CN112999153A

CN112999153A - Nano micelle carrying chemotherapeutic drug/photosensitizer and preparation method and application thereof

Info

Publication number: CN112999153A
Application number: CN202110257471.0A
Authority: CN
Inventors: 杨红; 张米娅; 陈华兵; 陈亮; 柯亨特; 邓益斌; 庞海燕; 朱洁
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-06-22
Anticipated expiration: 2041-03-09
Also published as: CN112999153B

Abstract

The invention discloses a nano micelle carrying chemotherapeutic drugs/photosensitizers, and a preparation method and application thereof, and aims to combine the effects of phototherapy and chemotherapy to prepare a micelle (ID-M) carrying indocyanine green (ICG) and adriamycin (DOX) together, characterize the prepared micelle, and investigate the in vitro anti-tumor effect of the micelle at a cellular level. The micelle nanoparticles prepared by the method have the advantages of mild conditions, small particle size, uniform dispersion, round shape, good chemical stability and illumination stability, and high active oxygen generation capacity under the irradiation of near-infrared light.

Description

Nano micelle carrying chemotherapeutic drug/photosensitizer and preparation method and application thereof

Technical Field

The invention relates to a nano-drug technology, in particular to a micelle nano-particle with double treatment effects of encapsulating chemotherapeutic drugs (such as adriamycin) and photosensitizers (such as indocyanine green) and a preparation method thereof.

Background

Cancer is a global major disease, and chemotherapeutic drugs are the main treatment means of cancer, but have the problems of high toxicity, poor selectivity, strong side effects and the like. Even if the method of drug combination therapy or combined application of surgery and chemotherapy and the like is adopted, the curative effect is still poor. The effect of the medicine on tumor can be effectively improved by carrying the medicine by using novel medicine delivery systems such as liposome, micelle and the like.

Doxorubicin (DOX) is widely used as an anti-tumor chemotherapeutic drug, but has the problems of cardiotoxicity and the like. The main principle of phototherapy, which is a novel method for treating tumors in recent years, is that photosensitizers generate heat and active oxygen after being irradiated by excitation light, thereby killing cancer cells. Indocyanine green (ICG) is currently the only photosensitizer approved by the FDA for marketing, and illumination can produce both PTT and PDT therapeutic effects. And has very low toxicity in the absence of illumination, but is unstable in water and is easy to be eliminated in vivo.

Existing studies co-entrap ICG and DOX with different types of vectors, including: different drug delivery systems are formed by biologically purified carriers (such as human serum albumin, erythrocyte membranes and the like), chemically synthesized carriers (responsive polymers, polymers for modifying target heads and the like) and inorganic nanoparticle carriers (mesoporous silica, gold nanocages, MOF structures and the like), the particle size is mostly between 100 and 200nm, and the ideal release effect and the effect of killing tumor cells can be achieved under certain conditions. However, the preparation process of these nanoparticles is relatively complex, the biologically purified carrier needs complex extraction and purification, and the chemically synthesized high molecular polymer has potential toxicity and complex synthesis steps, and the inorganic nanoparticles have poor biocompatibility and certain toxicity.

The micelle administration system has obvious advantages. It can raise the bioavailability of insoluble medicine, reduce toxic side effect, improve in vivo stability and prolong circulation time, and has slow release effect and passive targeting effect on tumor cell. The micelle is prepared by using the polymer material which is mature in synthesis process and is already on the market and the polyethylene glycol-b-polycaprolactone PEG-PCL as a carrier material, and is beneficial to further research, development and clinical application. The ICG/DOX micelle with the particle size of 221nm is prepared by the prior art at the temperature of 37 ℃. However, there are the following drawbacks: 1) the particle size is too large. Generally, the <200nm is more beneficial for the micelle to avoid the interference of an endothelial reticulum system RES and reach a tumor site in a targeted way; 2) the preparation temperature is high, ICG requires low temperature storage, which affects stability.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a micelle carrying chemotherapeutic drugs/photosensitizers, and a preparation method and application thereof. Based on the fact that after chemotherapeutic drug DOX enters cells, lysosome escape is achieved under the synergistic effect of photosensitizer ICG, and the chemotherapeutic drug enters into cell nuclei to exert chemotherapeutic effect, the nanoparticle coated with DOX and ICG is designed, 1) in the nanoparticle determination, a fluorescence spectrophotometer is adopted to determine the content of DOX, an ultraviolet-visible spectrophotometer is adopted to determine the content of ICG, and the result is guaranteed to be reliable; 2) reliable results were obtained in vitro and in cell experiments, using free drug as well as micelles encapsulating ICG and DOX alone as controls. Therefore, the invention adopts the PEG-PCL which is a commercially available carrier material, realizes the preparation of the ICG/DOX co-entrapped micelle (ID-M) with the particle size of less than or equal to 100 nm at room temperature (25 ℃), has the advantages of simple preparation process, mild preparation conditions, controllable particle size, good biocompatibility, good tumor targeting property, good retention property and the like, and integrates the preparation and application of the nano-micelle which realizes the efficient killing of tumor cells by a photochemical treatment means, effectively combines with the light treatment to promote the chemotherapeutics to enter the nucleus to exert curative effect.

The invention adopts the following technical scheme:

a nano micelle carrying chemotherapeutic drug/photosensitizer is obtained by polymer-encapsulated chemotherapeutic drug and photosensitizer; the hydrated particle size of the nano micelle carrying the chemotherapeutic drug/photosensitizer is 10-160 nm; preferably, the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.

The preparation method of the nano micelle carrying the chemotherapeutic drug/photosensitizer comprises the following steps of mixing a chemotherapeutic drug solution, a photosensitizer solution and a polymer solution, and then carrying out ultrasonic treatment to obtain a solution A; dropwise adding the solution A into water, and carrying out ultrasonic treatment to obtain a solution B; and dialyzing, ultrafiltering and centrifuging the solution B to obtain the nano micelle carrying the chemotherapeutic drug/photosensitizer.

The invention discloses the application of the nano micelle carrying the chemotherapeutic drug/photosensitizer in the preparation of tumor treatment drugs; the polymer PEG-PCL is selected as a carrier, and the chemotherapeutic drug and the photosensitizer are simultaneously encapsulated, so that the dual targeting treatment effect of combining chemotherapeutic toxicity and phototoxicity on tumors in a nano drug-loaded platform is realized, and the effects of chemotherapy and/or photodynamic treatment and/or photothermal treatment are achieved.

In the invention, the preparation of the nano micelle carrying the chemotherapeutic drug/photosensitizer is carried out at room temperature.

In the present invention, the solvent in the chemotherapeutic drug solution, photosensitizer solution and polymer solution is DMSO (dimethyl sulfoxide).

In the invention, the mass ratio of the photosensitizer to the chemotherapeutic drug to the polymer is (0.1-2.0) to (0.1-2.0) (0.8-16), preferably (0.5-1.5) to (4-12), and most preferably, the mass ratio of the photosensitizer to the chemotherapeutic drug is 1: 1; for example, the mass ratio of the photosensitizer to the chemotherapeutic drug to the polymer is 1: 6-10.

In the invention, the volume ratio of the solution A to the water is 1: 5-15.

In the invention, the ultrasonic treatment time is 5-10 minutes.

In the invention, the concentration of the chemotherapeutic drug solution is 1mg/200 mu L; the concentration of the photosensitizer solution is 1mg/200 mu L; the concentration of the polymer solution is 6-10 mg/200. mu.L, preferably 8 mg/200. mu.L.

In the invention, the rotating speed of ultrafiltration centrifugation is 3500-6000 r.min^-1。

Dissolving DOXHCl in a DMSO solution at room temperature, adding triethylamine for desalting, and performing ultrasonic treatment to form a DMSO solution of DOX; dissolving ICG in DMSO, and performing ultrasonic treatment to form a DMSO solution of ICG; mixing a DMSO solution of PEG-PCL with a DMSO solution of DOX and a DMSO solution of ICG, dripping the mixture into water after ultrasonic treatment, performing ultrasonic treatment again to obtain a mixed solution containing micelles, and performing ultrafiltration and concentration to obtain a micellar solution ID-M encapsulating chemotherapeutic drugs and photosensitizer. The desalted adriamycin and indocyanine green are jointly encapsulated in the hydrophobic cavity of the micelle to obtain the micelle with the particle size not more than 160nm, and the micelle is used as a new antitumor medicament, so that the toxicity and the targeting of the medicament to tumors are enhanced, the synergistic effect is exerted, the toxic and side effects of the whole body are reduced, and the combined treatment of the tumors is realized.

In the invention, the chemotherapeutic drug is adriamycin, and the photosensitizer is indocyanine green; the structural formulas of the adriamycin (DOX) and the indocyanine green (ICG) are respectively as follows:

the micelle prepared by the PEG-PCL material sold in the market has ideal particle size, illumination stability, micelle release and tumor cell killing effect, and has a wide application prospect in tumor treatment. And the mechanism of the anti-tumor effect is investigated at the cell level, and the micelle entrapping two drugs is proved from different directions of photo-thermal, singlet oxygen, cytotoxicity, intracellular drug distribution and the like, so that the micelle has the anti-tumor synergistic effect

The micelle nanoparticle prepared by the invention has high tumor cell uptake and large killing effect on tumor cells, has synergistic effect in combination with chemotherapy/photodynamic therapy under the condition of near-infrared illumination, treats injured cells and enhances the chemotherapy effect by photodynamic therapy, shows high-efficiency low-toxicity anti-tumor effect of chemotherapy and photodynamic therapy, and is a safe and effective nano new preparation.

Drawings

Figure 1 morphology characterization of nanoparticles: transmission electron micrograph of 1-A micelle; the dynamic particle size of the 1-B micelle and a distribution map thereof; zeta potential diagram of 1-C micelles.

FIG. 2 is a graph of the photostability of micelles.

FIG. 3 is a graph of the cumulative amount of drug released from the micelle.

FIG. 4 is a graph showing the amount of micelle uptake by breast cancer cells 4T 1.

FIG. 5 is a graph showing the cytotoxicity results of micelles against 4T1 cells.

FIG. 6 is a fluorescence image of active oxygen generated in cells promoted by micellar irradiation.

FIG. 7 is a diagram showing that micellar irradiation promotes disruption of lysosomal membrane.

FIG. 8 fluorescence of drug nucleation before and after micelle illumination.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention without, however, limiting it. The micelle nanoparticle carrying the chemotherapeutic drug/photosensitizer is called micelle for short. The carrier of the invention is PEG-PCL, in particular to PEG₁₁₅-b-PCL₆₀Commercially available or prepared according to conventional methods available, such as "research on environmentally responsive nanoparticles for tumor imaging and therapy"; the ultrasonic device is KQ-100DE, ultrasonic instruments of Kunshan city, Inc., and the ultrasonic power of the embodiment is 100W.

Example one

Weighing 8mg PEG₁₁₅-b-PCL₆₀Dissolving in 200 mL DMSO, and performing ultrasonic treatment for 5 minutes to obtain a polymer solution; weighing ICG 1mg, adding 200 mL of DMSO, and performing ultrasonic dissolution for 5 minutes to obtain a photosensitizer solution; separately weighing1mg DOX & HCl is dissolved in 200 mL DMSO, 3 mL triethylamine is added, and the mixture is sonicated for 5 minutes to obtain a desalted adriamycin solution.

Preparation of micelles:

micelles are prepared by a liquid dispersion method. Mixing the three solutions, and continuing to perform ultrasonic treatment for 5 min to obtain a uniform solution A. And (3) dropwise adding the solution A into water (2 drops per second) while carrying out ultrasonic treatment on another 6 mL of deionized water, and carrying out ultrasonic treatment on the solution A for 10 minutes to obtain a micelle primary solution B, wherein the whole operation process is room temperature (25 ℃). Dialyzing solution B (intercepting molecular weight of dialysis bag MWCO: 3500 Da) overnight, changing deionized water every 2 h for dialysis, placing in ultrafiltration centrifuge tube, and dialyzing at 5000 r.min^-1The mixture is ultrafiltered and centrifuged at the rotating speed for 10 minutes to obtain a micelle solution ID-M which is carried by ICG and DOX together and is a nano micelle solution carrying chemotherapeutic drugs/photosensitizers, and the micelle solution is placed for 48 hours without turbidity.

The drug loading and encapsulation efficiency are calculated according to conventional methods, such as Drug Loading (DL) calculated according to the formula DL% = (mass of free drug + mass of encapsulated drug)/(mass of free drug + mass of encapsulated drug + mass of carrier) x100%, and Encapsulation Efficiency (EE) calculated according to the formula EE% = (mass of encapsulated drug)/(mass of free drug + mass of encapsulated drug) x 100%. The encapsulation efficiency is respectively as follows: ICG 96.1 +/-1.4%, DOX 93.9 +/-3.2%, and the drug loading rates are respectively as follows: ICG 17.5 +/-2.4 percent and DOX 9.1 +/-1.8 percent.

In addition, in the method, single-encapsulated ICG micellar solution (I-M), single-encapsulated DOX micellar solution (D-M) and blank micellar solution (B-M) are prepared by the same method without adding ICG, DOX or ICG/DOX.

Morphology characterization of micelles:

(1) transmission electron microscopy characterization of micelles: and dripping 20 mu L of micelle solution ID-M onto a copper mesh carbon supporting film, putting the copper mesh carbon supporting film into a dryer, volatilizing water, and observing the shape by using a 120 kV Transmission Electron Microscope (TEM). The results are shown in FIG. 1-A. The result shows that the prepared nanoparticles are regular round and have the particle size of 41.0 +/-5.9 nm.

(2) The particle size and the distribution of the micelle are characterized: taking the newly prepared micelle solution ID-M, diluting conventionally, performing ultrasonic treatment for 10 min, mixing uniformly, taking 1 mL, and analyzing the particle size and distribution by using a laser scattering particle size analyzer. The results are shown in FIG. 1-B. The result shows that the prepared micelle is in unimodal distribution, the average hydrated particle size is 80.4 +/-14.3 nm (the particle size containing a surface hydrated layer), the polydispersity index (PDI) is 0.252 +/-0.015, the interception of endothelial reticulocytes is avoided, the intrasomal long circulation is formed, and the passive targeting effect is exerted; as shown in FIG. 1-C, the micelle is negatively charged and has a Zeta potential of-17.23. + -. 0.66 mV. Is beneficial to the circulation of the micelle in vivo and is not easy to be removed.

Investigating the photo-thermal stability of the micelle:

the ICG concentration is 10mg × mL^-10.5 mL of the ID-M micelle solution of (1.5W. times.cm) using a 785 nm laser^-2) The light was illuminated for 5 min and the solution temperature was recorded every 30 s. Turning off the laser, cooling the solution to room temperature, irradiating again for 5 min under the same conditions, and repeating the above steps for 5 times. The results are shown in FIG. 2, the first three temperature increases steadily reach 36.5 + -1.1^oC (temperature difference: 11.5 +/-1.1)^oC) And the photo-thermal stability is good.

Examination of the in vitro release behavior of micelles:

the examination was carried out by dialysis. Respectively mixing 80 mg × mL^-1The DOX concentration ID-M and free I/D solution was placed in dialysis bag (MWCO: 3500 Da), then placed in PBS at pH 7.4 PBS, pH 5.0 simulating physiological environment and environment in tumor cell lysosome, and mixed at 37^oC、120 r×min^-1Dialyzing in a constant temperature oscillator. Respectively taking release solutions at different time points, supplementing an equal volume of fresh buffer solution, detecting the content of DOX in the buffer solution by a fluorescence method, calculating the cumulative release amount, and inspecting the release rule. Free ICG/DOX (free I/D) was prepared by first mixing both in a volume of DMSO (5% DMSO) and then adding deionized water under sonication to form a clear system.

Fig. 3 is a graph of cumulative release. Table 1 shows that the zero order equation, the first order equation, the Higuchi equation and the Weibull equation are respectively adopted to fit the data related to the cumulative release amount of DOX, so as to obtain the release behavior results of ID-M and free I/D in PBS (pH 5.0 PBS) and PBS (pH 7.4): cumulative release of Free (Free) I/D in PBS at pH 7.4 over 24 h was74.8 +/-7.0 mu g, and the cumulative release amount of 24 h in PBS with the pH value of 5.0 is 77.3 +/-2.6 mu g; the cumulative release of ID-M at pH 7.4 in PBS for 24 h was 23.1. + -. 5.2. mu.g, and the cumulative release at pH 5.0 in PBS for 24 h was 47.5. + -. 4.7. mu.g. The release rules of the micelle drug and the free drug are fitted and then accord with the Weibull equation, the drug is loaded by the micelle and has obvious slow release effect, the release is very slow in the PBS with the pH value of 7.4 for simulating the normal physiological environment, the release is accelerated by the PBS for simulating the environment pH value of 5.0 in the tumor cell lysosome, and the T is_1/2The time is shortened from 105.36 h to 22.85 h, and the time is shortened by 78.3%. The ID-M can deliver DOX to the lysosome of the tumor cells in a targeted mode, release and play a role, and the leakage of the precursor is less when the target site is reached.

TABLE 1 statistical table of micelle release profiles

5. Examination of cell uptake:

4T1 cells grown logarithmically were seeded in 6-well plates (cell density 5' -10)⁵One/well), placed in a cell incubator (37)^oC，5% CO₂) After 24 h incubation, ICG concentrations of 8mg × mL were added^-1The control was incubated for 24 h with Free ICG + DOX mixture (Free I/D) and the cells were washed with PBS, and the cells were pipetted into PBS and counted; the cells were crushed by ultrasound (500W, 120 s), the cell crushing solution was added to DMSO, and the concentrations of ICG and DOX taken in by the cells were measured by an ultraviolet spectrophotometer and a fluorescence spectrophotometer, respectively. FIG. 4 shows that the drug-loaded micelle-loaded ID-M provides ICG uptake into cells in an amount of 1.72. + -. 0.02. mu.g/10⁶The amount of DOX taken up by each cell was 1.38. + -. 0.11. mu.g/10⁶One cell, significantly higher than the free ICG uptake of 0.82 +/-0.02 mug/10⁶The amount of free DOX per cell was 0.54. + -. 0.03. mu.g/10⁶(ii) individual cells; the uptake of DOX was increased 2.0-fold and the uptake of ICG was increased 1.6-fold. And the cell uptake of the corresponding drugs of the ID-M group is almost the same as that of the D-M group and the I-M group, and the ID-M keeps the advantages of each single entrapped micelle.

Cytotoxicity examination of micelles:

to investigate the toxicity of micelles to tumor cells, logarithmically grown 4T1 cells were seeded in 96-well plates (cell density 5' 10)⁴Each is multiplied by mL^-1) Incubation 37^oC，5% CO₂Adding different concentrations of I/D-M and Free I/D, changing culture medium 24 h after administration, and irradiating with 785 nm laser (1.5W × cm)^-23 min). After further incubation for 24 h, the medium was changed and 20 mL of 5 mg × mL was added^-1MTT solution, and incubation for 4 h. The liquid in the well plate was aspirated, DMSO was added, shaken and mixed well, and absorbance value (Abs) at 490 nm was measured with a microplate reader. Calculation of cell survival (%) = Abs_{Experimental group}/Abs_{Control group}And' 100 percent; inputting the survival rate and administration concentration of each group into IC₅₀A calculator for calculating to obtain IC₅₀A value; and, calculating the synergy index CI, CI = +, when CI<0.8, the synergistic treatment effect is obvious.

As can be seen from the cell viability in fig. 5, the blank micelle was hardly toxic to the cells. The result of calculation is that before the illumination, only DOX chemotherapy effect is achieved, and the cell survival rate under the high concentration of the ID-M group is 24.8 +/-2.2 mg multiplied by mL^-1The cell survival rate of Free I/D is 34.5 + -8.5 mg × mL^-1Calculating to obtain IC₅₀（mg×mL^-1): I-M has little toxicity over the range of dosing concentrations<<Free I/D group 3.02<<D-M group 1.44 ≈ ID-M group 1.35. After illumination, the composition has both DOX chemotherapy and ICG phototoxicity, IC₅₀（mg×mL^-1): I-M group 4.72<<D-M group 1.83<Free I/D group 1.21<<ID-M group 0.72. The cytotoxicity of ID-M is obviously increased after illumination, and the cell survival rate is 3.6 +/-0.3 mg multiplied by mL under high concentration^-1（IC₅₀46.67%) reduction in Free I/D cell viability 15.7. + -. 0.2 mg X mL^-1. Meanwhile, the cytotoxicity of the ID-M is statistically inspected, and has no significant difference (P) with other groups except the difference with the I-M under non-illumination>0.05). But under illumination ID-M (>1 mg×mL^-1) The difference (P) is very significant from each group<0.01), the anti-tumor effect is obviously enhanced. And the calculated synergy index CI of the ID-M is 0.62, and the CI conforms to the document pair CI<0.8 has remarkable synergyThe requirement of the same treatment effect indicates that the ICG and DOX co-entrapped ID-M has obvious antitumor synergistic effect when the two treatment means are combined.

Investigation of promotion of intracellular production of active oxygen:

4T1 cells in logarithmic growth phase were seeded in 12-well plates and ICG diluted with medium was added at a concentration of 4 mg X mL^-1I-D, D-M, ID-M and Free I/D, incubated for 24 h, washed three times with serum-Free medium and illuminated (785 nm, 1.5W × cm)^-2) DHE was added to the non-illuminated group for 30 min. The red fluorescence was observed with a fluorescence microscope. ICG promotes the production of Reactive Oxygen Species (ROS) which is an important indicator of its role in killing cells. Detecting ROS by a fluorescent probe dihydroethidium DHE, oxidizing the DHE which does not have fluorescence into oxidized ethidium in cells by the ROS, mixing the oxidized ethidium into DNA to generate red fluorescence, and observing the intensity of the fluorescence by using a fluorescence microscope to judge the amount of the ROS in the cells. As seen from the reactive oxygen species ROS fluorescence detection (fig. 6), fluorescence intensity (a.u.), before light irradiation: PBS group 0.2= Free I/D group 0.2= I-M group 0.2<D-M group 0.3= ID-M group 0.3, each group had almost only very weak fluorescence. After illumination: PBS group 0.4<D-M group 0.5<<Free I/D group 1.4<<I-M group 2.4<ID-M group 2.6. Thus, ID-M has the strongest ability to promote the generation of ROS in tumor cells and has the greatest phototoxicity.

Investigation of disruption of the lysosomal membrane to facilitate entry of the drug into the nucleus:

the detection was carried out by Acridine Orange (AO) fluorescent probe method, and 4T1 cells were seeded in 24-well plates and ICG diluted with medium was added to each plate at a concentration of 4 mg X mL^-1After incubation for 24 h in the solution of ID-M, Free I/D, I-M, D-M, the light group (785 nm, 1.5W × cm)^-2) Adding 10mg × mL together with the non-illuminated group^-1AO of (3) was incubated for 30 min. The cells in the well plate were observed for differential fluorescence using a fluorescence microscope. The Acridine Orange (AO) fluorescent probe can penetrate through a living cell membrane, enter acidic lysosomes in cells, and is protonated to generate red fluorescence, and green fluorescence is generated in neutral environments such as cell nucleus and the like. As is clear from the observation results of the fluorescence microscope in FIG. 7, the film rupture rates of the groups before the light irradiation were relatively close to each other. The ICG-containing sample groups all had reduced red fluorescence and green fluorescence after illuminationA phenomenon of fluorescence enhancement. After statistics, the lysosome membrane rupture rates of each group 30 min after illumination are respectively as follows: 1.3 percent of PBS group<D-M group 5.3%<Free I/D group 41.3%<I-M group 100% = ID-M group 100%. Therefore, ICG generates ROS after illumination, lysosome is promoted to rupture membranes, and DOX in ID-M is promoted to enter cell nucleus to play a role in killing tumor cells.

And (3) investigating the synergistic effect of promoting the chemotherapeutic drugs to enter cell nuclei by illumination:

4T1 cells were seeded on a confocal dish and ICG diluted with medium was added at a concentration of 4 mg X mL^-1The solution of (5) was incubated for 6 hours, and then irradiated with light (785 nm, 1.5W. times.cm)^-2) Incubation was continued for 6 h. With non-illuminated groups, 1mg × mL^-1Hoechest 33342 stained nuclei, incubated and washed three times with PBS, lysosomes were stained with 50 mM Lysotracker DND-26, incubated and washed, 1 mL of medium was added, and the state of cells in a confocal culture dish was observed with a laser confocal microscope.

DOX can enter the tumor cell nucleus to inhibit the synthesis of DNA and RNA, and the chemotherapeutical effect is judged according to the nuclear entering amount. FIG. 8 shows the DOX-in-kernel results in ID-M. The nuclei were stained blue, the lysosomes were stained green, and the DOX showed red fluorescence. DOX in the nucleus is reflected by the red-blue fluorescence co-localization rate, DOX in the lysosome is reflected by the red-green fluorescence co-localization rate, and the remaining DOX is in the cytoplasm. The red and blue co-localization rate is high, which indicates that much DOX enters the cell nucleus and the anti-tumor effect is good; the red and green co-localization rate is high, which indicates that much DOX exists in lysosomes and the effect is poor. As a result, examination of co-localization rate revealed that 92.5% of DOX was in lysosomes, 2.7% in nuclei, and 4.8% in cytoplasm before light irradiation; and at different time after illumination, the escape amount of the DOX from the lysosome into the cell nucleus is gradually increased until the DOX accounting for 82.7 percent of the total amount enters the cell nucleus after 12 hours after illumination, so that the medicine amount in the cell nucleus is increased by 29.6 times. Therefore, the cytotoxicity of ID-M is improved by illumination and is related to the increase of the drug amount entering cell nucleus, thereby proving that the ID-M can effectively inhibit the growth of tumor cells and has the synergistic effect of chemotherapy and phototherapy.

Comparative example 1

Weighing 8mg PEG₁₁₅-b-PCL₆₀Dissolving in 200 mL DMSO, and ultrasonic treating for 5 min to obtainTo a polymer solution; weighing ICG 1mg, adding 200 mL of DMSO, and performing ultrasonic dissolution for 5 minutes to obtain a photosensitizer solution; 1mg of DOX & HCl was further weighed and dissolved in 200 mL of DMSO, 3 mL of triethylamine was added, and the mixture was sonicated for 5 minutes to obtain a desalted adriamycin solution.

(1) Micelles are prepared by a liquid dispersion method. Mixing the three solutions, directly dripping into 6 mL of deionized water without ultrasonic treatment, dripping under ultrasonic treatment (2 drops per second), continuing ultrasonic treatment for 10 min to obtain a micelle primary solution B, wherein the whole operation process is room temperature (25 ℃). Dialyzing solution B (intercepting molecular weight of dialysis bag MWCO: 3500 Da) overnight, changing deionized water every 2 h for dialysis, placing in ultrafiltration centrifuge tube, and dialyzing at 5000 r.min^-1The rotating speed of the centrifugal pump is adopted for ultrafiltration and centrifugation for 10 minutes, the color of the supernatant is light, the lower layer precipitates a lot, and the color is dark, which indicates that the drug is separated out and the entrapment fails.

(2) Micelles are prepared by a liquid dispersion method. Mixing the three solutions, and continuing to perform ultrasonic treatment for 5 min to obtain a uniform solution A. And adding the solution A into water while carrying out ultrasonic treatment on another 6 mL of deionized water (the solution A is added within 1 second and is not added dropwise), and carrying out ultrasonic treatment for 10 minutes to obtain a micelle initial solution B, wherein the whole operation process is room temperature (25 ℃). Dialyzing solution B (intercepting molecular weight of dialysis bag MWCO: 3500 Da) overnight, changing deionized water every 2 h for dialysis, placing in ultrafiltration centrifuge tube, and dialyzing at 5000 r.min^-1The rotating speed of the centrifugal pump is adopted for ultrafiltration and centrifugation for 10 minutes, the color of the supernatant is light, the lower layer precipitates a lot, and the color is dark, which indicates that the drug is separated out and the entrapment fails.

(3) Micelles are prepared by a liquid dispersion method. The whole operation process is room temperature (25 ℃), mixing the three solutions, and continuing to perform ultrasonic treatment for 5 min to obtain a uniform solution A. Adding 6 mL of deionized water into water dropwise (2 drops per second) while performing ultrasound treatment, performing direct dialysis (MWCO: 3500 Da) overnight, changing deionized water every 2 hr, dialyzing in ultrafiltration centrifuge tube at 5000 r.min^-1The rotating speed of the centrifugal pump is adopted for ultrafiltration and centrifugation for 10 minutes, the color of the supernatant is light, the lower layer is precipitated much, and the color is dark, which indicates that the medicine is separated out and the entrapment is lostFail.

In contrast, the entrapment effect of the comparative method (3) is better than that of the comparative methods (1) and (2), but the entrapment effects of the three comparative methods are much lower than that of the drug loading method of the invention, and the method of the first embodiment of the invention has very little lower-layer precipitation after ultrafiltration.

Comparative example No. two

Micelle ID-M can also be prepared by adopting a film dispersion method, and the specific scheme is as follows: dissolving ICG, DOX and PEG-PCL in dichloromethane, ultrasonically mixing in a distillation flask, spin-drying in a rotary evaporator, adding distilled water dropwise under ultrasonic condition, dissolving the thin film on the wall of the distillation flask, and 1500 r.min^-1Centrifuging for 10 min, and filtering with 220 nm filter membrane to obtain micelle solution. The particle size of the micellar solution prepared by the method is 35.1 +/-2.8 nm, but turbidity is easy to generate, and the micellar solution is turbid after being placed for 10 minutes, which indicates instability.

Comparative example No. three

Adding 8mg PEG₁₁₅-b-PCL₆₀1mg ICG and 1mg DOX & HCl are dissolved in 600 mL DMSO, 3 mL triethylamine is added, and the mixture is subjected to ultrasonic treatment for 5 minutes to obtain a uniform solution A. And (3) dropwise adding the solution A into water (2 drops per second) while carrying out ultrasonic treatment on another 6 mL of deionized water, and carrying out ultrasonic treatment on the solution A for 10 minutes to obtain a micelle primary solution B, wherein the whole operation process is room temperature (25 ℃). Dialyzing solution B (intercepting molecular weight of dialysis bag MWCO: 3500 Da) overnight, changing deionized water every 2 h for dialysis, placing in ultrafiltration centrifuge tube, and dialyzing at 5000 r.min^-1The mixture is ultrafiltered and centrifuged for 10 minutes at the rotating speed to obtain a micelle solution ID-M which is jointly encapsulated by ICG and DOX, and the particle size of the micelle solution ID-M is analyzed by a laser scattering particle size analyzer, wherein the average hydrated particle size is 212.4 +/-12.7 nm.

The prior art has the problems of high preparation temperature and large particle size of nano-drugs, and the design of the invention is not more than 37 for better realizing the combined application of photosensitizer ICG and chemotherapeutic drug DOX^oAnd C, preparing the PEG-PCL entrapped ICG/DOX micelle with the particle size of less than 200nm at room temperature, fully exerting the synergistic effect of the two treatment means, and laying a foundation for further research and development of a novel high-efficiency low-toxicity preparation. The present invention relates to nano medicine technologyThe micelle nanoparticle is a micelle nanoparticle which is used for encapsulating and carrying a chemotherapeutic drug (such as adriamycin) and a photosensitizer (such as indocyanine green) and has double treatment effects, is prepared, is used as a new antitumor medicament, enhances the toxicity and targeting property of the medicament to tumors, plays a synergistic effect, reduces the toxic and side effects of the whole body, and further realizes the combined treatment of the tumors.

Claims

1. A nano micelle carrying chemotherapeutic drugs/photosensitizers is characterized in that the nano micelle is obtained by coating chemotherapeutic drugs and photosensitizers with polymers; the hydrated particle size of the nano micelle carrying the chemotherapeutic drug/photosensitizer is 10-160 nm.

2. The nanomicelle according to claim 1, wherein the chemotherapeutic agent is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.

3. The chemotherapeutic drug/photosensitizer loaded nanomicelle according to claim 1, characterized in that a chemotherapeutic drug solution, a photosensitizer solution, a polymer solution are mixed and then sonicated to obtain solution a; dropwise adding the solution A into water, and carrying out ultrasonic treatment to obtain a solution B; and dialyzing, ultrafiltering and centrifuging the solution B to obtain the nano micelle carrying the chemotherapeutic drug/photosensitizer.

4. Use of the chemotherapeutic drug/photosensitizer loaded nanomicelle of claim 1 for the preparation of a medicament for the treatment of tumors.

5. The method for preparing a chemotherapeutic drug/photosensitizer loaded nanomicelle according to claim 1, comprising the steps of mixing a chemotherapeutic drug solution, a photosensitizer solution, and a polymer solution, followed by sonication to obtain a solution a; dropwise adding the solution A into water, and carrying out ultrasonic treatment to obtain a solution B; and dialyzing, ultrafiltering and centrifuging the solution B to obtain the nano micelle carrying the chemotherapeutic drug/photosensitizer.

6. The method for preparing chemotherapeutic drug/photosensitizer loaded nanomicelles according to claim 5, wherein the preparation of the chemotherapeutic drug/photosensitizer loaded nanomicelles is performed at room temperature; the mass ratio of the photosensitizer to the chemotherapeutic drug to the polymer is (0.1-2.0) to (0.8-16).

7. The method for preparing nano-micelle carrying chemotherapeutic drug/photosensitizer as claimed in claim 5, wherein the volume ratio of the solution A and water is 1: 5-15.

8. The method for preparing nano-micelle carrying chemotherapeutic drug/photosensitizer according to claim 5, wherein the time of the ultrasonic treatment is 5-10 minutes.

9. The method for preparing nano-micelle carrying chemotherapeutic drug/photosensitizer according to claim 5, wherein the rotation speed of the ultrafiltration centrifugation is 3500-6000 r-min^-1。

10. The method for preparing the nano-micelle carrying the chemotherapeutic drug/photosensitizer as claimed in claim 5, wherein the chemotherapeutic drug is doxorubicin, the photosensitizer is indocyanine green, and the polymer is PEG-PCL.