CN115350280A

CN115350280A - Bionic nano-drug co-loaded with indocyanine green and inhibitor and preparation method thereof

Info

Publication number: CN115350280A
Application number: CN202211019704.4A
Authority: CN
Inventors: 王夺; 刘军杰; 张坤; 罗涛
Original assignee: Guangxi Medical University Affiliated Tumour Hospital
Current assignee: Guangxi Medical University Affiliated Tumour Hospital
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2022-11-18
Anticipated expiration: 2042-08-24
Also published as: CN115350280B

Abstract

The invention provides a bionic nano-drug co-loaded with indocyanine green and an inhibitor and a preparation method thereof, and relates to the technical field of drug synthesis. The bionic nano-drug co-loaded with indocyanine green and the inhibitor comprises a carrier, indocyanine green and the inhibitor. The invention firstly prepares FRMONs carrier, then carries ICG and PDE5i to establish acoustic dynamic engineering nano platform, the carrier with bell-shaped structure enables double scattering in the particles to generate a large amount of active oxygen in the process of acoustic dynamics, rich ROS accumulation can directly kill tumor cells, release antigen and activate systemic immune response, and simultaneously the jointly loaded PDE5i inhibitor can also release NO to promote the normalization of blood vessels.

Description

Bionic nano-drug co-loaded with indocyanine green and inhibitor and preparation method thereof

Technical Field

The invention relates to the technical field of drug synthesis, in particular to a bionic nano-drug co-loaded with indocyanine green and an inhibitor and a preparation method thereof.

Background

With the rapid development of nano science and technology, various organic or inorganic materials are made into nano structures for the biomedical field, and especially, the nano materials are used as drug carriers for disease treatment and are receiving more and more extensive attention.

The breast cancer is one of the most common tumors in clinic, the incidence rate of the breast cancer is increased year by year, and the health of human beings is seriously threatened. Triple negative breast cancer is high in malignancy degree, high in treatment difficulty and early in metastasis time, and the chance of surgical treatment is lost when the triple negative breast cancer is found to be in the middle and late stages. Therefore, the development of drugs having antitumor activity is an important problem to be solved at present.

Disclosure of Invention

In view of the above, the invention provides a bionic nano-drug co-loaded with indocyanine green and an inhibitor and a preparation method thereof, wherein a large amount of Reactive Oxygen Species (ROS) can be generated in an acoustic dynamic process by carrying a sonodynamic engineering nano-platform, wherein double scattering in particles is realized by a carrier with a bell-type structure. Abundant ROS accumulation can directly kill tumor cells, release antigens and activate systemic immune responses, and in addition, co-loaded phosphodiesterase 5 inhibitors (PDE 5 i) can release NO to promote vascular normalization.

A bionic nano-drug co-loaded with indocyanine green and an inhibitor is characterized in that,

comprises a carrier, indocyanine green and an inhibitor;

the carrier is FRMONs;

the inhibitor is a phosphodiesterase 5 inhibitor;

the medicine carrying rate of indocyanine green in the bionic nano-medicament is 4-5%; the drug loading rate of the phosphodiesterase 5 inhibitor is 7-8%;

the preparation method of the carrier comprises the following steps:

(1) Preparation of solid silica nanospheres (s-SiO) ₂ )：

Mixing deionized water, an ammonia solution and absolute ethyl alcohol, and then preheating for 30-40 minutes in a constant-temperature water bath at the temperature of 30 ℃ at the stirring speed of 300 rpm; slowly adding tetraethyl orthosilicate into the mixed solution, and carrying out hydrolysis reaction for 45-50 minutes at the temperature of 30 ℃ and the stirring speed of 300rpm to obtain solid silicon dioxide nanospheres;

(2) Coating treatment of solid silicon dioxide nanospheres:

adding a mixture of TEOS (tetraethyl orthosilicate) and PDES (phosphodiesterase) to the solid silica nanospheres prepared in the step (1), and coating the nanospheres with the hydrophobic hybrid layer for 1 hour at a stirring speed of 450-500 rpm at 25-30 ℃; then centrifuged at 12000rpm for 10 minutes at 25 ℃, the supernatant was discarded, and washed 3 times with deionized water; centrifuging at 5000rpm for 5min, removing supernatant, and collecting nanometer precipitate;

(3) Synthesis of FRMONs (fluorinated bell-type mesoporous organosilicon nanoparticles):

na is mixed with ₂ CO ₃ Dissolving in deionized water to prepare Na ₂ CO ₃ Dissolving, and then re-dispersing the nano precipitate collected in the step (2) in Na by ultrasonic vibration ₂ CO ₃ In solution; etching at 60 deg.C under 250rpm stirring speed for 7-10 min, centrifuging at 3000rpm for 5min to obtain FRMON, adding deionized water to resuspend FRMON, centrifuging at 5000rpm, washing FRMON twice, and storing for use.

Preferably, the volume ratio of the deionized water, the ammonia solution, the anhydrous ethanol and the tetraethyl orthosilicate in the step (1) is (5) from 1.5 to 2:3 to 5.

Preferably, in the mixture of TEOS and PDES in the step (2), the volume ratio of TEOS to PDES is 2.5-3:1-1.5.

Preferably, the volume ratio of the mixture of TEOS and PDES to the solid silica nanospheres in step (2) is 3.5-4.5 mL: 20-25 mg.

Preferably, na is used in step (3) ₂ CO ₃ The concentration of the solution is 0.06-0.07 g/mL.

Preferably, na is used in the step (3) ₂ CO ₃ The volume mass ratio of the solution to the nano-precipitate is 50-60 mL: 25-30 mg.

Preferably, in the re-suspension process in the step (3), the volume-to-mass ratio of deionized water to FRMON is 20-30 mL: 20-25 mg.

Preferably, the method comprises the following steps:

s1, preparing an indocyanine green (ICG) solution and a phosphodiesterase 5 inhibitor (PDE 5 i) solution respectively; mixing indocyanine green solution and phosphodiesterase 5 inhibitor according to the weight ratio of 1-2: 1-2 to obtain a mixed solution;

s2, under the condition of continuously stirring at 500rpm, adding the FRMON sample into the mixed solution obtained in the step S1 by using a microtiter plate within 8 hours to obtain a FRMON dispersion, reacting at room temperature for 24 hours, centrifuging at 5000rpm for 5 minutes, collecting precipitate, washing with absolute ethyl alcohol twice, and washing with deionized water once to obtain the indocyanine green and inhibitor co-loaded bionic nano-drug; the volume mass ratio of the absolute ethyl alcohol, the deionized water and the precipitate is 25mL: 10-15 mL:5mg;

s3, resuspending the bionic nano-drug co-loaded with the indocyanine green and the inhibitor and storing the resuspension in a PBS (phosphate buffer solution) solution, wherein the volume-to-mass ratio of the PBS solution to the bionic nano-drug is 25-30 mL:5mg.

Preferably, the concentration of the indocyanine green solution in the step S1 is 10mg/mL; the concentration of the phosphodiesterase 5 inhibitor solution is 20mg/mL.

Preferably, the concentration of the FRMON dispersion in step S2 is 5mg/mL.

Compared with the prior art, the invention has the following beneficial effects: the bionic nano-drug co-loaded with indocyanine green and the inhibitor is prepared by establishing a sonodynamic engineering nano-platform, wherein the carrier with a bell-type structure enables double scattering in particles to generate a large amount of Reactive Oxygen Species (ROS) in a sonodynamic process. Abundant ROS accumulation can directly kill tumor cells, release antigens and activate systemic immune responses, and in addition, co-loaded phosphodiesterase 5 inhibitors (PDE 5 i) can release NO to promote vascular normalization.

Drawings

FIG. 1 is a transmission electron micrograph of FRMON;

FIG. 2 is a FRMON BET analysis plot;

FIG. 3 is a graph of ultraviolet absorption spectroscopy analysis of FRMON;

FIG. 4 DLS particle size plots of nanoparticles loaded with different drugs;

figure 5 is a graph of ZETA potential changes of nanoparticles after loading with different drugs;

FIG. 6 is a chart of the infrared spectra of nanoparticles loaded with different drugs;

FIG. 7 is a release profile of ICG in ICG/PDE5i @ FRMON;

FIG. 8 is a release profile of PDE5i in ICG/PDE5i @ FRMON;

fig. 9 is a schematic diagram of acoustic refraction of FRMON nanoparticles after sonication;

FIG. 10 is a diagram of ESR signals of different nano-drugs (TEMP as capture agent);

FIG. 11 is a graph of DPBF consumption for different nanomedicines;

FIG. 12 is a fluorescent staining pattern of ROS and NO in tumor cells treated with different groups of nano-drugs;

FIG. 13 shows the results of semi-quantitative analysis of ROS in tumor cells after treatment with different groups of nano-drugs;

FIG. 14 shows the results of semi-quantitative analysis of NO in tumor cells treated with different groups of nano-drugs;

FIG. 15 is a colony lens of tumor cells after different groups of nano-drugs have been treated;

FIG. 16 shows the quantitative results of tumor colonies after treatment of tumor cells with different groups of nano-drugs;

FIG. 17 shows the results of Polymerase Chain Reaction (PCR) assay for normal vascular genes and vascular normalization genes;

FIG. 18 shows the variation of calreticulin content in different treatment groups;

FIG. 19 shows the variation of the amount of high mobility proteins in different treatment groups;

FIG. 20 shows the change in ATP content in different treatment groups;

FIG. 21 shows the result of flow cytometry detection of the maturation effect of dendritic cells;

FIG. 22 shows the results of quantitative analysis of the maturation effect of dendritic cells;

FIG. 23 is a graph of tumor volume change following tumor treatment in live mice;

FIG. 24 is a mouse model survival curve following subcutaneous tumor implantation in mice.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

A bionic nano-drug co-loaded with indocyanine green and an inhibitor and a preparation method thereof comprise the following steps:

(1) Preparation of solid silica nanospheres (s-SiO) ₂ )：

Deionized water (DIW, 5 mL), ammonia solution (1.6 mL), and absolute ethanol (36 mL) were gently mixed in a wild-mouth bottle and then preheated in a thermostatic water bath at 30 ℃ for 30 minutes with a stirring speed of 300 rpm. Then, 3mL of tetraethyl orthosilicate (TEOS) was slowly added to the above mixed solution with a pipette, and hydrolysis was carried out at 30 ℃ and a stirring speed of 300rpm for 45 minutes to obtain 24mg of solid silica nanospheres;

(2) Coating solid silicon dioxide nanospheres:

dropwise adding a mixed solution containing 2.5ml of TEOS and 1ml of LPDES to the solid silica nanoball prepared in step (1) using a microtiter meter, and coating the nanoball having the hydrophobic hybridization layer at 25 ℃ for 1 hour at a stirring speed of 450 rpm; subsequently, the mixed solution was centrifuged at 25 ℃ for 10 minutes (speed: 12000 rpm) to discard the supernatant, and washed 3 times with deionized water (25 mL), centrifuged again for 5 minutes (speed: 5000 rpm), the supernatant was discarded, and 26.2mg of the precipitate was collected;

(3) Synthesis of FRMONs:

mixing 3.18g of Na ₂ CO ₃ Dissolving in 50ml of LDIW, and vibrating the nano precipitate collected in step (2) by ultrasonicDynamic re-dispersion in Na ₂ CO ₃ In solution. After etching at 60 ℃ for 7 minutes at a stirring speed of 250rpm, the final FRMON 20.5mg was harvested by centrifugation at 3000rpm for 5 minutes, followed by addition of 25mLDIW to resuspend FRMON, washing FRMON twice at 5000rpm for 5 minutes each, and stored for future use;

(4) ICG/PDE5i @ FRMON Synthesis

S1, preparing an indocyanine green solution and a phosphodiesterase 5 inhibitor solution respectively, wherein the concentration of the indocyanine green solution is 10mg/mL; the concentration of the phosphodiesterase 5 inhibitor solution is 20mg/mL; mixing indocyanine green solution with phosphodiesterase 5 inhibitor according to the ratio of 1:2 to obtain a mixed solution;

s2, under the condition of continuously stirring at 500rpm, adding a FRMON sample into the mixed solution by using a microtiter plate within 8h to obtain a FRMON dispersion with the concentration of 5mg/mL, and reacting for 24h at room temperature; centrifuging at 5000rpm for 5min, and collecting precipitate with mass of 5mg; washing with 25mL of absolute ethyl alcohol twice, and washing with 15mL of deionized water once; obtaining the bionic nano-drug (ICG/PDE5i @ FRMON) carrying the indocyanine green and the inhibitor together;

and S3, re-suspending the nano-drug and storing the nano-drug in 25ml of PBS solution for further use.

The drug loading rates for ICG and PDE5i in ICG/PDE5i @ FRMON were 4.86% and 7.11%, respectively.

Comparative example 1

A bionic nano-drug carrying indocyanine green and a preparation method thereof comprise the following steps:

(1) The same as example 1;

(2) The same as example 1;

(3) The same as example 1;

(4) Synthesis of ICG @ FRMON:

an indocyanine green solution was prepared at a concentration of 10mg/mL, an FRMON sample was added to the above indocyanine green solution, and indocyanine green-loaded biomimetic nano-drugs (icg @ FRMON) were prepared according to step (4) of example 1, and the nano-drugs were resuspended and stored in a PBS solution for further use. In the ICG @ FRMON, the drug loading rate of ICG is 4.86%.

The following experimental analyses were performed on the nano-drugs prepared in example 1 and comparative example 1:

(1) The result of transmission electron microscope analysis of FRMON is shown in figure 1, and as can be seen from figure 1, the invention prepares a monodisperse FRMON carrier with the particle size of 200 nm.

(2) The BET analysis of FRMON showed that it has a double size mesoporous structure (4 nm and 11 nm) as shown in FIG. 2, and as can be seen from FIG. 2; this determines that FRMON can act as a support to hold both ICG and PDE5i by electrostatic interaction.

(3) The results of ultraviolet absorption spectroscopy analysis of FRMON are shown in FIG. 3, from which FIG. 3 characteristic absorption spectra of ICG (860 nm) and PDE5i (290 nm) can be observed, demonstrating successful loading of ICG and PDE5i.

(4) The particle size of the nanoparticles after loading different drugs is analyzed, a DLS particle size diagram is shown in figure 4, and as can be seen from figure 4, after loading ICG/PDE5i together, no obvious change of the particle size of FRMONs is found, which indicates that the size of the material is not influenced after loading the drugs.

(5) Fig. 5 is a graph showing the change of ZETA potential of nanoparticles after loading different drugs, fig. 6 is a graph showing the infrared spectrum of nanoparticles after loading different drugs, and it can be seen from fig. 5 and fig. 6 that the loading of ICG and PDE5i changes the surface ZETA potential and FTIR vibration intensity.

(6) FIG. 7 is the release profile of ICG in ICG/PDE5i @ FRMON and FIG. 8 is the release profile of PDE5i in ICG/PDE5i @ FRMON. Fig. 7 and 8 show that in the pH response mode, the release of ICG and PDE5i is favored by specific acidic tumor microenvironment (tumor acidic microenvironment closer using the pH 6.5 model), which can be further accelerated by local ultrasound irradiation (US).

(7) Fig. 9 is a schematic diagram showing acoustic refraction after the FRMON nanoparticles are subjected to ultrasound, and it can be seen from fig. 9 that two scattering convex interfaces (i.e. the inner core and the outer shell surfaces) in the FRMON carrier can realize two times of ultrasonic backscattering.

(8) Fig. 10 is an ESR signal diagram (taking TEMP as a capture agent) of different nano-drugs, and it can be seen from fig. 10 that a signal of obvious singlet oxygen appears under the guidance of ultrasound, indicating the generation of singlet oxygen.

(9) FIG. 11 is a graph of consumption of DPBF, a reagent that reacts with and is consumed by ROS, by various nanomedicines, such that the production of ROS can also be detected laterally. The lower the DPBF, the higher the ROS content generated is demonstrated.

(10) Grouping experiments were performed, G1: control (PBS), G2: FRMON (US), G3: ICG @ FRMON, G4: ICG @ FRMON (US), G5: ICG/PDE5i @ FRMON and G6: ICG/PDE5i @ FRMON (US). Different groups of nano-drug tumor cell level tests are adopted, the fluorescence staining patterns of tumor cell ROS and NO are shown in figure 12, the semi-quantitative analysis result of tumor cell ROS is shown in figure 13, and the semi-quantitative analysis result of tumor cell NO is shown in figure 14. As can be seen from fig. 12-14, the most produced (singlet oxygen) ROS in the group comprised by US and ICG, ICG @ frmon (US) (G4) and ICG/PDE5i @ frmon (US) (G6) produced more ROS, while PDE5i provided the strongest NO (nitric oxide) production capacity for ICG/PDE5i @ frmon (US).

(11) Grouping according to the experimental mode of experiment (10), performing clone formation test, and forming tumor colony light mirror image as shown in FIG. 15 and quantitative result of tumor colony as shown in FIG. 16 after tumor cell treatment. As can be seen from FIGS. 15-16, of these, ICG @ FRMON (US) (G4) and ICG/PDE5i @ FRMON (US) (G6) exert the most potent inhibitory effect to inhibit colony formation of 4T1 tumor cells. ICG @ FRMON (US) (G4) and ICG/PDE5i @ FRMON (US) (G6) were demonstrated to exert the greatest antitumor effect.

(12) The results of detecting normal vascular genes and vascular normalization genes by Polymerase Chain Reaction (PCR) using PDE5i @ FRMON nanomedicine are shown in FIG. 17. As can be seen from fig. 17, the three genes (TGFB 1, ANGPT1 and SIPR 1) representing vascular maturation of Human Umbilical Vein Endothelial Cells (HUVEC) were upregulated in the PDE5 i-related group, and were more significantly upregulated especially upon application of ultrasound. While the important angiogenic genes of the other three genes (VEGFA, EGF and ANGPT 2) were unchanged. This phenomenon indicates that PDE5i does promote vascular normalization without interfering with normal blood vessels.

(13) Grouping by way of experiment (10), exploring the effects of inducing immunogenic death of tumor cells in vitro, and examining some markers of immunogenic death, wherein figure 18 is the variation of Calreticulin (CRT) content in different treatment groups; FIG. 19 shows the variation of the amount of high mobility protein (HMGB 1) in different treatment groups; FIG. 20 shows the change in ATP levels in the different treatment groups. Since ROS can directly determine immunogenic death, this intragranular double-scatter enhanced ROS production in US and ICG-containing groups (ICG @ FRMON (US) (G4) and ICG/PDE5i @ FRMON (US) (G6)) results in significant upregulation of three immunogenic death markers including Calreticulin (CRT), high mobility protein (HMGB 1) and ATP.

(14) The dendritic cells were grouped in the manner of experiment (10) and the maturation effect of the dendritic cells was examined, and the flow cytometry results are shown in FIG. 21 and the quantitative analysis results are shown in FIG. 22. As can be seen from FIGS. 21 and 22, two groups of ICG @ FRMON (US) (G4) and ICG/PDE5i @ FRMON (US) (G6) induced dendritic cell maturation, confirming that they were able to potently activate immunity.

(15) Grouping according to the way of experiment (10), for the tumor treatment of the living mice, the change curve of the tumor volume is shown in FIG. 23, and from FIG. 23, ICG/PDE5i @ FRMON (US) (G6) shows the best treatment effect and has statistical significance.

(16) The tumor models were subcutaneously transplanted into mice grouped according to the experiment (10), and the survival curves of the mouse models are shown in FIG. 24. As can be seen from FIG. 24, the ICG/PDE5i @ FRMON (US) (G6) after the treatment exhibited the longest survival time and survival rate, and had statistical significance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. A bionic nano-drug co-loaded with indocyanine green and an inhibitor is characterized in that,

comprises a carrier, indocyanine green and an inhibitor;

the carrier is FRMONs;

the inhibitor is a phosphodiesterase 5 inhibitor;

the medicine carrying rate of indocyanine green in the bionic nano-medicine is 4-5%; the drug loading rate of the phosphodiesterase 5 inhibitor is 7-8%;

the preparation method of the carrier comprises the following steps:

(1) Preparing solid silicon dioxide nanospheres:

mixing deionized water, ammonia solution and absolute ethyl alcohol, and then preheating for 30-40 minutes in a constant-temperature water bath at the temperature of 30 ℃ at the stirring speed of 300 rpm; slowly adding tetraethyl orthosilicate into the mixed solution, and carrying out hydrolysis reaction for 45-50 minutes at the temperature of 30 ℃ and the stirring speed of 300rpm to obtain solid silicon dioxide nanospheres;

(2) Coating treatment of solid silicon dioxide nanospheres:

adding a mixture of TEOS and PDES to the solid silica nanospheres prepared in the step (1), and coating the nanospheres with the hydrophobic hybridization layer for 1 hour at a stirring speed of 450-500 rpm at 25-30 ℃; then centrifuged at 12000rpm for 10 minutes at 25 ℃, the supernatant was discarded, and washed 3 times with deionized water; centrifuging at 5000rpm for 5min, removing supernatant, and collecting nanometer precipitate;

(3) Synthesis of FRMONs:

na is mixed with ₂ CO ₃ Dissolving in deionized water to prepare Na ₂ CO ₃ Dissolving the solution, and then re-dispersing the nano precipitate collected in the step (2) in Na by ultrasonic vibration ₂ CO ₃ In solution; etching at 60 deg.C under 250rpm stirring speed for 7-10 min, centrifuging at 3000rpm for 5min to obtain FRMON, adding deionized water to resuspend FRMON, centrifuging at 5000rpm, washing FRMON twice, and storing for use.

2. The biomimetic nano-drug loading indocyanine green and the inhibitor together according to claim 1, wherein the volume ratio of the deionized water, the ammonia solution, the anhydrous ethanol and the tetraethyl orthosilicate in the step (1) is 5: 3 to 5.

3. The indocyanine green and inhibitor-co-loaded biomimetic nano-drug according to claim 1, wherein in the mixture of TEOS and PDES in the step (2), the volume ratio of TEOS to PDES is 2.5-3:1-1.5.

4. The indocyanine green and inhibitor-co-loaded biomimetic nano-drug according to claim 1, wherein the volume-to-mass ratio of the mixture of TEOS and PDES in step (2) to the solid silica nanospheres is 3.5-4.5 mL: 20-25 mg.

5. The biomimetic nano-drug co-loaded with indocyanine green and the inhibitor according to claim 1, wherein the Na in the step (3) ₂ CO ₃ The concentration of the solution is 0.06-0.07 g/mL.

6. The indocyanine green and inhibitor co-loaded biomimetic nano-drug according to claim 1, wherein the Na in step (3) is ₂ CO ₃ The volume mass ratio of the solution to the nano-precipitate is 50-60 mL: 25-30 mg.

7. The indocyanine green and inhibitor-co-loaded biomimetic nano-drug according to claim 1, wherein in the resuspension process in step (3), the volume-to-mass ratio of deionized water to FRMON is 20-30 mL: 20-25 mg.

8. The preparation method of the indocyanine green and inhibitor co-loaded bionic nano-drug according to any one of claims 1 to 7, characterized by comprising the following steps:

s1, preparing an indocyanine green solution and a phosphodiesterase 5 inhibitor solution respectively; mixing indocyanine green solution and phosphodiesterase 5 inhibitor according to the weight ratio of 1-2: 1-2 to obtain a mixed solution;

s2, under the condition of continuously stirring at 500rpm, adding a FRMON sample into the mixed solution obtained in the step S1 by using a micro-titrator within 8h to obtain a FRMON dispersion, reacting at room temperature for 24h, centrifuging at 5000rpm for 5min, collecting precipitate, washing with absolute ethyl alcohol twice, and washing with deionized water once to obtain the bionic nano-drug co-loaded with indocyanine green and the inhibitor; the volume mass ratio of the absolute ethyl alcohol, the deionized water and the precipitate is 25mL: 10-15 mL:5mg;

9. The preparation method of the indocyanine green and inhibitor-co-loaded biomimetic nano-drug according to claim 8, wherein the concentration of the indocyanine green solution in the step S1 is 10mg/mL; the concentration of the phosphodiesterase 5 inhibitor solution is 20mg/mL.

10. The indocyanine green and inhibitor-co-loaded biomimetic nano-drug according to claim 1, wherein the concentration of the FRMON dispersion in step S2 is 5mg/mL.