CN107007835A - Carry Prussian blue targeted nano compound and preparation method thereof - Google Patents

Abstract

The disclosure of the invention Prussian blue targeted nano compound of load, including its internal package have and on PEGylation folate-targeted lactic acid/co-glycolic acid shell of prussian blue nano particle, PEGylation folate-targeted lactic acid/co-glycolic acid shell taxol are distributed with.The technical problem to be solved in the present invention is to provide a kind of achievable multi-modal imaging of optoacoustic/magnetic resonance, and then the treatment of Image-guided is realized, complex treatment tumour improves Prussian blue targeted nano compound of load of oncotherapy curative effect and preparation method thereof.

Description

Prussian blue-loaded targeting nanocomposite and preparation method thereof

technical field

The invention relates to the field of polymer compounds, in particular to a targeting nanocomposite loaded with Prussian blue and a preparation method thereof.

Background technique

Breast cancer is one of the most common malignant tumors in women, accounting for 7-10% of all kinds of malignant tumors in the whole body. It is the second leading cause of tumor-related death, second only to lung cancer, and seriously affects women's body and mind. Health and even life-threatening. Therefore, the diagnosis of breast cancer is particularly important, and the diagnosis of breast cancer depends on the development of imaging technology, and further affects the treatment and prognosis of breast cancer. Mammography is one of the most effective means of diagnosing breast cancer in recent years, but there is still a risk of radiation. Ultrasound and magnetic resonance (MRI) also play an important role in the diagnosis, staging, and follow-up of breast cancer without the risk of radiation; however, limited spatial resolution and poor contrast affect the role of ultrasound in the early diagnosis of breast cancer. effect. Magnetic resonance (MRI) has the advantages of high spatial resolution and no tissue penetration limitations, but has the disadvantages of relatively low sensitivity and is expensive. Photoacoustic (PA) imaging (PAI) is an emerging non-invasive imaging method, which has the advantage of high resolution compared with ultrasound imaging; Deoxygenated hemoglobin) does not absorb light well, and PAI has relative limitations in detecting deep tissues. Therefore, the use of near-infrared absorbing substances as contrast agents for photoacoustic imaging to detect deep tissues has aroused widespread interest.

In summary, by combining photoacoustic imaging and magnetic resonance imaging with multimodal imaging, high resolution and high sensitivity can be achieved in order to detect deep tissue to achieve safe and radiation-free early diagnosis of breast cancer, real-time treatment monitoring and purpose of improving prognosis. However, to achieve efficient multimodal imaging, multifunctional multimodal molecular probe contrast agents are particularly important. Molecular probes based on nanomaterials can combine a variety of different imaging methods, overcome their respective limitations, and provide deeper information for tissues or diseases more intuitively and visually. Therefore, novel multimodal contrast agents for photoacoustic/magnetic resonance imaging have become a research hotspot in recent years.

Photothermal therapy (Photothermal therapy, PTT) is a non-invasive and minimally invasive tumor treatment method. There have been many studies in recent years. It uses photothermal substances to convert near-infrared light into heat to "burn" tumor cells. However, in photothermal therapy, the uneven distribution of heat generated may lead to incomplete tumor ablation leading to recurrence and metastasis. Combining photothermal therapy and chemotherapy can significantly increase the efficiency of comprehensive treatment of tumors. However, traditional chemotherapeutic drugs are non-specific to cancer cells. While killing cancer cells, they also cause damage to normal cells and tissues, which leads to serious side effects throughout the body, reduces curative effect, and may lead to drug resistance. . From the perspective of avoiding these shortcomings, the nanotechnology developed in recent years can be combined with chemotherapy drugs, so that anti-tumor drugs can selectively act on tumor cells, control drug release, prolong the circulation time of drugs in the body, and reduce systemic side effects; and, Highly targeted drugs and controlled-release drugs can be localized in the original tumor and metastases, and highly aggregated in the tumor site, selectively killing tumor cells, improving the effect of tumor treatment, and reducing systemic side effects. Among them, targeting is divided into passive targeting and active targeting, both of which can enable drug delivery to the tumor area. Active targeting is based on the combination of antigen and antibody to actively target tumor cells, which can increase the concentration of drugs at the tumor site and reduce the accumulation in healthy tissues and organs throughout the body, thereby increasing the therapeutic effect. Therefore, the combination of photothermal therapy and active targeting nano-chemotherapy will greatly improve the therapeutic effect of tumors.

Nano-diagnosis and treatment The further application and development of nano-medicine for advanced diagnosis and treatment can control the release of diagnostic and therapeutic substances, improve the efficacy of diagnosis and treatment, and reduce toxic and side effects. There are many kinds of diagnostic and therapeutic nanoparticles, but there are many limitations, including non-specific accumulation, insufficient circulation time, poor distribution in vivo, poor biodegradation, toxicity and so on. Ideal diagnostic and therapeutic nanoparticles can not only selectively and quickly accumulate in diseased tissues, realize multimodal imaging, improve effective treatment, but also are safe, non-toxic, non-immunogenic, and economical. Prussian blue nanoparticles (PB NPs) are a classic drug approved by the US Food and Drug Administration for the treatment of clinical radiation exposure. In recent years, PB NPs have attracted the attention of many researchers because of their high conductivity, outstanding stability, good biocompatibility, controllable particle size, and easy surface activation. Moreover, PB NPs can be used as photothermal substances for photothermal therapy due to their good photothermal conversion efficiency. However, there is still a gap between PB NPs and ideal nanoparticles for diagnosis and treatment, because the Prussian blue-based nanoparticles studied so far are all naked, without encapsulation or surface modification, and are easily cleared by the body over time. Moreover, most of these Prussian blue-based nanocomposites cannot achieve comprehensive therapeutic effects combining photothermal therapy and chemotherapy because they do not carry drugs. Although individual studies have designed Prussian blue nanomaterials to be loaded with drugs, how to make Prussian blue nanomaterials have an active targeting function so that Prussian blue and drugs can accurately reach the tumor area and achieve precise treatment is still a research hotspot.

Based on the above purposes, if a photoacoustic/magnetic resonance multimodal imaging can be designed, and then image-mediated therapy can be realized, the synthesized targeting nanocomposite can absorb near-infrared light energy and convert it into heat, improving The local temperature of the tumor, combined with chemotherapy drugs, can achieve comprehensive treatment of tumors and improve the efficacy of tumor treatment.

Contents of the invention

The technical problem to be solved by the present invention is to provide a Prussian blue-carrying targeting nanocomposite and its its preparation method.

In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions: Prussian blue-targeted nanocomposites, including PEGylated folic acid-targeted lactic acid/glycolic acid copolymer shells wrapped with Prussian blue nanoparticles inside, PEGylated folic acid-targeted The lactic acid/glycolic acid copolymer shell is embedded with paclitaxel.

Multifunctional nanocomposites are composed of various nanomaterials and have been applied in the diagnosis and treatment of diseases. The components of these nanocomposites include biomedical materials that can be used for imaging, such as organic dyes, quantum dots, magnetic resonance contrast agents, CT contrast agents, etc., and diagnostic substances, such as antitumor drugs, DNA, siRNA, etc. etc. At the same time, there are also carriers and some surface modifications together. Among them, if biomedical materials are to be safely delivered to target tissues, they must be compatible with the physiological environment. Many biomedical materials are hydrophobic and are easily cleared by the immune system in the body, so they cannot function well. Therefore, it is very important to choose an appropriate carrier so that it is compatible with the physiological environment.

The Prussian blue-loaded targeting nanocomposite of the technical solution of the present invention, PEG is the abbreviation of polyethylene glycol, PEGylated folic acid targeting lactic acid/glycolic acid copolymer is abbreviated as PLGA-PEG-FA, and the PLGA-PEG-FA is wrapped inside Prussian blue nanoparticles, referred to as PB NPs, the outer shell of PLGA-PEG-FA is inlaid with paclitaxel, that is, PTX, to form a Prussian blue-loaded targeting nanocomposite, referred to as PLGA-PB-PTX-PEG-FA nanocomposite, which can realize light Acoustic/magnetic resonance multimodal imaging, and then realize image-mediated therapy. The synthesized targeted nanocomposite can absorb near-infrared light energy, convert it into heat, increase the local temperature of the tumor, combine with chemotherapy drugs, realize comprehensive treatment of tumors, and improve the curative effect of tumor treatment. It has been found through research that the nanocomposite of the present invention can successfully reach the targeted tumor area through the vascular endothelial gap, and has good photothermal conversion ability. , the photothermal conversion efficiency of the nanocomposite is increased. In the case of four cycles of laser switching, the temperature rises and falls stably, showing good photothermal conversion characteristics and photothermal stability, and can be used as a good photothermal material; A multifunctional drug delivery system with high drug loading to achieve controlled and sustained release of drugs.

Further, the PEGylated folic acid targeting lactic acid/glycolic acid copolymer shell is polylactic acid glycolic acid PLGA with folic acid FA linked via polyethylene glycol PEG.

Furthermore, its shape is spherical; the particle diameter is 236.6±55.04nm; the potential is -24.44±1.7mV.

Further, the encapsulation efficiency of the paclitaxel is 77.82%, and the drug loading is 7.22%.

Further, there is an absorption peak in the wavelength range of 500-900nm.

Furthermore, there are absorption peaks at 1756.23 cm ^-1 and 2086 cm ^-1 , and a characteristic peak at 2927 cm ^-1 .

Further, the half-life of its laser irradiation is 12h.

The above are the properties and confirmation of the Prussian blue-loaded targeting nanocomposite of the present invention.

Another technical solution of the present invention, the preparation method of the Prussian blue targeted nanocomposite, includes the preparation of Prussian blue nanoparticles and the preparation steps of the nanocomposite, wherein the preparation steps of the nanocomposite are as follows:

(1) Weigh 5 mg of paclitaxel and 50 mg of PEGylated folic acid-targeted lactic acid/glycolic acid copolymer and put them into a beaker;

(2) Add 2 mL of dichloromethane into the beaker, seal the mouth of the beaker, shake it with an oscillator to dissolve it, and obtain A solution;

(3) Mix 200 μL, 100 mg/mL Prussian blue nanoparticles and 100 μL 4% polyvinyl alcohol, add the mixed solution dropwise to solution A, vibrate for 2 minutes in an ice bath, and a blue primary emulsion appears;

(4) Pour it into 5mL of 4% polyvinyl alcohol solution, and vibrate again for 2 minutes in an ice bath to obtain a light blue complex emulsion;

(5) mechanical stirring was volatilized to dichloromethane for 2 hours;

(6) Wash with deionized water several times and collect after centrifugation to obtain microspheres;

(7) Store the microsphere sample in a 4°C refrigerator to obtain the Prussian blue-targeted nanocomposite.

The preparation method of the Prussian blue-loaded targeting nanocomposite of the present invention uses PEGylated folic acid (folic acid, FA)-linked PLGA as a film-forming material, and adopts a double emulsification method to prepare loaded Prussian blue nanoparticles (PB NPs) and paclitaxel (Paclitaxel, PTX) PLGA-PB-PTX-PEG-FA targeting nanocomplex. The particle size, potential, surface morphology, etc. of the complex were detected; the ultraviolet absorption spectrum, infrared spectrum and structural characteristics of each component were detected by ultraviolet-visible spectrophotometry, Fourier transform infrared spectrometer, and laser confocal microscope; 0.647W/cm ² , 808nm laser irradiation was used to detect its photothermal properties in vitro; the encapsulation efficiency and drug loading of paclitaxel in nanocomposites were detected by high performance liquid chromatography. The PLGA-PB-PTX-PEG-FA targeting nanocomposite with spherical shape, regular shape, uniform size and stable properties was successfully prepared. The nanocomposite has good optical absorption properties and photothermal conversion characteristics, high drug loading capacity, and has good targeting ability to MBA-MD-231 breast cancer cells in vitro. Combined with laser irradiation, it has good in vitro The ability to combine chemotherapy and photothermal treatment of tumor cells.

Further, the preparation method of described Prussian blue nanoparticles is:

A. Add 0.5mmol 98mg citric acid to 20mL 1.0mmol/L FeCl ₃ aqueous solution, stir at 60°C to obtain solution a;

B. Then, add 20mL of 1.0mM potassium ferrocyanide aqueous solution containing 0.5mmol citric acid dropwise into solution a to form a clear blue solution, and stir at 60°C for 1min;

C. Cool to room temperature, and then stir at room temperature for 30 minutes to obtain a dispersion;

D. Add 40 mL of acetone to the dispersion, and centrifuge at 28,000 rpm for 1 hour to form a precipitate of Prussian blue nanoparticles;

E. Purification: Prussian blue nanoparticle precipitation was dissolved with 20mL distilled water sonication, separated and centrifuged with 20mL acetone;

F, repeating the purification process several times;

G. Finally, dissolve the precipitated Prussian blue nanoparticles in double-distilled water, place them in a 14000MWCO dialysis bag, and then dialyze the dialysis bag in double-distilled water for 24 hours to obtain Prussian blue nanoparticles;

H. Put the obtained Prussian blue nanoparticles into a refrigerator at 4° C. for preservation.

The preparation method of the Prussian blue-loaded targeting nanocomposite of the present invention uses PEGylated folic acid (folic acid, FA)-linked PLGA as a film-forming material, and adopts a double emulsification method to prepare loaded Prussian blue nanoparticles (PB NPs) and paclitaxel (Paclitaxel, PTX) PLGA-PB-PTX-PEG-FA targeting nanocomplex. The particle size, potential, surface morphology, etc. of the complex were detected; the ultraviolet absorption spectrum, infrared spectrum and structural characteristics of each component were detected by ultraviolet-visible spectrophotometry, Fourier transform infrared spectrometer, and laser confocal microscope; 0.647W/cm ² , 808nm laser irradiation was used to detect its photothermal properties in vitro; the encapsulation efficiency and drug loading of paclitaxel in nanocomposites were detected by high performance liquid chromatography. The PLGA-PB-PTX-PEG-FA targeting nanocomposite with spherical shape, regular shape, uniform size and stable properties was successfully prepared. The nanocomposite has good optical absorption properties and photothermal conversion characteristics, high drug loading capacity, and has good targeting ability to MBA-MD-231 breast cancer cells in vitro. Combined with laser irradiation, it has good in vitro The ability to combine chemotherapy and photothermal treatment of tumor cells.

Description of drawings

Fig. 1 is the transmission electron microscope figure of Prussian blue nano-particle in the embodiment of the present invention;

Figure 2 is a scanning electron microscope image of Prussian blue nanoparticles;

Fig. 3 is the transmission electron micrograph of PLGA-PB-PTX-PEG-FA nanocomposite;

Fig. 4 is the scanning electron micrograph of PLGA-PB-PTX-PEG-FA nanocomposite;

Figure 5 is a particle size diagram of the PLGA-PB-PTX-PEG-FA nanocomposite detected by a Malvern laser particle size analyzer;

Fig. 6 is the potential distribution diagram of Malvern laser particle size analyzer detecting PLGA-PB-PTX-PEG-FA nanocomposite;

Figure 7 is a UV spectrophotometric diagram of PLGA-PB-PTX-PEG-FA nanocomposite and PB NPs;

Fig. 8 is the Fourier transform infrared spectrogram of PLGA-PB-PTX-PEG-FA nanocomposite, PB NPs and PLGA-PTX-PEG-FA;

Fig. 9 is the proton nuclear magnetic resonance spectrum of PLGA-PEG-FA;

Figure 10 is the paclitaxel sustained release curve in vitro of the PLGA-PB-PTX-PEG-FA nanocomposite with or without laser irradiation;

Figure 11 is the magnification of PLGA-PB-PTX-PEG-FA nanocomposite targeting MDA-MB-231 cells in vitro × 600 times;

Figure 12 is the in vivo targeting effect of the PLGA-PB-PTX-PEG-FA nanocomposite on MDA-MB-231 transplanted tumors, towards the group and the non-targeting group (the tumor is circled);

Fig. 13 is the in vitro fluorescence imaging diagram of each tissue;

Figure 14 is a thermal infrared imaging image of the tumor site in the tumor-bearing nude mice of the PLGA-PB-PTX-PEG-FA nanocomposite+laser irradiation group and the control group;

Fig. 15 is a graph showing the relative tumor volume trend of nude mice in each treatment group.

detailed description

The preparation method of the present invention carrying the Prussian blue targeting nanocomposite, the specific steps are as follows:

1. Preparation of targeted nanocomposites loaded with Prussian blue

1. Preparation of Prussian blue nanoparticles

a. Add 0.5mmol citric acid (98mg) into 20mL FeCl ₃ (1.0mmol/L) aqueous solution, stir at 60°C to obtain solution a;

b. Then, add 20mL potassium ferrocyanide K ₄ [Fe(CN) ₆ ] (1.0mM) aqueous solution containing an equivalent amount of 0.5mmol citric acid dropwise to the above solution a solution, a clear blue solution appears, at 60°C Stir for 1 minute;

c, the solution is cooled to room temperature, and then stirred at room temperature for 30 minutes to obtain a dispersion;

d. Add the same volume of 40mL acetone to the dispersion, and centrifuge at 28000rpm for 1 hour to form a precipitate of Prussian blue nanoparticles;

e. Prussian blue nanoparticles were precipitated and then dissolved by sonication with 20 mL of distilled water, separated and centrifuged with an equal volume of 20 mL of acetone;

f. Repeat the purification process several times, five to ten times;

g. Finally, the precipitated Prussian blue nanoparticles were dissolved in double distilled water, placed in a 14000MWCO dialysis bag, and the breathable bag was dialyzed in double distilled water for 24 hours to remove excess citrate and other small particles, and then the dialyzed Prussian blue nanoparticles (PB NPs) were stored in a refrigerator at 4°C.

2. Preparation of targeted nanocomposites loaded with Prussian blue (PLGA-PB-PTX-PEG-FA)

Using the double emulsion method, paclitaxel-loaded targeting nanocomplexes were prepared. Specific steps are as follows:

(1) Weigh 5 mg paclitaxel and 50 mg PLGA-PEG-FA of PEGylated folic acid-targeted lactic acid/glycolic acid copolymer (PLGA-PEG-FA, 50:50) into a beaker;

(2) Add 2 mL of dichloromethane (CH ₂ Cl ₂ ) into the beaker, seal the mouth of the beaker with plastic wrap, and vibrate with an oscillator to completely dissolve the polymer material and paclitaxel;

(3) Add a mixture of 200 μL Prussian blue nanoparticles PB NPs (100 mg/mL) and 100 μL 4% polyvinyl alcohol PVA dropwise to the above solution, and vibrate for 2 minutes in an ice bath with a power of 130W*80%. blue primary emulsion;

(4) Quickly pour into 5mL PVA solution (4%) of polyvinyl alcohol, and fully sonicate for 2 minutes again under ice bath conditions, with a power of 130W*40%, to obtain a light blue complex emulsion;

(5) Stir mechanically for 2 hours until the CH ₂ Cl ₂ is fully volatilized;

(6) The microspheres were collected after repeated washing and centrifugation with deionized water, and the Prussian blue targeting nanocomposite was obtained;

(7) The samples prepared above were stored in a refrigerator at 4°C.

Experimental control group 1: prepared by the same method, the difference is: PLGA-PTX-PEG-FA nanoparticles without Prussian blue (without adding Prussian blue nanoparticles in step (3));

Experimental control group 2: prepared by the same method, the difference is: PLGA-PB-PEG-FA nanoparticles without paclitaxel (step (1) without paclitaxel);

Non-targeting group: prepared by the same method, except that PLGA-PEG without folic acid was used in step 1.

When conducting cell activity experiments and in vivo fluorescence experiments, it is also necessary to add fluorescent dyes, cell membrane red fluorescent probe DiI or cell membrane green fluorescent probe DIR to prepare DiI or DIR-loaded PLGA-PB-PTX-PEG-FA nanocomposites in step 1 Avoid light during preparation and storage.

2. Detection of general properties, photothermal characteristics, encapsulation efficiency and drug loading of paclitaxel, and sustained release experiment in vitro of the Prussian blue-loaded targeting nanocomposite (PLGA-PB-PTX-PEG-FA).

1. Basic properties of Prussian blue nanoparticles and Prussian blue-loaded targeting nanocomposites (PLGA-PB-PTX-PEG-FA nanocomposites):

(1) The surface morphology and structural characteristics of Prussian blue nanoparticles PB NPs and PLGA-PB-PTX-PEG-FA nanocomposites were observed by transmission electron microscopy and scanning electron microscopy;

(2) Malvern laser caliper and surface potential meter to measure the particle size, distribution and surface potential of PLGA-PB-PTX-PEG-FA nanocomposites;

(3) The absorption spectra of PLGA-PB-PTX-PEG-FA nanocomposites and Prussian blue nanoparticles are detected by ultraviolet-visible spectrophotometry;

(4) using a Fourier transform infrared spectrometer to detect the infrared spectra of PLGA-PB-PTX-PEG-FA nanocomposites, Prussian blue nanoparticles and PLGA-PTX-PEG-FA;

(5) Each component of the PLGA-PB-PTX-PEG-FA nanocomposite was detected by laser confocal microscope. 5.0 mL of 1.12 mg/mL citrate-protected Prussian blue nanoparticles was added to 0.55 mg (0.8 μmoles) 5-(aminoacetamido)fluorescein and 23 μg (120 nmols) N-(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride. The reaction mixture was stirred overnight and then dialyzed against a 3000MWCO membrane for one week to remove unreacted dye. PLGA-PB-PTX-PEG-FA nanocomposites were fabricated with dye-linked PB NPs and DiI-linked PLGA-PTX-PEG-FA. Confocal microscopy detected the fluorescence of the individual components of the PLGA-PB-PTX-PEG-FA nanocomposite at different emission wavelengths.

(6) Confirm the structure of PLGA-PEG-FA by ¹ H-NMR.

The test result is:

(1) As shown in Fig. 1 and Fig. 2, the prepared Prussian blue nanoparticles are shown in cube shape by transmission electron microscope and scanning electron microscope, and the particle diameter is about 40-50nm. As shown in Figure 3 and Figure 4, observed under the light microscope and scanning electron microscope, the PLGA-PB-PTX-PEG-FA nanocomposite is spherical, with smooth surface, uniform size and good dispersion.

(2) As shown in Figure 5 and Figure 6, the particle size of the PLGA-PB-PTX-PEG-FA nanocomposite detected by the Malvern laser particle size analyzer is (236.6±55.04) nm, and the surface potential is (-24.44±1.7) mV.

(3) As shown in Figure 7, the UV-Vis spectrophotometer detected an absorption peak in the wavelength range of 500-900nm in the PLGA-PB-PTX-PEG-FA nanocomposite and PB NPs, which was located around 702nm.

(4) As shown in Figure 8, the Fourier transform infrared spectrum results show that the absorption peaks of PLGA-PTX-PEG-FA and PB NPs are located at 1756.23cm ^-1 and 2086cm ^-1 , while PLGA-PB-PTX-PEG- The FA nanocomposite has the above two characteristic peaks at the same time. And, the PLGA-PB-PTX-PEG-FA nanocomposite has the characteristic peak of CH ₂ at 2927cm ^-1 .

(5) Laser confocal microscopy showed that PB NPs exhibited green fluorescence after excitation, PLGA-PTX-PEG-FA exhibited red fluorescence, and PLGA-PB-PTX-PEG-FA nanocomposites exhibited orange-yellow fluorescence.

(6) As shown in Figure 9, ¹ H-NMR is used to confirm the structure of PLGA-PEG-FA, and the chemical shift values are 1.0 and 4.9ppm. The peaks belong to the hydrogen protons of _-CH3 and -CH- on PLGA respectively, that is, peak 1 and peak 4; the peak with a chemical shift value of 3.4ppm belongs to the -CH ₂ - hydrogen proton on PEG, namely peak 2; the peak with a chemical shift value of 4.1ppm belongs to the aromatic amine hydrogen proton on folic acid, namely peak 3. The small peaks with chemical shift values of 6.5 and 6.8ppm were assigned to the aromatic nucleus protons of folic acid, namely peak 5 and peak 6.

2. In vitro photothermal properties of PLGA-PB-PTX-PEG-FA nanocomposites

Prepare various concentrations of PLGA-PB-PTX-PEG-FA nanocomposites (0.625mg/mL, 1.25mg/m, 2.5mg/mL, 5mg/mL, 10mg/mL, 20mg/mL), PLGA-PTX- PEG-FA (20 mg/mL).

(1) Put 20mg/mL PLGA-PB-PTX-PEG-FA nanocomposite, 200uL each of PLGA-PTX-PEG-FA and double distilled water in a 24-well plate, use 808nm, output power 0.647W/cm ² Laser irradiation for 10 min.

(2) Put 200uL of the PLGA-PB-PTX-PEG-FA nanocomposites with different concentrations prepared above into a 24-well plate, and irradiate with 808nm laser with an output power of 0.647W/cm ² for 10 minutes.

(3) Place 200uL of 20mg/mL PLGA-PB-PTX-PEG-FA nanocomposites in a 24-well plate, and the output powers are 0.328W/cm ² , 0.647W/cm ² , and 1.218W/cm ² , respectively. 808nm laser irradiation for 10 minutes.

(4) The 5mg/mL PLGA-PB-PTX-PEG-FA nanocomposite was irradiated with 808nm laser with an output power of 0.647W/cm ² for 50s, 380s off as a cycle, and four cycles of irradiation.

The temperature change caused by the above treatment was detected by an infrared imager, and the temperature was recorded every 10s.

The test result is:

(1) After laser irradiation, the temperature of the PLGA-PB-PTX-PEG-FA nanocomposite rises significantly. The initial temperature is about 27.5°C. After about 100s, the temperature rises to 70.2°C and then maintains at 70.2°C- After 520s, the temperature continued to rise to 90.4°C. However, the temperature of double distilled water group and PLGA-PTX-PEG-FA group rose to about 41°C due to laser irradiation, but did not continue to rise; as shown in Table 1-1-1:

Table 1-1-1 Temperature changes of different groups of reagents 0.647W/cm ² laser irradiation for 10 minutes

(2) After different concentrations of PLGA-PB-PTX-PEG-FA nanocomposites are irradiated by laser, it can be seen that each group has different degrees of temperature rise, showing a positive correlation between the rising temperature and the concentration of nanocomposites, as shown in Table 1-1-2 shows:

Table 1-1-2 Temperature changes of different concentrations of PLGA-PB-PTX-PEG-FA nanocomposite 0.647W/ ^cm2 laser irradiation for 10 minutes

(3) After 20mg/mL PLGA-PB-PTX-PEG-FA nanocomposites are irradiated with different power lasers, the temperature rises to varying degrees, showing a positive correlation between rising temperature and laser power, as shown in Table 1-1- 3 shows:

Table 1-1-3 Temperature changes of 20mg/mL PLGA-PB-PTX-PEG-FA nanocomposites irradiated with 0.328, 0.647 and 1.218W/cm ² lasers for 10 minutes

(4) After the 5mg/mL PLGA-PB-PTX-PEG-FA nanocomposite was irradiated by the laser switch for four cycles, it can be seen that the temperature rises in the same trend, and there is no obvious temperature peak drop, as shown in Table 1-1-4 :

Table 1-1-4 5mg/mL PLGA-PB-PTX-PEG-FA nanocomposite is irradiated with 0.647W/cm ² laser for 50s, the laser is turned off for 380s, and the temperature change of four cycles

time(s) 0 50 430 480 860 910 1290 1340 1720 temperature(℃) 24.5 52.1 26.7 55.1 26.7 56.0 26.7 55.8 26.5

3. Encapsulation efficiency, drug loading and sustained release in vitro of paclitaxel

1) Encapsulation efficiency and drug loading of PLGA-PB-PTX-PEG-FA nanocomposite paclitaxel

The encapsulation efficiency and drug loading of paclitaxel were detected by high performance liquid chromatography (HPLC), and the specific steps were as follows:

(1) Dissolve the paclitaxel powder with methanol, and prepare 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 75 μg/mL, 90 μg/mL, 100 μg/mL, and 200 μg/mL solutions respectively;

(2) The high-performance liquid chromatograph detects the sample injection, and the results are drawn into a standard curve;

(3) Dissolve 50 mg of PLGA-PB-PTX-PEG-FA nanocomposite (5 mg of paclitaxel) in a mixture of 1 mL of DMSO and 2 mL of dichloromethane, mix well until the microspheres are completely dissolved, take 0.1 mL and dilute with 0.9 mL of DMSO, After filtering with a 0.22 μm PVDF membrane, it is detected on the computer (chromatographic conditions: detection wavelength 227nm, mobile phase is acetonitrile: water (45:55, V/V), injection 10 μ L), and the encapsulation efficiency and loading of paclitaxel are calculated according to the following formula: dose:

Encapsulation efficiency (%)=drug content in the nanocomposite/total drug delivery amount×100%;

Drug loading (%)=amount of drug encapsulated in the nanocomposite/total weight of the nanocomposite×100%.

2) In vitro sustained release experiment of PLGA-PB-PTX-PEG-FA nanocomposite

The in vitro sustained-release properties of PLGA-PB-PTX-PEG-FA nanocomposites were detected by dialysis, and were divided into laser irradiation group and control group. The specific steps were as follows:

(1) Take 50mg of PLGA-PB-PTX-PEG-FA nanocomposite precipitate and dissolve it in 0.5mL deionized water, put 0.2mL in the laser irradiation group into a 24-well plate, expose to 808nm, output power 0.647W/cm ² After 10 min of laser irradiation, 1 mL of deionized water was added. For the control group, 0.2 mL of the above-mentioned nanocomposite solution was added to 1 mL of deionized water.

(2) Put the two groups in dialysis bags (molecular weight cut-off: 8000Da), and then completely immerse them in 150mL slow-release medium (0.01% Tween-80, 0.02% sodium azide, 30% ethanol) in a blue bottle.

(3) Place the blue-necked bottle in a constant temperature shaker for continuous shaking (37°C, 120rpm);

(4) 2h, 4h, 6h, 8h, 12h, 1d, 2d, 3d, and 4d after the above steps are completed, take out 1mL of dialysate, and then add 1mL of fresh sustained-release medium;

(5) Store the slow-release sample in a -20°C refrigerator, and use HPLC to detect the paclitaxel content in the sample after the sampling is completed;

(6) Calculate the cumulative release percentage (%) of paclitaxel in the PLGA-PB-PTX-PEG-FA nanocomposite at different time points, and draw the drug release curve over time.

The test result is:

The encapsulation efficiency of paclitaxel in the PLGA-PB-PTX-PEG-FA nanocomposite was 77.82%, and the drug loading was 7.22%.

As shown in Figure 10, the release time curve of paclitaxel in the PLGA-PB-PTX-PEG-FA nanocomposite within 4 days in vitro can be seen, the half-life of the drug in the laser irradiation group and the control group were 12h and 48h, respectively. Irradiation accelerated drug release. In the laser irradiation group, paclitaxel in the nanocomposite was released suddenly on the first day, and the release rate slowed down after 1 day. In contrast, the drugs in the control group were released slowly, showing a gradual upward trend. On the 4th day, the cumulative drug release in the laser irradiation group and the control group was 79.26% and 65.73%, respectively.

3. In vitro targeting and cytotoxicity experiments of Prussian blue-loaded targeting nanocomplexes

1. Cell targeting experiments

Human breast cancer cell line MDA-MB-231. The cells were routinely cultured in a constant temperature incubator at 37°C with 5% CO ₂ , and the medium formula was DMEM medium containing 10% fetal bovine serum, 0.1 mg/mL streptomycin, and 100 U/mL penicillin. The medium was changed routinely, and the cells were digested with 0.25% trypsin every other day for subculture.

The cells in the logarithmic growth phase were seeded in laser confocal culture dishes at a density of 1×10 ⁴ /dish. Incubate for 24 hours until the cells adhere to the wall and show growth.

The experiments were divided into 3 groups:

Targeting group (PLGA-PB-PTX-PEG-FA), non-targeting group (PLGA-PB-PTX-PEG), receptor blocking group;

3 petri dishes for each group. An equal amount of DiI-labeled corresponding reagent was added to each group, and an appropriate amount of folic acid antibody was added in advance to the receptor blocking group, and after co-incubating for 1 h, the targeting nanocomplex was added. After mixing, put it in a _CO2 incubator and incubate for 20 minutes, then take out the culture dish, wash it repeatedly with PBS buffer, wash away the free nanocomposites, then add 1.5 mL of 4% paraformaldehyde to each culture dish, and incubate for 10 minutes. Discard the paraformaldehyde and wash with PBS three times. Add DIO40uL/dish and incubate for 10 minutes. Discard the DIO and wash with PBS three times. Add DAPI 40uL/dish and incubate for 10 minutes. Discard the DIO and wash with PBS three times. Drop a little PBS to keep the dish dry, and observe the combination of nanocomposites and cells in each group under CLSM.

The test results are as follows: observed by the laser confocal microscope, the nanocomplex labeled with the red fluorescent probe DiI of the cell membrane exhibits red fluorescence, the green fluorescent probe DiR of the cell membrane can label the breast cancer cell membrane with green fluorescence, and DAPI can label the breast cancer cell nucleus Fluorescent blue. As shown in Figure 11, it can be observed that in the MDA-MB-231+ targeting group, more red fluorescent (b) nanocomplexes are tightly bound around the MDA-MB-231 cell membrane, indicating that the targeted nanocomposites The drug has the unique ability to actively target breast cancer cells. However, almost no red fluorescent nanocomplexes were observed around the cells of the MDA-MB-231+ non-targeting group and the receptor blocking group.

2. Cytotoxicity experiment

The MDA-MB-231 cells in the logarithmic growth phase were planted in a 96-well plate with a cell density of 8×10 ³ per well, and cultured in a 37°C, 5% CO ₂ constant temperature incubator for 24 hours.

The experimental group is the group with cells and adding reagents, the negative control group is the group with cells but no reagents, and the blank control group is the group without cells and no reagents;

In each group, 6 parallel wells were set up. Grouped as follows: PLGA-PB-PTX-PEG-FA group, PLGA-PB-PEG-FA group, free PTX group, PLGA-PB-PTX-PEG-FA+NIR group, PLGA-PB-PEG-FA+NIR group , NIR group. Choose to add various reagents (10 mg/mL, 100 μL/well, paclitaxel concentration is about 0.7 mg/mL) of the above mixed medium to each well, and incubate for 2 h without adding any reagents in the NIR group. Finally, take out the 96-well plate, discard the medium in the well, wash with PBS and add 50 μL/well of fresh medium. Among them, PLGA-PB-PTX-PEG-FA+NIR group, PLGA-PB-PEG-FA+NIR group and NIR group were irradiated with 808nm laser for 45s with an output power of 0.693W/cm ² per well. Finally, each well was supplemented with enough fresh medium and placed in an incubator for 24 hours of incubation. After 24 hours, 10 μL of CCK8 was added to each well, and after incubation for 1 hour, the absorbance value (OD value) of each well was detected at 450 nm with a microplate reader. Calculate the cell viability rate of the experimental group with the following formula:

Cell viability rate (%)=(OD value of experimental group-blank control group)/(average OD value of control group-OD value of blank control group)×100%.

The test result is:

The cell viability (%) of each treatment group measured by CCK8 method is shown in Table 1-2-1. Table 1-2-1 shows the effect of each group on the activity of paclitaxel after incubation with MDA-MB-231 cells under the same concentration of paclitaxel (C _[PTX] ≈0.7mg/mL). The results showed that the targeted nanocomposite combined with laser irradiation group (PLGA-PB-PTX-PEG-FA+NIR) had the strongest toxic effect on cells, which was 22.57±4.47%, compared with other groups, P<0.05, The difference is statistically significant. Compared with the paclitaxel group alone (47.52±3.47%), the cell activity of the targeted nanoparticle group combined with the laser group (PLGA-PB-PEG-FA+NIR) was low (32.00±3.73%), and the targeted nanocomposite The cell viability of the object group (PLGA-PB-PTX-PEG-FA) was higher (60.01±3.85%). While the cell viability of PLGA-PB-PEG-FA group and NIR group was higher than 90%, which indicated that the toxicity of PB NPs and polymer itself was very low, almost negligible, and laser irradiation alone had no effect on cell viability, It shows that the decrease in cell activity caused by the combination of the reagent group and the laser irradiation group is the decrease in cell activity caused by the photothermal effect of the reagent after laser irradiation.

Table 1-2-1 Effects of different treatment groups on the activity of MDA-MB-231 cells

In summary: PLGA-PB-PTX-PEG-FA targeting nanocomplex has good ability to target breast cancer MDA-MB-231 cells, which provides an experimental basis for subsequent in vivo targeting experiments and further targeted tumor imaging and therapy. Basic and theoretical basis; PLGA-PB-PTX-PEG-FA targeted drug-loaded nanocomposites assisted by laser irradiation can combine chemotherapy and photothermal effects to maximize the toxic effect and minimize the cell survival rate. The formed nanocomposite has good biological safety, has a chemotherapeutic effect, can kill tumor cells, can be combined with photothermal effect, combined with targeting effect, can target the tumor site, reduce the toxic and side effects of systemic chemotherapy, and kill tumor cells to the greatest extent , which laid a solid experimental foundation for the follow-up in vivo comprehensive treatment.

4. In vivo targeting experiment of targeting nanocomposites loaded with Prussian blue

Select MDA-MB-231 cells in logarithmic growth phase, digest with 0.25% trypsin, centrifuge at 1000r/min for 5min, dilute to 1×10 ⁷ /mL with sterile PBS after centrifugation, count with Plate count. Locally disinfect the back skin of nude mice with iodophor, then gently lift the buttocks and back skin of nude mice by an assistant, and inject 0.15 mL subcutaneously with a 1 mL syringe to form a skin mound. After the inoculation, put them back into the cage and observe them every 1-2 days.

Ten MDA-MB-231 breast cancer xenograft tumor model nude mice were selected for small animal in vivo fluorescence imaging, and were randomly divided into two groups: targeting nanocomposite (DIR/PLGA-PB-PTX-PEG-FA) group and non-targeting To the nanocomposite (DiR/PLGA-PB-PTX-PEG) group, 5 rats in each group. Anesthetized by intraperitoneal injection of 1% sodium pentobarbital before imaging. After anesthesia, the in vivo fluorescence imaging of small animals was performed before injection, and then the targeted nanocomplex and non-targeted nanocomposite were injected slowly through the tail vein. The dosage for each injection was 0.2 mL (20 mg/mL). At 2h, 4h, 6h and 24h, images were collected using a small animal in vivo fluorescence imaging system. Background fluorescence is filtered out for better observation. The local fluorescence intensity of tumors in the two groups was compared and observed, and the images were quantitatively analyzed by Living Image software system. The in vivo targeting efficiency of the two groups of tumor regions was represented by total radiation efficiency×10 ⁷ (total radiant efficiency×10 ⁷ ). After 24 hours of imaging, the nude mice were killed, and the tumor, heart, liver, spleen, lung, kidney, and brain were dissected for in vitro fluorescence imaging, and the in vitro fluorescence intensity of each part was calculated.

The test result is:

As shown in Figure 12, the in vivo fluorescence results of small animals showed that there was no fluorescence in the two groups before injection, but after the tail vein injection of the targeted nanocomposite, the targeted group could see strong red fluorescence in the tumor, spleen, brain, and spine (circled), marking the aggregation of DiR-labeled targeting nanocomplexes, and the strongest fluorescence at 1 h after injection. As time went on, the fluorescence intensity of the tumor, spine, and brain gradually decreased, while the fluorescence intensity of the liver area gradually increased. At 24 hours after the injection of the targeted nanocomplex, a small amount of fluorescence was still visible in the tumor area, spleen and liver area. In contrast, after tail vein injection of non-targeted nanocomplexes, there was no fluorescence in the tumors of the non-targeted group, but fluorescence was seen in the liver area and disappeared after 24 hours. Living Image software systematically analyzed the fluorescence intensity of the tumor area in the targeting group and the non-targeting group, as shown in Table 2-1-1. The fluorescence intensity of the tumor area in the targeting group was significantly higher than that in the non-targeting group, P<0.05, and the difference was statistically significant. Moreover, the fluorescence in the tumor area of the targeting group was the strongest 1 h after the tail vein injection of the targeting nanocomplex, and then gradually decreased, but the fluorescence was still visible in the tumor area at 24 h, which was significantly higher than the fluorescence intensity of the tumor area before injection.

As shown in Figure 13, in vivo fluorescence showed that the liver, spleen, and lungs of the two groups showed fluorescence, but only the tumors in the targeted group showed fluorescence, and the tumors in the non-targeted group showed no fluorescence aggregation (a). The fluorescence intensity of each part in vitro is shown in Table 2-1-2. The fluorescence intensity of isolated tumor and spleen tissue in the targeting group was significantly higher than that in the non-targeting group, P<0.05, and the difference was statistically significant.

Table 2-1-1 Fluorescence intensity of in vivo tumor sites in the targeting group and non-targeting group (×10 ⁷ )

point in time targeting group non-targeted group pre 2.22±0.02 2.30±0.06 1h 395.95±9.26 23.43±12.01* 2 hours 264.00±41.15 23.86±11.53* 4 hours 209.30±48.51 24.23±11.14* 6 hours 174.60±37.19 24.65±11.24* 24 hours 147.30±28.28 26.43±11.17*

Note: *P<0.05 compared with the target group.

Table 2-1-2 Ex vivo fluorescence intensity of each tissue in the targeting group and non-targeting group (×10 ⁷ )

Note: *P<0.05 compared with the target group.

In summary: The method of injecting DiR-labeled targeting and non-targeting nanocomplexes through the tail vein was injected into nude mice through the tail vein, and small animals were used before and 1h, 2h, 4h, 6h and 24h after injection. An in vivo fluorescence imaging system was used to observe the biological distribution of nanocomplexes with fluorescent probes in tumors of nude mice and in vivo. The results showed that after tail vein injection of nanocomplexes for 1 h, the distribution of targeting nanocomplexes in tumor tissue was significantly higher than that of non-targeting nanocomplex group, which indicated that PLGA-PB-PTX-PEG-FA targeting nanocomplexes were well Properties of active targeting of breast cancer cells in vivo. After 24 hours of tail vein injection of the nanocomposite, fluorescence was still visible in the tumor area of the targeting group, indicating that the PLGA-PB-PTX-PEG-FA targeting nanocomplex has active targeting and EPR (enhanced permeability) because of its nanoparticle size. and retention effect) effect caused by passive targeting. After the tail vein injection of PLGA-PB-PTX-PEG non-targeted nanoparticles, due to the lack of folic acid targeting group, most of the non-targeted nanoparticles will be metabolized by the liver before reaching the tumor area, so there is no fluorescence accumulation in the tumor area. Fluorescent imaging is mainly concentrated in the liver area. Quantitative fluorescence intensity indicators showed that the tumor fluorescence intensity in the targeting group was significantly higher than that in the non-targeting group, and the difference was statistically significant, which confirmed the good in vivo targeting ability of the PLGA-PB-PTX-PEG-FA targeting nanocomposite.

5. Photoacoustic/magnetic resonance multimodal imaging experiments enhanced by targeting nanocomposites loaded with Prussian blue

1. In vitro photoacoustic imaging

The PLGA-PB-PTX-PEG-FA nanocomposite and PLGA-PTX-PEG-FA were prepared into different concentrations of solutions (2.5mg/mL, 5mg/mL, 10mg/mL, 20mg/mL), and double distilled Water was used as a control group for photoacoustic imaging. Add each group of reagents to the 2.5% gel model, use Photoacoustic imaging system to collect photoacoustic images of nanoparticles without Prussian blue (PLGA-PTX-PEG-FA), nanocomposites with Prussian blue (PLGA-PB-PTX-PEG-FA) and double distilled water. The photoacoustic value of each sample was measured using the software that comes with the photoacoustic system.

The test results are: the results of in vitro photoacoustic imaging show that compared with the nanoparticles without PB NPs (PLGA-PTX-PEG-FA) and double distilled water without photoacoustic imaging, the PLGA-PB-PTX-PEG-FA nano The photoacoustic imaging of the composite is obvious, and with the increase of the concentration of the PLGA-PB-PTX-PEG-FA nanocomposite, the photoacoustic imaging is gradually enhanced, and the photoacoustic value is also gradually increasing, as shown in Table 2-2-1 .

Table 2-2-1 In vitro photoacoustic values of PLGA-PB-PTX-PEG-FA nanocomposites, PLGA-PTX-FA-PEG and double distilled water at various concentrations (mean ± standard deviation, n = 3)

2. In vitro magnetic resonance imaging

The PLGA-PB-PTX-PEG-FA nanocomposite and PLGA-PTX-PEG-FA were prepared into different concentrations of solutions (2.5mg/mL, 5mg/mL, 10mg/mL, 20mg/mL), and double distilled Water was used as a control group for magnetic resonance imaging. Put them into EP tubes with a diameter of 1cm, put them into a water container, and use a Philips Achieva 3.0T magnetic resonance scanner in the Netherlands to collect T1*WI images, using a head coil. The scanning parameters are: repetition time (TR) 494ms, echo Time (TE) 10ms, flip angle (Flip angle) 90°, slice thickness 1.0mm, field of view (FOV) 190mm. Each group of samples was repeated three times. The system software was used to measure the signal intensity (Signal intensity, SI _NPs ) of the region of interest (ROI) of each hole, and the signal intensity of water (SI _water ) was used as a comparison, and the signal intensity of each sample was calculated by the following formula (Percentageofsignal intensity enhancement, PSIE).

PSIE = (SI _NPs - SI _water )/SI _water x 100%.

The test result is:

In T1*WI imaging, compared with the nanocomposite without PB NPs (PLGA-PTX-PEG-FA) and double distilled water without T1 enhanced signal, the PLGA-PB-PTX-PEG-FA nanocomposite enhanced MRI T1 imaging is obvious. And with the increase of the concentration of PLGA-PB-PTX-PEG-FA nanocomposite, its signal intensity enhancement rate (PSIE) gradually increased, compared with the PLGA-PTX-PEG-FA group, P<0.05, the difference was statistically significant Meaning, as shown in Table 2-2-2. It shows that the nanocomposite prepared in this experiment has positive reinforcement effect, the higher the concentration of PLGA-PB-PTX-PEG-FA nanocomposite, the more obvious its positive reinforcement effect.

Table 2-2-2 The signal intensity enhancement rate of each concentration of PLGA-PB-PTX-PEG-FA nanocomposite, PLGA-PTX-FA-PEG and double distilled water (mean ± standard deviation, n = 3)

Note: *P<0.05 compared with PLGA-PB-PTX-PEG-FA.

3. Enhanced in vivo photoacoustic imaging

Fifteen nude mice with successfully constructed MDA-MB-231 breast cancer xenografts were selected for photoacoustic imaging in vivo, and were randomly divided into three groups: targeting nanocomposite (PLGA-PB-PTX-PEG-FA) group, non-targeting nanocomposite (PLGA-PB-PTX-PEG-FA) group, To nanocomposite (PLGA-PB-PTX-PEG) group and normal saline group (NS group), 5 rats in each group. Anesthetized by intraperitoneal injection of 1% sodium pentobarbital before imaging. After anesthesia, in vivo photoacoustic imaging was performed before the injection, and then the targeted nanocomposites, non-targeted nanocomposites and normal saline were injected slowly through the tail vein, each injection volume was 0.2 mL (20 mg/mL). Images were collected with a photoacoustic imager at 1h, 2h, 4h, 6h and 24h. Keep the same scanning section before and after enhancement, compare and observe the local photoacoustic signal intensity of the tumor in the three groups, select the tumor ROI, and use the software that comes with the instrument to perform quantitative analysis of the photoacoustic signal on the images before and after the reagent injection. The photoacoustic signal ratio (The ratio of PA value, 100%) of each sample was calculated using the following formula, and a graph of the photoacoustic signal ratio over time was drawn.

The ratio of PA value = PA _pose / PA _pre × 100%.

The test result is:

use Photoacoustic imaging system, real-time dynamic observation of tumor development in nude mice. By comparing various time points before and after injection of contrast agent, the results showed that in the targeted group, after injection of the targeted nanocomplex, the photoacoustic signal in the tumor area was most obvious at 1 h, and then gradually decreased, but there was still an obvious photoacoustic signal at 24 h. In the non-targeting group, the photoacoustic signal was not significantly enhanced 1h-6h after injection of the non-targeting nanocomposite, and a weak photoacoustic signal was seen 24h after injection. In the normal saline group, there was no significant change in the photoacoustic images before and after contrast. Comparing the photoacoustic signal ratios of the three treatment groups before and after injection of the reagents in each group, it can be seen that the photoacoustic signal ratios of the non-targeting group were slightly enhanced, but compared with the normal saline group, P>0.05, the difference was not statistically significant. The enhancement in the targeted group was very obvious, and the photoacoustic signal ratio of the targeted group was compared with the other two groups, P<0.05, and the difference was statistically significant, as shown in Table 2-2-3.

Table 2-2-3 Photoacoustic ratio of tumor tissue in each group (mean ± standard deviation, n = 5)

Note: Compared with saline group, ^# P<0.05; compared with target group, ^▲ P<0.05.

4. Enhanced in vivo magnetic resonance imaging

Fifteen nude mice with successfully constructed MDA-MB-231 breast cancer xenografts were selected for photoacoustic imaging in vivo, and were randomly divided into three groups: targeting nanocomposite (PLGA-PB-PTX-PEG-FA) group, non-targeting nanocomposite (PLGA-PB-PTX-PEG-FA) group, To nanocomposite (PLGA-PB-PTX-PEG) group and normal saline group (NS group), 5 rats in each group. After intraperitoneal injection of 1% pentobarbital sodium for anesthesia, the Philips 3.0T superconducting magnetic resonance instrument and small animal coils were used to perform plain scanning in the prone position before the injection, and then the targeted nanocomposites and non-targeted nanocomposites were injected slowly through the tail vein respectively. For the nanocomposite and normal saline, the injection volume was 0.2mL (20mg/mL) for each mouse, and enhanced scans were performed at 1h, 2h, 4h, 6h and 24h after successful injection. Automatic shimming was routinely performed before scanning, and T1WI sequence was applied. The scanning parameters were as follows: TR=3200ms, TE=80ms, slice thickness=2mm, FOV=60mm×60mm, NSA=10. Use the system software to select ROI, measure the signal intensity (Signal intensity, SI) SI _tumor and SI _muscle of the nude mouse tumor and thigh muscle region of interest in the same layer before and after enhancement at each time point, and calculate the relative signal intensity (SIr) and Signal intensity enhancement rate (Percentage of signal intensity enhancement, PSIE):

SIr＝SI _tumor /SI _muscle

PSIE=(SIr _post -SIr _pre )×100%/SIr _pre

When selecting ROI, try to avoid structures such as necrotic areas and cystic changes at the same site and at the same level. The size of the ROI should be consistent. The ROI at different positions should be measured 3 times, and the average value should be taken.

The test result is:

MRI imaging was performed on the MDA-MB-231 breast cancer xenograft tumor nude mouse model, and the plain scan tumor showed a relatively uniform slightly high-intensity shadow, with a clear boundary with the surrounding tissue. Before and after injection in the normal saline group, there was no significant change in the signal intensity of the tumor. After PLGA-PB-PTX-PEG in the non-targeting group and PLGA-PB-PTX-PEG-FA in the targeting group were injected through the tail vein, the signal intensity of the tumor tissue in the non-targeting group slightly changed at 24 hours with the prolongation of scanning time. However, the signal intensity enhancement rate in the tumor area was not significantly different from that in the normal saline group, P>0.05. In the targeting group, the signal intensity of the tumor tissue was most obviously enhanced at 1 hour, and then decreased, but the signal intensity of the tumor tissue was still significantly enhanced at 24 hours. And the signal intensity enhancement rate of the tumor area in the targeting group was compared with the other two groups, P<0.05, the difference was statistically significant, as shown in Table 2-2-4.

Table 2-2-4 Comparison of MRI signal intensity enhancement rate before and after tail vein injection of contrast medium in MDA-MB-231 transplanted tumor in nude mice

Note: Compared with saline group, ^# P<0.05; compared with target group, ^▲ P<0.05.

The PLGA-PB-PTX-PEG-FA nanocomposite prepared by the invention has good photoacoustic and MRI imaging capabilities in vitro. The present invention also compares the nanoparticle PLGA-PTX-PEG-FA without wrapping PB NPs, which cannot be imaged by photoacoustics and MRI, indicating that PB NPs is the main imaging material, and also shows that the PLGA-PB-PTX-PEG-FA prepared by the present invention Nanocomposites have successfully encapsulated PB NPs without affecting their photoacoustic and MRI imaging capabilities. As shown in the results, the signal intensity enhancement percentage of the 20mg/mL PLGA-PB-PTX-PEG-FA nanocomposite was 153.15±0.76%, which was much higher than the 22.11±0.17% of the PLGA-PTX-PEG-FA group, It was confirmed that the nanocomposite can be used as a contrast enhancement agent for MRI.

In summary, the experiment proves that the PLGA-PB-PTX-PEG-FA targeting nanocomplex prepared by the present invention can target breast cancer tumor cells by specifically binding to the folate receptor on the surface of MDA-MB-231 cells , to enhance photoacoustic and magnetic resonance imaging of breast cancer xenografts, and has the potential to become a photoacoustic/magnetic resonance multimodal imaging enhancer. The nanocomposite can not only realize tumor molecular imaging, but after being targeted to the tumor site, combined with laser, it can promote the release of paclitaxel carried in the target area, which is expected to realize targeted comprehensive treatment under real-time monitoring of imaging technology, and further realize early diagnosis of tumors Targeted therapy with visualization.

6. Comprehensive treatment of breast cancer in nude mice with Prussian blue targeting nanocomposites

1. Combined laser irradiation on tumor surface temperature changes

When the MDA-MB-231 transplanted tumors in nude mice grew to about 1.0 cm, 35 tumor-bearing mice were selected and randomly divided into 7 groups, 5 in each group. The experimental groups were: PBS control group (control), paclitaxel drug group (PTX), drug-free nanoparticle group (PLGA-PB-PEG-FA), targeting nanocomposite group (PLGA-PB-PTX-PEG- FA), drug-free nanoparticles+laser irradiation group (PLGA-PB-PEG-FA+NIR), targeted nanocomposite+laser irradiation group (PLGA-PB-PTX-PEG-FA+NIR) and simple Laser irradiation group (NIR). The concentration of paclitaxel injection alone was 1.4mg/mL (equivalent to the drug concentration in the 20mg/mL nanocomplex), and the concentration of other reagent groups was 20mg/mL, and each nude mouse was injected with 0.2mL of the corresponding reagent. The treatment plan is: each group will be given tail vein injection of corresponding reagents, and according to the results of the second part of imaging, the PLGA-PB-PEG-FA+NIR and PLGA-PB-PTX-PEG-FA+NIR groups will be injected 1 hour after the injection of reagents. Left and right, the tumor area was irradiated with 808nm laser with an output power of 0.647W/cm ² for 10min. In the laser irradiation group, only the above parameters were used to irradiate the tumor area for 10 minutes. The temperature change caused by the above treatment was detected by an infrared imager, and the temperature was recorded every 10 s. Plot a histogram of temperature versus time.

(1) Observe the living conditions of nude mice before treatment and on the 3rd, 6th, 9th, 12th, 15th, 18th, and 21st days after treatment, take pictures, measure the long and short diameters of tumors with a vernier caliper, and substitute the following formula for calculation Tumor volume (TV):

TV=(L×S ² )/2

Among them, L is the longest diameter of the tumor, and S is the shortest diameter of the tumor. The relative tumor volume (RTV) was calculated according to the tumor volume:

RTV = TV/TV ₀

Where TV is the tumor volume at each time point, and TV ₀ is the tumor volume before treatment. Relative tumor growth curves were plotted against the calculated relative tumor volume values. After 21 days of treatment, the nude mice were sacrificed by neck dislocation, and the tumor mass was removed and weighed. The tumor inhibition rate (Tumor inhibition rate, TIR) was calculated using the following formula.

TIR=(1-average tumor mass in treatment group/average tumor mass in control group)×100%

(2) After sacrificing nude mice, the necrotic border tissue of each group was dissected and fixed with 4% paraformaldehyde. After paraffin embedding and sectioning, some of them were used for HE staining, and some of them were detected by immunohistochemical PCNA method Proliferation and tumor cell apoptosis were detected by TUNEL method. The hearts, livers, spleens, lungs, and kidneys of nude mice in the PLGA-PB-PTX-PEG-FA+NIR group and the control group were dissected, fixed with 4% paraformaldehyde, embedded in paraffin, sliced, and used for HE staining. HE staining was observed with a low power lens (×100 times). In PCNA and TUNEL methods, brown-yellow or brown granules with coloring intensity higher than the background in the nucleus are positive signs. Use a high-power lens (×400 times) to observe the number of positive cells in each section, randomly select 5 fields of view for each section, and use the following formula to calculate:

PCNA: Proliferating index (Proliferating index, PI) = number of positive cells/total number of cells × 100%.

TUNEL: Apoptotic index (Apoptotic index, AI) = number of positive cells/total number of cells × 100%.

The test results are: as shown in FIG. 14 , 1 hour after tail vein injection of the PLGA-PB-PTX-PEG-FA targeting nanocomposite, the tumor area was irradiated with laser light. After 60s, the infrared camera can see that the tumor area changes from blue-purple to red in the center (A) to yellow (B), green (C), and blue (D), indicating that the temperature of the tumor area has increased, and then the center of the tumor area is red (A) The halo continues to expand, suggesting a gradual increase in temperature. In the laser irradiation group, the tumor area turned light blue after 60 seconds, suggesting that the temperature had risen a little, about 40°C. By 10 minutes, the tumor area had maintained a light blue halo (E). The main temperature changes are shown in Table 3-1. After injecting the targeted nanocomposites into the tail vein and irradiating with laser for 10 minutes, the temperature of the tumor surface rose rapidly, from 34.88±0.75°C to 52±3.05°C, while the surrounding tissues of the tumor The temperature detected by the infrared camera is only 39°C. In the laser irradiation group (Control), after 10 minutes of laser irradiation, the temperature only rose to 41±0.14°C, as shown in Table 3-1.

Table 3-1 The temperature change of the tumor site in the tumor-bearing nude mice of the targeted nanocomposite + laser irradiation group and the control group (°C, mean ± standard deviation, n = 5)

time(s) PLGA-PB-PTX-PEG-FA+NIR control 0 34.88±0.75 36.00±0.71 60 44.43±5.06 40.20±1.00 120 47.13±5.53 40.65±0.07 180 48.60±5.31 40.75±0.21 600 52.00±3.05 41.00±0.14

2. Tumor growth curve and quality tumor inhibition rate

The nude mice in the paclitaxel treatment group experienced transient fatigue, less activity, and poor food intake after administration. Nude mice in other groups had no obvious adverse reactions. It can be seen from the experimental conclusion that the tumors of the tumor-bearing nude mice in the PLGA-PB-PTX-PEG-FA+NIR group formed scabs 3 days after treatment, and shrunk significantly after 12 days of treatment. Significant growth. The other groups of tumors all had different degrees of growth. Among them, the NIR group, the PLGA-PB-PEG-FA group and the control group had a significant increase in tumor growth, and the tumor growth in the PLGA-PB-PTX-PEG-FA group was higher than that in the PTX group. Slowly, the relative tumor growth in the PLGA-PB-PEG-FA+NIR group was slower than the above two groups, and the most obvious tumor volume reduction was in the PLGA-PB-PTX-PEG-FA+NIR group.

The relative tumor growth curves of MDA-MB-231 breast cancer tumors in nude mice in each treatment group are shown in Figure 15, and the relative tumor volumes are shown in Table 3-2. The relative tumor volume of the nude mice in the group showed a gradual increase trend with time. The growth curves of the control group, PLGA-PB-PEG-FA and NIR groups were the steepest, followed by the PTX group, and the growth curves of the PLGA-PB-PTX-PEG-FA group and PLGA-PB-PEG-FA+NIR group increased more gently The relative tumor volume increased slowly. However, the relative tumor growth curve of the PLGA-PB-PTX-PEG-FA+NIR group showed a gradual downward trend, and the relative tumor volume was significantly reduced. Except for the NIR group, the PLGA-PB-PEG-FA group and the control group, P<0.05 was compared between the other treatment groups, and the difference was statistically significant. The tumor inhibition rate of each treatment group is shown in Table 3-3. It can be seen that the PLGA-PB-PTX-PEG-FA+NIR group showed an obvious tumor inhibition effect, and the quality tumor inhibition rate was 98.86%, and the tumor inhibition rate of other groups decreased sequentially . Compared with the PLGA-PB-PEG-FA group and NIR group and the PLGA-PB-PTX-PEG-FA+NIR group, the quality tumor inhibition rate is very low, and the tumor growth can hardly be inhibited, and the tumor growth is obvious.

Table 3-2 Relative tumor volume of nude mice in each treatment group (mean ± standard deviation)

Note: Compared with PLGA-PB-PTX-PEG-FA+NIR, *P<0.05; compared with the control group, #P<0.05.

Table 3-3 Tumor inhibition rate (100%) of each treatment group nude mice

group Tumor inhibition rate (100%) PLGA-PB-PTX-PEG-FA+NIR 98.86% PLGA-PB-PEG-FA+NIR 86.59% PLGA-PB-PTX-PEG-FA 74.39% PTX 65.85% PLGA-PB-PEG-FA 3.25% NIR 2.44% control 0.00%

3. Tumor cell proliferation

The results of immunohistochemical PCNA showed that the expression of different numbers of brownish yellow or brown PCNA positive cells could be seen in MDA-MB-231 tumor tissues of each group, and the proliferation index (PI) of each group was calculated. The results showed that as shown in Table 3-4, the number of positive cells in the PLGA-PB-PTX-PEG-FA+NIR group was the least, the proliferation index was significantly lower than that of other groups, and the cell proliferation index in the control group was the highest. Except for the NIR group, the PLGA-PB-PEG-FA group and the control group, P<0.05 was compared between the other treatment groups, and the difference was statistically significant.

4. Tumor cell apoptosis

The TUNEL test takes the appearance of brownish-yellow granules in the nucleus as a positive standard, and the morphological signs of apoptotic cells are brownish-yellow nuclei, condensed and concentrated chromatin. The staining results are shown in Table 3-4, different numbers of positive cells can be seen in the tumors of nude mice in each treatment group, and the number of apoptotic cells in the PLGA-PB-PTX-PEG-FA+NIR group is the most, and the apoptosis index is ( 87.06±3.01)%, except for NIR group, PLGA-PB-PEG-FA group and control group, P<0.05 in pairwise comparison of other treatment groups, the difference was statistically significant.

Table 3-4 Proliferation index (PI) and apoptosis index (AI) of tumor tissue in nude mice in each treatment group (mean ± standard deviation, 100%)

group PI (100%) AI (100%) PLGA-PB-PTX-PEG-FA+NIR 17.62±3.27 ^# 87.06±3.01 ^# PLGA-PB-PEG-FA+NIR 29.76±2.35* ^# 66.16±5.91* ^# PLGA-PB-PTX-PEG-FA 48.88±6.74* ^# 48.85±3.98* ^# PTX 60.05±4.44* ^# 37.81±4.45* ^# PLGA-PB-PEG-FA 81.40±2.33* 19.91±3.18* NIR 81.97±5.08* 18.36±5.21* control 84.79±3.08* 17.53±7.49*

Note: Compared with PLGA-PB-PTX-PEG-FA+NIR, *P<0.05; compared with the control group, ^# P<0.05.

5. HE staining

The results of HE staining of tumors in each treatment group were: PLGA-PB-PTX-PEG-FA+NIR group tumor HE staining showed a large amount of necrosis, and the red sheet-like unstructured area was seen under the microscope, and no tumor cells were seen in it. The degree of HE staining necrosis of tumors in the PLGA-PB-PEG-FA+NIR group, PLGA-PB-PTX-PEG-FA group, and PTX group was successively reduced, and the cell morphology in the tumor area of the control group, PLGA-PB-PEG-FA, and NIR group was basically the same. Normal, nuclear staining is normal. Compared with the heart, liver, spleen, lung, and kidney of the PLGA-PB-PTX-PEG-FA+NIR group and the control group, there was no obvious abnormality in HE staining.

In summary: In the experiment, 1 hour after the tail vein injection of the targeted nanocomplex, the tumor area was irradiated with laser for 10 minutes. The temperature of the tumor surface rose from 34.88±0.75°C to 52±3.05°C, which was enough to “burn” the tumor. The temperature of the tissue around the tumor was only 39°C, indicating that the targeted photothermal therapy was safe and did not damage the surrounding tissue. However, the temperature of the laser irradiation group (Control) only rose to 41±0.14°C after 10 minutes of laser irradiation, and this temperature could not kill tumor cells. The tumor continued to grow, and also ruled out the possibility that such a low-energy near-infrared laser generated enough heat by itself, further indicating that the PLGA-PB-PTX-PEG-FA targeting nanocomplex was really targeted to the tumor The area, after being irradiated by the laser, generates enough heat to kill tumor cells. The treatment measures of each experimental group were only implemented once, and the laser energy was only 0.647W/cm ² , indicating that the prepared targeting nanocomposites have good targeting effect and photothermal energy, and the drug loading capacity is high, which can accumulate in the tumor Area, combined with photothermal and chemotherapy effects, increases tumor treatment effect. The tumor pictures and relative tumor growth curves of nude mice during follow-up treatment also showed this point, and the PLGA-PB-PTX-PEG-FA+NIR group could significantly inhibit the growth of tumors. The PLGA-PB-PEG-FA+NIR group is second only to the PLGA-PB-PTX-PEG-FA+NIR group, and can also inhibit tumor growth, which is attributed to the photothermal effect of PLGA-PB-PEG-FA. Compared with PTX, PLGA-PB-PTX-PEG-FA can be more endocytosed by tumor cells because of its tumor-targeting effect and nano-drug delivery function, so that the local drug concentration in the tumor is higher than that of free PTX, thereby Play a greater anti-tumor effect than PTX. The above experimental results are similar to those reported in the literature related to other PB NPs. However, according to the experimental results, PLGA-PB-PEG-FA cannot inhibit tumor growth, which also shows the biological safety of the nanocomposite without chemotherapeutic drugs. The NIR group also could not inhibit the tumor growth, which ruled out the factor of the laser self-heating leading to the death of the tumor cells as above. Our results confirmed that the as-prepared PLGA-PB-PTX-PEG-FA targeting nanocomplex could serve as an ideal nanomaterial for comprehensive chemotherapy and photothermal tumor therapy in vivo.

PCNA and TUNEL methods were used to detect cell proliferation and apoptosis, and the experimental results also confirmed the above point of view. The proliferation index (PI) and apoptosis index (AI) of tumor cells in the PLGA-PB-PTX-PEG-FA+NIR group were the lowest. The tumor HE staining showed that the necrotic area was the largest in the PLGA-PB-PTX-PEG-FA+NIR group, and the pathological sections showed that the full field of view was almost full of amorphous necrotic cells. This is consistent with the results of PCNA and TUNEL, further confirming the ability of PLGA-PB-PTX-PEG-FA targeting nanocomplexes to effectively treat tumors comprehensively. Moreover, HE stained sections of heart, liver, spleen, lung, and kidney of nude mice in the PLGA-PB-PTX-PEG-FA+NIR group had no significant changes compared with the control group after 21 days of follow-up, indicating that the prepared targeted nanocomposite Good biosecurity. In summary, the prepared PLGA-PB-PTX-PEG-FA targeting nanocomposite has the ability to be a photoacoustic, magnetic resonance multimodal contrast agent and an image-mediated combination of chemotherapy and photothermal comprehensive tumor therapy targeting. Nano matter.

For those skilled in the art, under the premise of not departing from the technical solution of the present invention, some deformations and improvements can also be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the effect and effect of the implementation of the present invention. Patent utility.

Claims (9)

1. carry Prussian blue targeted nano compound, it is characterised in that have prussian blue nano particle including its internal package Inlayed on PEGylation folate-targeted lactic acid/co-glycolic acid shell, PEGylation folate-targeted lactic acid/co-glycolic acid shell It is embedded with taxol.

2. according to claim 1 carry Prussian blue targeted nano compound, it is characterised in that：Described PEGylation folic acid Targeting lactic acid/co-glycolic acid shell is to be connected with folic acid FA by polyethylene glycol PEG on polylactic-co-glycolic acid PLGA.

3. according to claim 2 carry Prussian blue targeted nano compound, it is characterised in that：Its profile is spherical in shape；Institute 236.6 ± the 55.04nm of particle diameter stated；Current potential is -24.44 ± 1.7mV.

4. according to claim 3 carry Prussian blue targeted nano compound, it is characterised in that：The bag of described taxol Envelope rate is 77.82%, and drugloading rate is 7.22%.

5. according to claim 4 carry Prussian blue targeted nano compound, it is characterised in that：In 500~900nm wavelength There is an absorption crest in scope.

6. according to claim 5 carry Prussian blue targeted nano compound, it is characterised in that：In 1756.23cm^-1、 2086cm^-1There is absworption peak, in 2927cm^-1Existing characteristics peak.

7. according to claim 6 carry Prussian blue targeted nano compound, it is characterised in that：What its laser was irradiated partly declines Phase is 12h.

8. the preparation method of the Prussian blue targeted nano compound of load according to claim 1~7 any one, its feature It is：The preparation process of preparation and nano-complex including prussian blue nano particle, wherein, the preparation step of nano-complex It is rapid as follows：

(1) 5mg taxols are weighed and 50mg PEGylations folate-targeted lactic acid/co-glycolic acid is put into beaker；

(2) 2mL dichloromethane is added into beaker, rim of a cup is sealed, oscillator vibration dissolves it, obtains solution A；

(3) by 200 μ L, 100mg/mL prussian blue nano particle and 100 μ L 4% polyvinyl alcohol, by mixed liquor by It is added dropwise into solution A, sound and vibration 2 minutes under ice bath, blue colostric fluid is presented；

(4) pour into 5mL4% poly-vinyl alcohol solutions, sound and vibration 2 minutes again under condition of ice bath obtain light blue double emulsion；

(5) mechanical agitation 2 hours to dichloromethane volatilizees；

(6) repeatedly washed with deionized water, collected after centrifugation, obtain microballoon；

(7) microsphere sample is put into 4 DEG C of refrigerators and preserved, Prussian blue targeted nano compound must be carried.

9. the preparation method according to claim 8 for carrying Prussian blue targeted nano compound, it is characterised in that：Described The preparation method of prussian blue nano particle is：

A, 0.5mmol 98mg citric acids are added to 20mL 1.0mmol/L FeCl₃In the aqueous solution, 60 DEG C of stirrings obtain solution a；

B, then, 1.0mM ferrocyanide aqueous solutions of potassium of the 20mL comprising 0.5mmol citric acids is added dropwise in a solution and presented For limpid blue solution, 60 DEG C of stirring 1min；

C, room temperature is cooled to, 30min is stirred at room temperature, dispersion liquid is obtained；

D, 40mL acetone added to dispersion liquid, 28000rpm centrifugation 1h form prussian blue nano particle precipitation；

E, purifying：Prussian blue nano particle precipitation 20mL distilled water Sonication dissolves, with 20mL acetone separations and from The heart；

F, repeatedly purge process；

G, again by after purification prussian blue nano particle precipitation be dissolved in distilled water, be placed in 14000MWCO bag filters, then will Bag filter is placed in distilled water the 24h that dialyses, and obtains prussian blue nano particle；

H, by obtained prussian blue nano particle be put into 4 DEG C of refrigerators preserve.