction concentrations, and the results showed that the drug loading rate (5.75%) and the encapsulation rate (94.85%) of paclitaxel were the best when the reaction concentration was 80 mg/mL.
TABLE 1 drug loading and encapsulation efficiencies of nanomaterials after PTX reactions at different concentrations
PTX(mg/mL) Loading Efficiencies(％) Encapsulation Efficiencies(％)
13 0.98 97.84
40 3.04 97.32
65 4.91 97.17
80 5.75 94.85
As shown in FIGS. 1A-C, BSA-Gd2O3、BSA-Gd2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb nanoparticles are spherical, uniform in particle size, good in dispersity and free of obvious aggregation phenomenon.
As shown in the figure D-F, the particle size of the nano probe prepared by the invention is about 38 nm.
As shown in fig. 2, the Zeta potential change indicates successful assembly for each reaction step.
The drug release profile as shown in figure 3 shows that 37% of PTX is released within the first 24 hours and then an additional 3% is slowly released after 4 days, showing a slow release trend overall.
Following Gd in the material as shown in FIG. 4A3+The concentration is increased, and the corresponding T1WI signal is gradually increased; 4B represents BSA-Gd2O3And BSA-Gd2O3T1 relaxation rate r of PTX1Are respectively 11.62mM-1s-1、10.71mM-1s-1。
As shown in FIGS. 5A-B, BSA-Gd increases with storage time2O3PTX and BSA-Gd2O3The hydrated particle size and the magnetic resonance signal intensity of the/PTX @ Anti-HE4mAb do not show obvious changes, which indicates that the BSA-Gd2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb has good stability.
As shown in FIG. 6, different concentrations of BSA-Gd2O3HO-8910 ovarian cancer cells and EA.After co-culturing hy926 cells for 24 hours, the cells were incubated with BSA-Gd-not added2O3The two cells were found to have good biocompatibility.
As shown in FIG. 7, different concentrations of PTX, BSA-Gd2O3PTX and BSA-Gd2O3PTX @ Anti-HE4mAb and EA.After hy926 cells are co-cultured for 24 hours, compared with the cells of a control group which is only added with PBS, the nano probe BSA-Gd prepared by the invention is found2O3the/PTX @ Anti-HE4mAb was not significantly toxic to normal cells.
As shown in FIG. 8, different concentrations of PTX, BSA-Gd2O3PTX and BSA-Gd2O3The PTX @ Anti-HE4mAb was co-cultured with HO-8910 ovarian cancer cells for 24 hours, and then was combined with PTX and BSA-Gd2O3Comparison with PTX revealed that BSA-Gd2O3the/PTX @ Anti-HE4mAb has stronger killing effect on cancer cells.
As shown in FIGS. 9A-B, BSA-Gd prepared according to the present invention2O3PTX @ Anti-HE4mAb nano probe and BSA-Gd2O3MRI scan after co-incubation of/PTX and PBS with HO-8910 cells, BSA-Gd was found2O3The magnetic resonance enhancement effect of the/PTX @ Anti-HE4mAb is the best.
As shown in FIGS. 10A-C, BSA-Gd prepared according to the present invention2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb is injected into an ovarian cancer tumor-bearing nude mouse through tail vein, magnetic resonance imaging scanning is carried out at different time points, and T1 signal changes such as tumor and the like of the nude mouse are observedIt was found that BSA-Gd2O3The magnetic resonance enhancement effect of the/PTX @ Anti-HE4mAb is optimal; FIG. 10D shows that the nanoprobe is metabolized primarily by the liver system.
As shown in FIGS. 11A-D, BSA-Gd prepared according to the present invention2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb is periodically injected into an ovarian cancer tumor-bearing nude mouse through tail vein, the tumor size is periodically measured, the concurrent magnetic resonance imaging scanning is carried out, the tumor size change of the nude mouse is observed, and the BSA-Gd is found2O3The effect of the/PTX @ Anti-HE4mAb on inhibiting the growth of tumors in nude mice is better.
As shown in FIG. 12, no significant weight loss or animal death occurred in all three groups over the monitoring period, indicating that BSA-Gd2O3the/PTX @ Anti-HE4mAb has good treatment effect and no obvious toxic and side effect in vivo.
As shown in FIG. 13, 5 organs of heart, lung, liver, spleen and kidney of nude mice after treatment were separated and HE-stained to show no obvious organic lesions compared with the control group, which indicates that the BSA-Gd prepared2O3PTX and BSA-Gd2O3the/PTX @ Anti-HE4mAb had no significant toxic effect when applied.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any minor modifications, equivalent replacements and improvements made to the above embodiment according to the technical spirit of the present invention should be included in the protection scope of the technical solution of the present invention.

Claims (5)

1. Ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3A/PTX @ Anti-HE4mAb comprising the steps of:
a. BSA, Gd (NO) were weighed3)3·6H2O and NaOH, then mixing and stirring at constant temperature;
b. pouring the mixture obtained in the step a into a dialysis bag, and dialyzing the mixture in ultrapure water to obtain BSA-Gd2O3Nanoparticles;
c. concentrating the solution obtained in the step b by 10 times, filtering by using a filter, adjusting the pH of the solution to 9 by using NaOH after filtering, slowly adding PTX, performing ultrasonic action for 1.5 hours, and performing ultrafiltration after ultrasonic action to remove unreacted PTX to obtain BSA-Gd2O3PTX solution;
d. c, connecting the filtrate obtained in the step c with a monoclonal antibody HE4 antibody through an amide reaction, and finally performing ultrafiltration on the solution to remove unreacted impurities to obtain a nano probe BSA-Gd2O3/PTX@Anti-HE4 mAb。
2. The ovarian cancer diagnosis and treatment integrated nanoprobe BSA-Gd according to claim 12O3the/PTX @ Anti-HE4mAb, wherein in step a, the BSA is 250mg and the Gd (NO) is3)3·6H2O was 1mL of 50mM and NaOH was 1mL of 2M.
3. The ovarian cancer diagnosis and treatment integrated nanoprobe BSA-Gd according to claim 12O3the/PTX @ Anti-HE4mAb is characterized in that the total dialysis time in the step b is 48h, and water is changed every 6h and 8 times.
4. The ovarian cancer diagnosis and treatment integrated nanoprobe BSA-Gd according to claim 12O3the/PTX @ Anti-HE4mAb, wherein in step c the ultrafilter size is 0.45 μ M, the NaOH concentration is 0.2M, and the PTX concentration and amount are 13mg/mL and 10 μ L, respectively.
5. The ovarian cancer diagnosis and treatment integrated nanoprobe BSA-Gd according to claim 12O3the/PTX @ Anti-HE4mAb is characterized in that EDC is added into a monoclonal Anti-HE4 antibody in the step d, and NHS and BSA-Gd are added2O3PTX solution, and finally adding activated monoclonal anti HE4 antibody to the above mixture.
CN202010295841.5A 2020-04-15 2020-04-15 Ovarian cancer diagnosis and treatment integrated nano probe BSA-Gd2O3/PTX@Anti-HE4 mAb Active CN111298138B (en)

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CN112156192A (en) * 2020-09-29 2021-01-01 徐州医科大学 Composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof

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* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
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CN102735846A (en) * 2012-06-15 2012-10-17 河南生生医疗器械有限公司 Chemiluminescence immunodetection kit and detection method for ovarian cancer tumor marker HE4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112156192A (en) * 2020-09-29 2021-01-01 徐州医科大学 Composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof
CN112156192B (en) * 2020-09-29 2022-11-25 徐州医科大学 Composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof

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