CN111346226A

CN111346226A - Self-oxygen-generating nanoparticles and application thereof in mediating tumor photodynamic therapy

Info

Publication number: CN111346226A
Application number: CN202010169461.7A
Authority: CN
Inventors: 蒋冠; 侯晓阳; 杨春生
Original assignee: Affiliated Hospital of Xuzhou Medical University
Current assignee: Affiliated Hospital of Xuzhou Medical University
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-06-30

Abstract

The invention provides a novel self-oxygen-generating targeting nanoparticle, a preparation method thereof and application of the self-oxygen-generating targeting nanoparticle in mediating tumor photodynamic therapy. In vivo and in vitro antitumor experiments prove that the nano drug delivery system has stable property and can catalyze H₂O₂Generates oxygen and can target tumor cells,oxygen is generated locally in the tumor, the hypoxia state of the tumor tissue is relieved, and further the enhanced PDT effect can be exerted under the irradiation of NIR laser.

Description

Self-oxygen-generating nanoparticles and application thereof in mediating tumor photodynamic therapy

Technical Field

The invention belongs to the technical field of nano biomedicine, and particularly relates to novel self-oxygen-generating targeting nanoparticles, a preparation method thereof and application of the nanoparticles in mediating tumor photodynamic therapy.

Background

Photodynamic therapy (PDT) is a promising treatment, and compared to conventional therapies, PDT has the advantages of less trauma, low toxicity, good selectivity, high applicability, repeatable treatment, and the ability to work in conjunction with other treatments, such as surgery, to improve therapeutic efficacy, and has been used to treat a variety of diseases, including malignant tumors. PDT relies on excitation light of a specific wavelength as a light source to irradiate a photosensitizer, producing singlet oxygen in the presence of molecular oxygen, resulting in apoptosis or necrosis of tumor cells. Photosensitizers are the core of PDT, and conventional photosensitizers such as porphyrin and derivatives thereof have some defects including long half-life, slow metabolism, poor water solubility, easy aggregation, and limited tissue penetration depth due to short excitation wavelength, which limits their clinical application [ Pollack M, et al. safety of treating anti-pd-1in tissues with tissue-related transformed antibodies-cta-4 and anti-pd1in metallic melasma. Ann Oncol,2018,29: 250-.

With the development of photodynamic research, more and more novel photosensitizers are discovered. Near infrared fluorescent dyes such as ICG are one of the potential photosensitizers, and they have been found to have not only excellent fluorescence imaging capabilities, but also Photothermal (PTT) and PDT effects. The excitation wavelength of near infrared fluorescent dyes is in the near infrared region, which is advantageous to reduce interference from absorption and fluorescence of surrounding tissues, and longer near infrared light has superior tissue penetration, and near infrared fluorescent dyes are capable of generating large amounts of singlet oxygen under irradiation of Near Infrared (NIR) laser light, which makes them potential photosensitizers. IR820 is a novel near infrared fluorescent dye that, like ICG, is also useful for fluorescence imaging and PTT/PDT ]. Li et al co-entrapped docetaxel and IR820 in a nanocolloid, IR820 was able to produce singlet oxygen in conjunction with chemotherapy to effectively inhibit tumor growth under NIR laser irradiation [ Li W, et al.Mild photothermal therapy/photodynamic therapy/chemotherapy of Breast cancer by lyp-1modified docetaxel/IR820 co-loaded microorganisms. biomaterials,2016,106:119-133 ].

Photosensitizers exert the PDT therapeutic effect by transferring energy generated by photochemical action to surrounding molecular oxygen, generating strongly reactive singlet oxygen or other reactive oxygen species, and thus the molecular oxygen level at the treatment site is also one of the factors that limit the PDT efficacy. Since the growth and metabolism of tumor cells are rapid and oxygen supply is often insufficient, resulting in the hypoxic state of the tumor microenvironment, and oxygen is continuously consumed during PDT treatment to aggravate hypoxia, the shortage of substrate will reduce the yield of singlet oxygen, which ultimately affects the efficacy of PDT [ Yang ZL, Tian W, Wang Q, et al. Oxygen-imaging mesoporous expressed prussian blue nanoplatform for high hly effective photodynamic therapy of tumors. adv Sci Weinh ], 2018,5(5):1700847 ]. To address this problem, researchers have attempted various measures to increase the oxygen concentration around the photosensitizer. For example, Cheng et al use the principle that perfluorocarbons can accommodate higher oxygen content than the tumor microenvironment to load photosensitizers into perfluorocarbon nanodroplets, where abundant oxygen can ensure that photosensitizers produce sufficient active oxygen to achieve enhanced PDT treatment [ Cheng Y, Cheng H, Jiang C, et al. Guo et al encapsulate hemoglobin and photosensitizer ICG in liposomes, which can effectively deliver oxygen to the tumor site, reduce hypoxic conditions, and increase the tumor-inhibiting effect of PDT [ Guo X, Qu J, Zhu C, et al. synthetic respiratory of oxygen and photosensitive for enhancement of tumor formation and purification of photodynamic therapy. drug delivery, 2018,25(1):585-599 ]. Although the above method enhances the efficacy of PDT to some extent, it has several problems to overcome, such as complicated drug synthesis procedures, oxygen-carrying stability, and oxygen release efficiency in tumors, etc.

Current research has found that higher concentrations of hydrogen peroxide (H) are present in tumor tissue compared to normal tissue₂O₂) (concentration range: 100. mu.M-1 mM), and H₂O₂Under certain conditions, it can break down into water and oxygen, and is therefore a potential reservoir of oxygen in tumor tissue. Manganese dioxide (MnO)₂) Can be reacted with H₂O₂Under the condition of acid pH, oxygen is generated by reaction, and the reaction equation is as follows: MnO₂+H₂O₂+2H⁺→Mn²⁺+2H₂O+O₂×) and based on this principle, Xing et al synthesized MnO₂Nano particle as carrier for presenting photosensitizer to local tumor, MnO₂And H in tumor tissue₂O₂After the reaction, the oxygen content is increased, thereby enhancing the tumor killing effect of PDT [ Ai X, Hu M, Wang Z, et al. enhanced cellular inhibition by tissue killing hypoxia status and reproducing tumor-associated macrophages of human light-responsive conversion nanocrystals. bioconjugate Chem,2018,29(4):928-938 ].]. However, the problems of tedious synthetic steps and potential biological safety due to excessive manganese ion uptake still exist. Therefore, it is of great significance to find safe and efficient oxygen generators.

Disclosure of Invention

In view of the defect that the yield of main toxic substance ROS in photodynamic therapy is low due to hypoxia in tumor microenvironment, the invention aims to provide a novel self-produced oxygen targeting nanoparticle using a polylactic acid-glycolic acid copolymer as a carrier, thereby providing a new idea and a new method for relieving tumor hypoxia and improving PDT (photodynamic therapy) to treat malignant melanoma.

To achieve the above technical object, the inventors considered that Catalase (CAT) is a highly efficient enzyme produced in vivo by catalyzing H₂O₂(CAT：H₂O₂1:1,000,000) produce oxygen, increase local oxygen concentration in tumors, regulate tumor hypoxia. The combined use of CAT and a photosensitizer can significantly improve photosensitizer-mediated PDT effects. However, free CAT inactivation is easily caused by proteases present in the body, and free drugs including photosensitizers are difficult to effectively accumulate in tumors due to lack of tumor selectivity, resulting in limited oxygen production and PDT efficacy.

Therefore, the inventor further encapsulates free drugs such as CAT and the like in the drug, thereby isolating the drugs from contacting with molecules in vivo, improving the stability of the drugs and not affecting the organisms of the drugsAnd (4) learning functions. Based on the accumulation and assumption of previous studies, the present inventors co-entrapped IR820 and CAT in PLGA nanoparticles. In order to improve the capacity of targeting tumor cells, Hyaluronic Acid (HA) capable of targeting CD44 molecules is modified on the surface of the nanoparticle so as to construct HA-PLGA-CAT-IR820 nanoparticles (HCINP). When the drug-loaded nanoparticles target to enter tumor tissues, CAT in the drug-loaded nanoparticles can catalyze H₂O₂Oxygen is generated and released, the hypoxia of tumor tissues is overcome, and the local oxygen concentration is improved; IR820 converts oxygen to singlet oxygen under NIR laser irradiation for PDT therapeutic effect.

Specifically, the purpose of the invention is realized by the following technical scheme: the self-oxygen-generating nanoparticle for mediating tumor photodynamic therapy is of a core-shell spherical structure, IR820 and CAT are distributed inside the nanoparticle, PLGA forms an inner nanoparticle shell, the outermost layer is coated with HA serving as an outer shell, the HA is hyaluronic acid, the PLGA is polylactic acid-glycolic acid copolymer, the IR820 is novel indocyanine green, and the CAT is catalase. It should be noted that PLGA in the present invention is preferably a PLGA 50:50 block copolymer, which is a polymer approved by the food and drug administration as a pharmaceutical adjuvant, and has the characteristics of excellent biocompatibility, biodegradability, and easy modification.

In addition, the invention also provides a preparation method of the self-oxygen-generating nanoparticles for mediating tumor photodynamic therapy, which comprises the following steps:

(1) adding CAT and IR820 into PVA water solution with concentration of 0.7-2.0%, dissolving completely and mixing;

(2) weighing PLGA and dissolving the PLGA in an organic solvent to obtain a PLGA solution;

(3) dripping the solution obtained in the step (1) into the PLGA solution obtained in the step (2), and performing ultrasonic emulsification to obtain primary emulsion;

(4) adding HA into 1-4% polyvinyl alcohol aqueous solution, stirring to fully dissolve and uniformly mix, then dropwise adding the primary emulsion, and performing ultrasonic emulsification to obtain multiple emulsion;

(5) and (3) stirring the multiple emulsion obtained in the step (4) at room temperature in a dark place for 8-16h at the stirring speed of 200-400r/min, removing the redundant organic solvent, collecting the obtained clear solution, centrifuging the solution at 4 ℃ and 14000rpm for 18-23min by using a high-speed refrigerated centrifuge, collecting the precipitate, washing the precipitate for 2-4 times by using ultrapure water or PBS (phosphate buffer solution), and removing free drugs and impurities to obtain the self-oxygen-producing nanoparticles.

Further preferably, in the preparation method of the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, the amounts of IR820, CAT, PLGA and HA are respectively:

still further preferably, in the method for preparing the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, the amounts of IR820, CAT, PLGA and HA are respectively:

in a most preferred embodiment of the present invention, the method for preparing self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, wherein the amounts of IR820, CAT, PLGA and HA are respectively:

further preferably, in the method for preparing the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, the organic solvent in step (2) is selected from one of the following: dichloromethane, trichloromethane, tetrahydrofuran, ethyl acetate and acetone.

Further preferably, in the preparation method of the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, the technical parameters of the ultrasonic emulsification in the step (3) are as follows: 400-500W ultrasonic treatment for 1.5-2.5 min.

Further preferably, in the preparation method of the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy as described above, the technical parameters of the ultrasonic emulsification in the step (4) are as follows: and (3) carrying out ultrasonic treatment at 380W for 4-6min by using 320-.

Compared with the prior art, the invention has the following advantages and remarkable progress:

(1) the CD44 targeted self-oxygen-generating nano drug delivery system HCINP is successfully constructed, is spherical and has good monodispersity, the hydrated particle size is 187.4nm, the PDI is 0.125, and the surface charge is-16.3 mV; the encapsulation rates of IR820 and CAT in HCINP nanoparticles are respectively 27.6% and 19.1%; the stability measurement result shows that the HCINP nanoparticles have good stability in both aqueous solution and culture medium environments.

(2) The HCINP nanoparticles have good biocompatibility; after HSF cells and MV3 cells are treated by the nanoparticles, the tumor cells can take in a large amount of nanoparticles, and the intake of the nanoparticles is increased along with the increase of the material concentration; the nanoparticles enter cells by endocytosis and are positioned on lysosomes.

(3) The in vitro oxygen production capacity measurement result shows that the HCINP nanoparticles can catalyze H₂O₂Generating oxygen; the experiment result of the tumor-bearing mouse model shows that the HCINP nanoparticles injected into the tumor tissue through tail vein can utilize H₂O₂Generates oxygen, regulates the local hypoxia state of the tumor, and regulates the expression of HIF-1 α.

(4) In vitro experimental results show that the HCINP nanoparticles can exert enhanced PDT effect under NIR laser irradiation, and the effect is stronger than that of IR820+ NIR, CINP + NIR and HINP + NIR treatment groups. HCINP nanoparticles entering tumor tissue can exert enhanced PDT effect under NIR laser irradiation, the effect is stronger than that of IR820+ NIR, CINP + NIR and HINP + NIR treatment groups, and melanoma growth and recurrence are effectively inhibited.

(5) The results of mouse weight monitoring, blood biochemical and pathological detection show that HCINP + NIR treatment does not show obvious systemic toxic and side effects on tumor-bearing nude mice.

Drawings

FIG. 1: schematic diagram of synthesis and structure of HCINP nanoparticles.

FIG. 2: particle size (a) and surface charge (B) of the HCINP nanoparticles.

FIG. 3: CINP (A) and HCINP (B) TEM photographs of the nanoparticles.

FIG. 4: and detecting the modification of the HA on the nanoparticles by using a laser scanning confocal microscope.

FIG. 5: ultraviolet/visible/near infrared absorption spectra of HCINP nanoparticles.

FIG. 6: standard curves and linear regression equations; wherein, (A) an IR820 standard; (B) and (3) protein standard products.

FIG. 7: stability of HCINP nanoparticles in aqueous solution (A) and in different cell culture solutions (B).

FIG. 8: study of HCINP-catalyzed H with optical microscope (A) and ultrasonic imager (B)₂O₂The ability to generate oxygen.

FIG. 9: qRT-PCR (A) and immunofluorescence (B) examined the expression of CD44 in melanoma cells (note: P <0.05 compared to HSF group).

FIG. 10: the CCK-8 method is used for determining the cytotoxicity of the HCINP nanoparticles on HSF cells (A) and MV3 cells (B).

FIG. 11: the uptake of f-HCINP (f-NP) nanoparticles by MV3 cells was examined by inverted fluorescence microscopy.

FIG. 12: the targeted uptake of f-HCINP (f-NP) nanoparticles by HSF cells and MV3 cells was examined by inverted fluorescence microscopy.

FIG. 13: laser scanning confocal microscopy was used to detect uptake and intracellular localization of f-HCINP (f-NP) nanoparticles.

FIG. 14: the oxygen production capacity of HCINP Nanoparticles (NP) in tumor cells was examined by inverted fluorescence microscopy (A) and the average fluorescence intensity analysis (B) (note: P <0.05 compared to Ctr).

FIG. 15: the Live-Dead method is used for researching the enhanced PDT effect mediated by HCINP nanoparticles in vitro; wherein, a: ctr group, without adding drug and without irradiating laser; b: NIR group, only laser light is irradiated without drug; c: HCINP group, HCINP only; d: IR820+ NIR group, IR820 added and NIR laser irradiated; e: CINP + NIR group, adding CINP and irradiating NIR laser; f: a group of HINP + NIR, adding HINP and irradiating NIR laser; g: HCINP + NIR group, HCINP was added and NIR laser was irradiated.

FIG. 16 immunohistochemistry method investigated the regulation of HIF-1 α expression after HCINP nanoparticles entered tumor tissues (200 ×).

FIG. 17: the tumor-bearing model is used for researching the treatment effect of the HCINP nanoparticles on melanoma. A: a tumor growth curve; b: tumor gravimetric analysis after treatment was completed.

FIG. 18 histopathological examination of HCINP nanoparticles on major mouse organs (200 ×).

Detailed Description

The present invention will be described in further detail with reference to the following embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. In addition, the specific technical operation steps or conditions not indicated in the examples are performed according to the technical or conditions described in the literature in the field or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: synthesis, identification and characterization of targeted self-oxygen-producing nanoparticle HCINP

1 Material

PLGA (50:50,10kD) Seiranxi Biotech Ltd

PVA (30-70 kD), Sigma-Aldrich USA

2 method

2.1 Synthesis of HA-PLGA-CAT-IR820(HCINP) nanoparticles

(1) 1mg of CAT and 1mg of IR820 powder were accurately weighed using a balance and added to 0.2mL of a 1% aqueous PVA solution to be sufficiently dissolved and mixed.

(2) 10mg of PLGA powder was weighed out and dissolved in 2ml of LPCM.

(3) The solution obtained in the first step was dropped into a PLGA solution, and ultrasonic emulsification (ice bath, 50% power, 2min) was performed using an ultrasonic cell disruptor (900W) to obtain a primary emulsion.

(4) 0.5mg of HA powder was weighed into 10mL of 2% PVA aqueous solution and placed on a magnetic stirrer to be dissolved and mixed well.

(5) And (3) dropwise adding the primary emulsion into the solution obtained in the step (4), and putting the obtained solution under an ultrasonic cell disruptor for ultrasonic emulsification (ice bath, 39% power, 5min) to obtain the multiple emulsion.

(6) Subsequently, the resulting homogeneous emulsion was placed on a magnetic stirrer and stirred overnight at room temperature in the dark, and the organic solvent was evaporated.

(7) The next day, the resulting clear solution was collected by centrifugation (14000rpm, 20min) and the pellet was washed 3 times with ultrapure water or PBS to remove free drug and impurities.

(8) And finally adding 500 mu L of ultrapure water or PBS for resuspension to obtain the product, namely the HCINP nanoparticles, and storing the product in a refrigerator at 4 ℃ for later use.

2.2 Synthesis of Nano particles of HCNP, CINP and HINP

HA-PLGA-CAT (HCNP), PLGA-CAT-IR820(CINP) and HA-PLGA-IR820(HINP) nanoparticles were further synthesized according to the nanoparticle synthesis method mediated in 2.1, and the same procedure was used except that IR820, HA or CAT were not added during the synthesis process.

2.3 identification and characterization of HCINP nanoparticles

(1) The aqueous solution of HCINP was diluted with an appropriate amount of ultrapure water, and the hydrated particle size, PDI and Zeta potential of the nanoparticles were measured using a DLS particle size analyzer.

(2) Respectively taking aqueous solutions of HCINP and CINP, diluting the aqueous solutions with a proper amount, dropwise adding the aqueous solutions onto a copper net sprayed with a carbon film, and sucking the solutions dry by using filter paper after 10 min; dropping a drop of uranyl acetate on the copper mesh for dyeing for 1min, carefully sucking the redundant dye along the edge of the copper mesh by using filter paper, observing and photographing the sample by using a TEM after airing, and performing particle size analysis on the sample by using an electron microscope photo and NanoMeasurer1.2 software.

(3) Dissolving a certain amount of the cell membrane red fluorescent probe DiI in DCM, replacing HA with fluorescently-labeled HA-FITC, and synthesizing the nanoparticles according to the method except the other steps. The nanoparticles were observed and photographed using a laser scanning confocal microscope (DiI: 549nm at the maximum excitation wavelength and 565nm at the maximum emission wavelength; FITC: 494nm at the maximum excitation wavelength and 525nm at the maximum emission wavelength).

(4) The solutions of IR820, HCNP and HCINP were diluted with ultrapure water and the UV absorption spectra of each group of drugs were measured with a UV spectrophotometer.

2.4 encapsulation efficiency determination

2.4.1 encapsulation efficiency determination of IR820

(1) Standards were prepared using free IR820, and each concentration was set to 0, 1.5625, 3.125, 6.25, 12.5, 25, 50 μ g/mL, and a standard curve was prepared by measuring absorbance of the standard at a wavelength of 690 nm.

(2) Diluting the supernatant collected by centrifugation in the process of preparing HCINP nanoparticles, and then measuring the diluted supernatant together with a standard substance.

(3) And substituting the absorbance value measured by the sample into a regression equation obtained by standard curve fitting to calculate the drug concentration, multiplying the drug concentration by the dilution factor to obtain the drug concentration in the sample supernatant, and finally multiplying the total volume of the supernatant to obtain the free drug mass.

(4) The encapsulation efficiency of IR820 is × 100% drug initial mass-free drug mass/drug initial mass.

2.4.2 CAT encapsulation efficiency determination

(1) Mixing the reagent A and the reagent B according to the volume ratio of 50:1 to prepare a proper amount of BCA working solution, and uniformly mixing for later use.

(2) Preparing protein standard substance and diluting to final concentration of 0.5mg/mL, adding the standard substance into 96-well plate according to 0,1, 2, 4, 8, 12, 16, 20 μ L, and adding standard substance diluent to make up to 20 μ L, wherein the concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL respectively.

(3) The supernatant collected by centrifugation in the process of preparing HCINP nanoparticles was diluted and 20. mu.L of the diluted supernatant was added to a 96-well plate.

(4) 200 μ LBCA medium was added to each well and incubated in an incubator at 37 ℃ for 35 min.

(5) And (3) measuring the absorbances of the standard substance and the sample at the wavelength of 562nm by using a multifunctional microplate reader, and drawing a standard curve.

(6) Substituting the absorbance value measured by the sample into a regression equation obtained by standard curve fitting to calculate the concentration, multiplying the concentration by the dilution factor to obtain the drug concentration in the sample supernatant, and finally multiplying the total volume of the supernatant to obtain the free drug mass.

(7) The CAT encapsulation efficiency was × 100% based on the initial drug mass-free drug mass/initial drug mass

2.5 stability assay of HCINP

(1) The HCINP nanoparticles were diluted with ultrapure water in a suitable amount, and the hydrated particle size and PDI of the nanoparticles were measured and recorded at 0, 3, 6, 9, 12, and 15d, respectively, using a DLS particle size analyzer.

(2) The nanoparticles were added to PBS, DMEM + 10% FBS, respectively, diluted to the same concentration, and the hydrated particle sizes of the nanoparticles at 0, 24, and 48h were determined and recorded using a DLS particle size analyzer.

2.6 detection of the oxygen-generating Capacity of HCINP

Preparing a HINP solution with an IR820 concentration of 8 μ g/mL and two HCINP solutions, respectively, and adding a certain amount of H to the HINP and one HCINP solution₂O₂The final concentration of the solution was set to 100. mu.M, and the three solutions were incubated in an incubator at 37 ℃ for 1 hour, and the generation of bubbles in the three solutions was observed and photographed.

3 results

3.1 Synthesis of HCINP drug-loaded nanoparticles

As shown in figure 1, HCINP drug-loaded nanoparticles are prepared by a water phase/oil phase/water phase (W/O/W) double-emulsion solvent evaporation method. Setting the ratio of medicine to fat as 1:10, selecting dichloromethane as an organic phase, and taking PVA as an emulsifier. The IR820 and CAT are distributed in the core of the nanoparticle, a shell structure formed by PLGA is arranged outside the core, and HA is modified on the outermost layer.

3.2 particle size and surface Charge of HCINP

The hydrated particle size, PDI and surface charge of the nanoparticles were determined using the DLS method. The experimental results show that the average hydrated particle size of the HCINP nanoparticles is 187.4nm, the PDI is 0.125, and the narrow particle size distribution and the small PDI value indicate that the particle size of the nanoparticles synthesized by the method is uniform (FIG. 2A). The Zeta potential results show a surface charge of-16.3 mV for HCINP (FIG. 2B).

3.3 TEM Observation of morphology features of nanoparticles

Morphology features of CINP and HCINP nanoparticles were observed and compared separately using TEM. As shown in fig. 3A, CINP nanoparticles exhibit a typical core-shell structure. Whereas HCINP exhibits a core-shell structure on TEM photographs due to the surface coating with HA. The HCINP nanoparticles are spherical, have good monodispersity, and have an average particle size of 132.6nm (figure 3B).

3.4 detection of modification of nanoparticles by HA by laser scanning confocal microscope

In order to further verify that HA is successfully modified on the nanoparticles, the nanoparticles and HA are respectively labeled by a red fluorescent probe DiI and a green fluorescent probe FITC, fluorescent double-labeled nanoparticles are synthesized by the same synthesis method, and a laser scanning confocal microscope is used for observing and taking pictures. The experimental results show that green HA and red nanoparticles have a good co-localization relationship, and the green HA and the red nanoparticles are overlapped to show orange yellow fluorescence on a Merge picture (figure 4). The results show that HA is successfully modified on the nanoparticles, which is consistent with the results of TEM.

3.5 absorption Spectroscopy characterization of HCINP

An ultraviolet/visible/near-infrared absorption spectrum shows that the HCNP nano-particles have no absorption peak in the wavelength range of 400-1000 nm, the maximum absorption peak of free IR820 is 690nm, HCINP has larger absorption at 740nm and 830nm respectively, indicating that the IR820 is successfully entrapped, and the absorption peak of CAT in an aqueous solution is about 230nm (figure 5).

3.6 determination of drug content of nanoparticles

The encapsulation efficiency of the IR820 was measured by uv spectrophotometry, from the absorption spectrum in fig. 5, it was found that the absorption peak of the IR820 in the aqueous solution was 690nm, and none of the other components in the nanosystem had an absorption peak at this wavelength position, and the detection wavelength of the IR820 was not interfered, so this wavelength was selected as the detection wavelength of the IR820, the free IR820 was used to prepare a standard, a standard curve was drawn, and a linear fit was performed to obtain a regression equation where a is 0.0136C +0.0504, R2 is 0.9996, and the IR820 had a good relationship in the concentration range of 0 to 50 μ g/mL (fig. 6A), the amount of free drug was calculated from the equation, and the encapsulation efficiency of the IR820 was 27.6% was calculated from the equation (encapsulation efficiency (initial drug mass-free drug mass)/initial drug mass × 100%).

The encapsulation efficiency of CAT is measured by using a BCA method, the detection wavelength is 562nm, and IR820 and other components have no absorption peak at the wavelength position and do not interfere with the detection result. Drawing a standard curve, and performing linear fitting to obtain a regression equation: a is 0.8386C +0.1111, R2 is 0.9993 (fig. 6B). The amount of free drug was calculated according to the equation and the encapsulation efficiency of CAT was 19.1% calculated according to the encapsulation efficiency equation above.

3.7 stability assay of HCINP

The stability of the HCINP nanoparticles in aqueous solution was tested by measuring the change of hydration particle size and PDI of the HCINP nanoparticles at 0, 3, 6, 9, 12 and 15 d. The DLS measurement results showed that the average particle size and PDI of HCINP remained stable within 15d, indicating that the nanoparticles could be uniformly dispersed in the aqueous solution without aggregation and decomposition, and thus the nanoparticles had good stability in the aqueous solution (fig. 7A).

We then added equal amounts of HCINP to PBS, DMEM and DMEM + 10% FBS to simulate the state of nanoparticles in different physiological environments to study the stability of nanoparticles under cell culture conditions. As shown in fig. 7B, the hydrated particle size (185nm) of the nanoparticles in DMEM + 10% FBS was similar to that in aqueous solution and did not change significantly with time. The nanoparticles in PBS (194.4nm) and DMEM (195.7nm) had a slight increase in size, but were still less than 200nm and did not change over time. These results indicate that our synthesized HCINP nanoparticles can maintain good stability in aqueous and physiological solutions.

3.8 HCINP nanoparticles catalyze H₂O₂Generating oxygen

H₂O₂The oxygen generated by decomposition can generate bubbles in the aqueous solution, so the oxygen generation effect of the HCINP nanoparticles can be judged by detecting the generation of the bubbles. Each group of solutions reacted for a certain period of time was dropped on a glass slide, and it was observed by an optical microscope that HINP contained no CAT and thus was the same as H₂O₂No air bubbles are generated after the co-incubation; the HCINP group alone also did not bubble due to lack of catalytic substrate; and HCINP and H₂O₂After a period of co-incubation, a large number of bubbles were visible in the tube wall and solution, and the micrograph also showed the presence of a large number of bubbles of varying sizes, indicating the presence of oxygen (fig. 8A).

Ultrasonic imaging result display of HINP + H₂O₂No gas overload was found in the group and HCINP groupAcoustic signals, and HCINP + H₂O₂The panel had a strong echo signal between the walls of the tube indicating the presence of a large number of bubbles (FIG. 8B), consistent with the results of the optical microscope. These results prove that CAT is successfully entrapped into HCINP nanoparticles, has good enzymatic activity and can catalyze H₂O₂Oxygen is generated.

Example 2: research on in-vitro anti-tumor effect of targeting self-oxygen-generating nanoparticle HCINP

1 Material

Cell lines: human skin fibroblasts HSF were purchased from the institute of biotechnology, north beijing, and human skin melanoma cells MV3, M14, a375 were purchased from the shanghai cell bank of the chinese academy of sciences.

2 method

2.1 cell culture and cell count

2.1.1 preparation of reagents required for cell culture:

(1) PBS preparation: pouring the PBS powder into a beaker, and adding ultrapure water with the standard volume of 90% for dissolving; taking 5mL of ultrapure water to dissolve residual powder in the bag, pouring the powder into a beaker, and repeating the operation for three times; and (5) performing constant volume according to the standard volume.

(2) DMEM complete Medium DMEM basal medium was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (100 ×).

(3) Cell cryopreservation solution: the cell frozen stock solution needs to be prepared at present, DMSO is added into fetal calf serum according to the proportion of 9:1, and the fetal calf serum is placed in a refrigerator at 4 ℃ for standby.

2.1.2 cell recovery

(1) And taking out the cell freezing tube from the liquid nitrogen tank, quickly placing the cell freezing tube in a water bath kettle heated to 37 ℃, and placing the cell freezing tube on a clean bench after the cell freezing tube is melted.

(2) The vial was opened, the cell suspension transferred to a centrifuge tube using a pipette, 4mL complete medium was added, and centrifugation was performed at 1000rpm for 5 min.

(3) The supernatant was discarded, 5mL of fresh complete medium was added, and the cells were resuspended by gentle pipetting.

(4) The cell suspension was added to a petri dish, supplemented with medium, and then placed in a cell incubator for culture.

2.1.3 cell passages

(1) And taking the cells out of the incubator, placing the cells on a super clean bench, discarding the old culture medium, adding a PBS (phosphate buffer solution) solution to wash the cells, and sucking out the cells.

(2) After 1mL of trypsin solution was added, the cells were digested in an incubator, and immediately after the cells were partially rounded under a microscope, serum-containing medium was added to the petri dish to stop the digestion.

(3) The cells were gently pipetted down using a pipette, added to a centrifuge tube at 1000rpm, and centrifuged for 5 min.

(4) Discarding the supernatant, adding a proper amount of complete culture medium, gently blowing and beating the cells to resuspend the cells, respectively adding the cell suspension into a new culture dish, supplementing the culture medium, and putting the culture dish into a cell culture box for culture.

2.1.4 cell count

(1) The cells were digested in 2.1.3 steps, centrifuged and resuspended to a single cell suspension.

(2) The cell suspension to be detected is blown uniformly, then a small amount of liquid is sucked and slowly dripped along the edge of the cover glass, the suspension is fully filled under the cover glass, and bubbles are avoided.

(3) The number of cells in 4 large squares was counted.

(4) According to the formula, the cell number/mL is (the sum of the cell numbers of 4 big squares/4) × 10⁴。

2.1.5 cell cryopreservation

(1) Cells were changed one day before they were cryopreserved.

(2) Cells were digested and centrifuged as described in 2.1.3.

(3) The supernatant was discarded, and the pre-cooled cell culture medium was added, and the cells were gently pipetted to disperse the cells uniformly.

(4) The cell suspension was dispensed into 1mL aliquots of cryopreservation tubes.

(5) And (3) performing gradient cooling on the freezing tube, wherein the specific steps are that a refrigerator with the temperature of 4 ℃ is used for 10min, a refrigerator with the temperature of-20 ℃ is used for 30min, the refrigerator with the temperature of-80 ℃ is used for overnight, and the freezing tube is transferred into a liquid nitrogen tank for long-term storage the next day.

2.2 detection of CD44 expression levels in melanoma cells

2.2.1 qRT-PCR method for detecting CD44 expression level in melanoma cells

The relevant literature was consulted and the Primer design and specificity was tested using the Pubmed Primer-BLAST system, the Primer sequences being:

GAPDH upstream primer 5'-GGAGCGAGATCCCTCCAAAAT-3'

GAPDH downstream primer 5'-GGCTGTTGTCATACTTCTCATGG-3'

CD44 upstream primer 5'-GAAGAAAGCCAGTGCGTCTC-3'

CD44 downstream primer 5'-GTGCTCTGCTGAGGCTGTAA-3'

The above sequence was synthesized by Shanghai Biotechnology Ltd.

2.2.1.1 extraction of total RNA from cells:

(1) HSF, MV3, M14, a375 cell culture dishes in logarithmic growth phase were placed on ice, the medium was discarded and washed twice with PBS. Adding 1mLTRNzol total RNA extraction reagent to crack for 5min, and sucking liquid into a ribozyme-free EP tube after gently blowing with a gun head.

(2) 0.2mL of chloroform was added, the mixture was inverted and the mixture was left to stand for 5 min.

(3) Centrifuge at 12,000rpm at 4 ℃ for 15 min.

(4) Sucking 0.45mL of supernatant into a new EP tube, adding equal volume of isopropanol, standing at room temperature for 20min,

(5) centrifuge at 12,000rpm at 4 ℃ for 10min and discard the supernatant.

(6) Adding 1mL of 75% ethanol into an EP tube, washing the precipitate, standing for 1-2 min to make the precipitate fully contact, and fully dissolving the organic reagent.

(7) Centrifuging at 5,000rpm and 4 deg.C for 5min, pouring out liquid, placing the EP tube in the centrifuge again for instantaneous centrifugation, and discarding the residual liquid on the tube wall.

(8) And (3) placing the EP tube in an ultra-clean bench for drying for 3-5 min, adding 30-50 uL of clean-free water, and shaking to fully dissolve the precipitate.

(9) The absorbance of the sample at 260/280nm was measured using a ultramicrospectrophotometer and the RNA concentration and purity were calculated.

2.2.1.2 reverse transcription reaction:

(1) the RNA concentration determined in the above step was used for quantification, and 2. mu.g of total cellular RNA was collected and each experimental group was trimmed using Nuclear-free water.

(2) 2 μ L of AccuRTreaction Mix (4 ×) was added to the tube following the procedure of 5 × All-In-One RT Mastermix kit, Nuclean-free water was added to 8 μ L, and the tube was allowed to stand at room temperature for 5 min.

(3) To the tube was added 2. mu.L of AccuRT Reaction Stopper (5 ×).

(4) 5 × All-In-One RT MasterMix 4. mu.L, Nuclean-free water 6. mu.L were added sequentially on ice to make the total reaction system 20. mu.L.

(5) The reaction system was gently mixed and reacted on a PCR instrument under the following conditions: 10min at 25 ℃, 15min at 42 ℃ and 5min at 85 ℃, and taking out the PCR tube and storing in a refrigerator at-20 ℃.

2.2.1.3 qPCR reactions

(1) A qPCR reaction system is configured on ice according to the specification of an EvaGreen 2 × qPCRMastermix kit, 10 mu L of EvaGreen 2 × qPCR Mastermix is added into a tube, the upstream primer and the downstream primer are respectively 0.6 mu L and 2 mu L of cDNA, and the volume is fixed to 20 mu L by supplementing Nuclear-free water.

(2) The reaction program is executed on the real-time fluorescent quantitative PCR instrument: at 95 ℃ for 10min, and one cycle; 95 ℃ for 15s, 60 ℃ for 60s, for a total of 40 cycles.

(3) According to 2^－△△CtAnd calculating the expression condition of the target gene by using a formula.

2.2.2 immunofluorescence assay for CD44 expression in melanoma cells

(1) Plating HSF cells and MV3 cells in logarithmic growth phase, counting after trypsinization to 1 × 10⁵The culture medium is inoculated into a cell culture dish special for a confocal microscope, 1.5mL of the culture medium is inoculated into each hole, the culture medium is placed at 37 ℃ and 5 percent CO₂And culturing for 24h under saturated humidity condition.

(2) Fixing: the excess medium was aspirated off, washed three times with PBS at room temperature with shaking, the cells were covered with 4% paraformaldehyde, left at room temperature for 15min, and washed three times with PBS with shaking.

(3) Permeability: 0.5% Triton X-100 permeate was added to cover the cells, and the cells were left to stand at room temperature for 30min and washed three times with PBS.

(4) And (3) sealing: adding 0.5% BSA blocking solution to cover the surface of the specimen and blocking for 1h at 37 ℃.

(5) A first antibody: the CD44 monoclonal antibody was diluted 1:100 with BSA, coated on the specimen surface, incubated overnight at 4 ℃ and rewarmed for 1h at 37 ℃ the next day, and washed three times with PBS.

(6) Secondary antibody: fluorescent secondary antibodies were diluted 1:200 with BSA and the specimen surface was covered. Incubate 50min at 37 ℃ in the dark, and wash three times with PBS in the dark with shaking.

(7) Nuclear dyeing: adding DAPI solution to cover the surface of the specimen, standing at room temperature in the dark for 5min, and washing with PBS in the dark by oscillation for three times.

(8) Microscopic examination: and (4) observing and photographing by using a laser scanning confocal microscope. (AlexaFluor 488: the maximum excitation wavelength is 495nm and the maximum emission wavelength is 519 nm; after the DAPI is combined with the double-stranded DNA, the maximum excitation wavelength is 364nm and the maximum emission wavelength is 454 nm).

2.3CCK-8 method for determining cytotoxicity of HCINP nanoparticles on HSF cells and MV3 cells

(1) Taking HSF cells and MV3 cells in logarithmic growth phase, performing trypsinization and counting, and configuring into 3 × 10⁴And (3) inoculating the cells into a 96-well plate with 0.1mL of cell suspension per well, adding PBS (phosphate buffer solution) at the periphery of a medicine adding hole for sealing, and placing the medicine adding hole in an incubator for culturing so that the cells adhere to the wall.

(2) The old medium was aspirated, cell culture media containing different HCINP concentrations (1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL) were added, and control groups were set with 5 parallel wells per group and incubation was continued for 24 h.

(3) Add 10. mu.L of CCK-8 solution to each well and incubate at 37 ℃ for 2 h.

(4) And (5) measuring the absorbance of each hole at the wavelength of 450nm by using a microplate reader, and recording the experimental result.

2.4 detection of the uptake of drug-loaded nanoparticles by HSF cells and MV3 cells by an inverted fluorescence microscope

2.4.1 according to the first part of the method for synthesizing the nanoparticles, coumarin-6 is entrapped into the nanoparticles to prepare the nanoparticles with the fluorescent tracing function, and the nanoparticles are named as f-HCINP.

2.4.2 inverted fluorescence microscopy of HSF and MV3 cells for f-HCINP nanoparticle uptake

(1) The HSF cells and MV3 cells were digested and centrifuged as described above, and the cells were resuspended and counted in 2 × 10 medium⁵The cell density of each well is inoculated on a 6-well plate, and the plate is placed in an incubator to culture the cells to adhere to the wall.

(2) Adding culture medium containing f-HCINP nanoparticles with concentration of 4 μ g/mL, 8 μ g/mL and 16 μ g/mL respectively, setting control group, setting 3 multiple holes for each group, and placing into incubator for continuous culture for 6 h.

(3) The medium was discarded, washed three times with PBS, 4% paraformaldehyde was added to cover the cells, left at room temperature for 15min, and washed three times with PBS shaking.

(4) Adding DAPI solution to cover the cell surface, standing at room temperature in the dark for 5min, and washing with PBS in the dark for three times.

(5) The photographs were observed using an inverted fluorescence microscope.

2.5 laser scanning confocal microscopy of MV3 uptake and intracellular localization of f-HCINP nanoparticles by cells

(1) Collecting MV3 cells in logarithmic growth phase, digesting with pancreatin, counting, and configuring into 1 × 10⁵The cell suspension was inoculated into 1.5mL cell culture dishes for confocal microscope at 37 ℃ with 5% CO₂And culturing under saturated humidity condition.

(2) After the cells adhere to the wall, the old culture medium is discarded, the culture medium containing f-HCINP nanoparticles with the concentration of 8 mug/mL is added, a control group is arranged, each group is provided with 3 multiple holes, and the cells are put into an incubator to be continuously cultured for 6 hours.

(3) The medium was discarded, PBS was added to wash three times, 1mL of cell culture medium containing Lyso-Tracker Red was added, and the mixture was incubated in an incubator at 37 ℃ in the dark for 1 hour.

(4) The medium containing the dye was aspirated and gently washed three times with PBS.

(5) The staining solution was observed and photographed by a laser scanning confocal microscope (Lyso-Tracker Red staining solution: maximum excitation wavelength of 577nm, maximum emission wavelength of 590 nm; coumarin-6: maximum excitation wavelength of 466nm, maximum emission wavelength of 504 nm).

2.6 detection of the oxygen production Capacity of HCINP nanoparticles in tumor cells

(1) MV3 cells were digested as described above, centrifuged, resuspended and the cells counted at 2 × 10⁵The cell density of each well is inoculated on a 6-well plate, and the plate is placed at 37 ℃ and 5% CO₂And culturing under saturated humidity condition.

(2) Cells were added with [ Ru (dpp) ] at a concentration of 5. mu.M₃]Cl₂The medium of (4) was pretreated for 4 hours.

(3) Discarding the culture medium, adding culture medium containing HCINP nanoparticles with concentration of 4 μ g/mL and 8 μ g/mL respectively, setting control group with 3 multiple holes, and placing into incubator for further culture for 12 h.

(4) The medium was discarded and washed three times with PBS.

(5) Observation photographing was performed using an inverted fluorescence microscope, and Image J software was used for [ Ru (dpp)₃]Cl₂The fluorescence intensity of (2) was quantitatively analyzed.

2.7 in vitro study of HCINP nanoparticle-mediated enhanced PDT Effect

(1) Grouping experiments: a: ctr group, no medicine is added and NIR laser is not irradiated; b: NIR group, irradiating only NIR laser (3W/cm)²) (ii) a c: HCINP group, HCINP alone (8. mu.g/mL); d: IR820+ NIR set, IR820 (8. mu.g/mL) was added and NIR laser (3W/cm)²) (ii) a e: CINP + NIR group, CINP (8. mu.g/mL) was added and NIR laser (3W/cm)²) (ii) a f: HINP + NIR group, HINP (8. mu.g/mL) was added and NIR laser (3W/cm)²) (ii) a g: HCINP + NIR group, HCINP (8. mu.g/mL) was added while NIR laser (3W/cm)²)。

(2) MV3 cells were digested as described above, centrifuged, resuspended and the cells counted at 3 × 10⁴Cell density per well was seeded in 24-well plates and placed in cell culture incubator overnight for adherence.

(3) Discarding the old culture medium, adding culture medium containing corresponding drugs according to the grouping method, setting three multiple holes in each group, and placing the hole plate into an incubator to continue culturing for 12 h.

(4) The well plate was removed, the medium was discarded, and washed with PBS to remove the drug that did not enter the cells. Placing the pore plate under a laser for irradiation, and setting the irradiation power to be 3W/cm²The duration is 5 min.

(5) After treatment was complete, cells were stained by adding 2 μ M calcein (calcein-AM) and 4 μ M Propidium Iodide (PI) to each well, and the wells were placed in an incubator and incubated for 35 min.

(6) The photographs were taken by inverting the fluorescence microscope.

3 results

3.1 screening of high-expression melanoma cell line of CD44

We screened melanoma cells highly expressing CD44 by qRT-PCR for subsequent in vitro and in vivo experiments. The CD44 expression level of 3 commonly used melanoma cells MV3, M14, a375 was examined using human skin fibroblast HSF as a control group. The experimental result shows that compared with HSF cells, the expression level of CD44 in the three melanoma cells is increased, and the difference is statistically significant (P < 0.05); among them, CD44 was expressed in MV3 cells at the highest level (about 4.2-fold) (fig. 9A).

Immunofluorescence experiments prove that the expression level of CD44 in MV3 is obviously up-regulated compared with HSF cells, and is consistent with qRT-PCR results, and CD44 is mainly distributed on cell membranes (FIG. 9B). The characteristics are important theoretical bases for serving as drug targets, so that MV3 cells are selected as research objects in subsequent in vivo and in vitro experiments.

3.2 cytotoxicity assay of HCINP nanoparticles

In order to verify the biocompatibility of the HCINP nanoparticles, a series of nanoparticle solutions with concentrations are prepared to respectively treat HSF cells and MV3 cells, the cells are continuously cultured for 24 hours, and a CCK-8 solution is added for detection. The experimental results show that when the concentration of HCINP is lower than 8 mug/mL, the HCINP nanoparticles have no obvious cytotoxicity to HSF cells and MV3 cells. There was a slight decrease in cell viability at higher concentrations, and when the solubility of HCINP reached 16 μ g/mL, the cell viability of HSF cells and MV3 cells was 90.5% (fig. 10A) and 91.3% (fig. 10B), respectively. The cell viability of the two cells decreased similarly, but was still over 90%. To avoid this interference, we chose 8 μ g/mL as the experimental concentration in subsequent experiments.

3.3 uptake of Nanocarrier systems by tumor cells

In order to conveniently research the uptake and subsequent intracellular localization of tumor cells to the nanoparticles, the nanoparticles are labeled by coumarin-6, and the f-HCINP nanoparticles are synthesized. After exposing the nanoparticles to tumor cells for a period of time, DAPI stained the nuclei of the cells and cellular fluorescence was detected using an inverted fluorescence microscope and flow cytometer. The experimental results show that the uptake of f-HCINP nanoparticles by MV3 cells is obvious, and the uptake of the f-HCINP nanoparticles is increased along with the increase of the concentration, and the concentration of the f-HCINP nanoparticles is dependent (FIG. 11).

3.4 study of Targeted uptake action of tumor cells on drug-loaded nanoparticles

After HSF cells and MV3 cells are respectively treated by f-HCINP nanoparticles with the same concentration for a certain time, the uptake of the nanoparticles by the two cells is detected by an inverted fluorescence microscope and a flow cytometer. The experimental result shows that the green fluorescence in the HSF cells is darker, which indicates that the nano-particles are less in intake; whereas MV3 cells exhibited bright green fluorescence, indicating that MV3 cells were able to take up nanoparticles in large quantities (fig. 12). Therefore, the drug-loaded nanoparticles synthesized by the method have good tumor targeted uptake effect.

3.5 laser scanning confocal microscopy detection of nanoparticle uptake and intracellular localization

The laser scanning confocal microscope photograph shows (fig. 13) that there is no green fluorescence in the cells (Ctr group) that are not processed by the nanoparticles, but bright green fluorescence is visible in the cells after the f-HCINP nanoparticles are added for a period of time, and the bright green fluorescence is mainly distributed in cytoplasm around the cell nucleus, which indicates that the MV3 cells can take in a large amount of nanoparticles, and the result is consistent with the results of the inverted fluorescence microscope and the flow cytometer experiments. In order to study the intracellular localization of the nanoparticles, ER-Tracker Red was used to stain lysosomes, which were Red under a microscope, and the green f-HCINP nanoparticles and Red lysosomes were well overlapped (yellow) in the Merge photograph, which indicated that the nanoparticles entered the lysosomes.

3.6 detection of the oxygen production Capacity of HCINP nanoparticles in tumor cells

[Ru(dpp)₃]Cl₂Is a dissolved oxygen detection indicator, the fluorescence of which can be detected by oxygenThe molecule quenches, resulting in a decrease in fluorescence intensity. MV3 cells were treated with HCINP nanoparticles for 12h, then observed by inverted fluorescence microscopy and photographed. The experimental result shows that the cells of the control group (Ctr group) show bright red fluorescence, while the fluorescence intensity of the cells treated by adding HCINP nanoparticles is obviously weakened, and the fluorescence intensity is reduced along with the increase of the nanoparticle concentration (P)<0.05) (fig. 14A, B). These results indicate the presence of H in the cells₂O₂Can permeate into HCINP nano-particles, and then is catalyzed by CAT in the nano-particles to generate oxygen.

3.7 HCINP nanoparticle-mediated enhanced PDT Effect

The drugs in each group are added into a culture medium to treat tumor cells for 12h, laser light with 808nm is irradiated, and then the treated cells are stained by Calcein-AM/PI, live cells are stained into green, and dead cells are stained into red. The experimental results show that the Ctr group, the NIR group and the HCINP group have no obvious cell death and normal cell morphology, and show that the biocompatibility of the nanoparticles is good and is consistent with the CCK-8 experimental results. IR820 causes some cell death after NIR laser irradiation and most cells remain viable. The cell death numbers of the CINP + NIR group and the HINP + NIR group were further increased, but some cells remained and the morphology of the remaining cells was changed. The HCINP + NIR group showed almost complete cell death in the irradiated area with strong tumor killing effect (FIG. 15).

Example 3: research on antitumor effect of targeting self-oxygen-producing nanoparticle HCINP in tumor-bearing mice

1 Material

BALB/c-nu female nude mice, 6-8 weeks old, with a weight of about 19-21 g, were purchased from the animal center of Xuzhou medical university. Nude mice were housed in the SPF grade barrier system of xu zhou medical university. The feed and drinking water are sterilized in animal room and then fed to animals for free diet.

2 method

2.1 establishment of tumor-bearing mouse model

Collecting MV3 cells in logarithmic phase after digestion and centrifugation, washing with PBS 3 times, and making into 4 × 10⁷In the mixture/mL, 100. mu.L of the mixture was inoculated into the right hind limb of BALB/c nude mice. Raising in SPF environment for three weeks, and establishingTumor-bearing mouse model.

2.2 injecting the drug, taking the material, fixing, slicing and staining

When the tumor grows to 200-400 cm³The nude mice were then randomly divided into 3 groups of 3 mice each, control (Ctr), HINP and HCINP groups. Carrying out tail vein injection of the medicine: ctr group was injected with 100. mu.L of physiological saline; the HINP group and HCINP group were injected with the corresponding drugs at 1mg/kg, respectively. Mice were sacrificed 24h after drug injection, tumors were stripped, fixed with 10% neutral formaldehyde solution for 48h, paraffin sectioned, immunohistochemically stained, microscopically observed and photographed.

2.3 study of HCINP nanoparticle-mediated enhanced PDT Effect in the transplanted tumor model

(1) Grouping experiments: i: normal (no tumor), ii: control (ctr), III: NIR, IV: HCINP, V: IR820+ NIR, vi: CINP + NIR, vii: HINP + NIR, viii: HCINP + NIR

(2) When the tumor grows to 200-400 cm³The nude mice were then randomly divided into 7 groups (groups II-VIII), 5 mice per group.

(3) Carrying out tail vein injection of the medicine: the Control group and the NIR group were injected with 100. mu.L of physiological saline; IR820 groups were injected with 1mg/kg IR820 per injection; the other groups were injected with groups of nanoparticles containing 1mg/kg IR 820.

(4) The injection is performed 24h after the injection, the anesthesia is performed by intraperitoneal injection by chloral hydrate, the irradiation treatment is performed on the tumor part by a 808nm laser, and the irradiation speed is 3W/cm²，5min。

(5) Tumor growth was recorded, and mice were weighed and tumor volume was measured every two days for 14 consecutive days from the start of dosing. The volume is given by the following equation:

V(mm³)＝(d²×D)/2。

wherein D and D are the minor and major diameters of the tumor, respectively, in mm.

2.4 histopathological and blood Biochemical assays

2.4.1 histopathological examination

After 14 days of treatment, mice were sacrificed, and the major organs (heart, liver, spleen, lung, kidney) were dissected out and immersed in 10% neutral formaldehyde fixing solution for fixation for 48 h. Paraffin sections, HE staining, upright microscope observation and photography.

2.4.2 Biochemical detection of blood

Mice were bled from the eye before sacrifice and centrifuged at 4,000rpm for 6 min. The sample is sent to the clinical laboratory of affiliated hospital of Xuzhou medical university for biochemical blood detection, and the detection indexes comprise glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), Total Bilirubin (TBIL), urea nitrogen (BUN), Creatinine (CREA) and Uric Acid (UA).

3 results

3.1 HCINP nanoparticles improve tumor hypoxia

The expression level of HIF-1 α is regulated by the oxygen content of tumor tissue, the hypoxia condition of tumor microenvironment can be reflected by detecting the expression level of HIF-1 α in the tumor tissue, the immunohistochemical experiment result shows that HIF-1 α in the tumor tissue of Ctr group and HINP group is in high expression state, the average positive area of quantitative analysis is 1522 and 1503 respectively, no obvious difference exists between the two, the average positive area of HIF-1 α of HCINP group is 1092, compared with Ctr group and HINP group, the expression level is obviously reduced (figure 16), the result shows that HCINP nano-particle can play the role of self-oxygen production in the tumor tissue, and improve the hypoxia of tumor tissue.

3.2 enhanced PDT Effect mediated by drug-loaded nanoparticles

PDT efficacy was assessed by comparing tumor volume and tumor weight in different groups of mice. Tumor growth curves show that tumor volumes in the control and HCINP groups increase rapidly with time. The NIR group showed a slightly slower growth rate but still showed a fast growth trend. Tumor growth was inhibited in the IR820+ NIR group, indicating that free IR820 partially inhibited tumor growth under NIR irradiation, but rapidly due to limited PDT effect. The tumor growth rates were significantly slower in the CINP + NIR and HINP + NIR groups than in the IR820+ NIR groups. Tumors in mice in the HCINP + NIR group almost completely disappeared at day 14 (fig. 17A). After the treatment, the tumors of each group of mice were stripped and weighed, and the results showed that there was a difference between the tumors of each group, consistent with the tumor growth curve (fig. 17B).

3.3 systemic safety testing of HCINP nanoparticle therapy

To assess the biological safety of HCINP nanosystems treatment, we further studied the body weight changes of mice during treatment as well as the biochemical level of blood and histopathology of major organs (heart, liver, spleen, lung, kidney) after the end of treatment. The results of the experiment show that the body weight of the mice did not significantly decrease in all treatment groups during the treatment. No significant pathological changes were shown in the pathological sections of heart, liver, spleen, lung, kidney after 14 days of HCINP + NIR treatment compared to the Normal group (non-inoculated tumors) (fig. 18). In addition, blood biochemical examination results showed no significant differences in the values for aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), Total Bilirubin (TBIL), urea nitrogen (BUN), Creatinine (CREA), Uric Acid (UA) compared to the Normal group, indicating no significant systemic toxicity after treatment (table 1). These results indicate that HCINP nanoparticle-mediated PDT treatment has good biological safety and potential clinical application value.

TABLE 1 blood biochemistry results of Normal group and HCINP + NIR treatment group

EXAMPLE 1 and EXAMPLE 2 initial exploration has been conducted on the nature and function of HCINP nanoparticles, and example 3 further investigated using animal tumor-bearing models to investigate HCINP mediated antitumor effects in vivo and whether systemic toxicity is present after treatment.first we examined whether drug-loaded nanoparticles could improve tumor local hypoxia status HIF-1 is a nuclear protein regulated by oxygen levels ubiquitous in the body, consisting of HIF-1 α subunit and HIF-1 β subunit, involved in tumor cell proliferation, metastasis, immunity, drug resistance and tumor angiogenesis, thus HIF-1 is a potentially potent cancer treatment, and studies have found that inhibition of HIF-1 expression could inhibit tumor development and progression and enhance target sensitivity chemotherapy and PDT treatment, wherein HIF-1 α expression level is regulated by tumor tissue hypoxia by the mechanism that HIF-1 α is rapidly regulated by intracellular oxygen-dependent ubiquitin protein in normoxic stateThe result of HIF-1 α immunohistochemistry of tumor tissue shows that HIF-1 α of Ctr group and HINP group is in high expression state, and has no obvious difference between the two, and the expression of HIF-1 α of HCINP group is reduced, the result shows that HCINP nano-particles are injected into tumor-bearing mice through tail vein, HCINP can target into tumor tissue to catalyze local H of tumor₂O₂Generates and releases oxygen, increases the oxygen concentration in the tumor microenvironment, and improves the hypoxic state of tumor tissues.

The increased oxygen in the tumor tissue provides an abundant substrate for PDT treatment, producing more singlet oxygen, thereby increasing the anti-tumor effect of PDT. Animal experiment results show that the tumor volume in the control group and the HCINP group is increased rapidly, which indicates that HCINP alone has no influence on tumor growth. The NIR group showed a slight decrease in tumor growth rate, probably due to laser stimulation, but still showed a rapid growth trend overall. The tumor growth in the IR820+ NIR group was inhibited, indicating that free IR820 can exert photodynamic therapy effect under NIR laser irradiation to partially inhibit tumor growth, but due to lack of targeting of IR820, the concentration of drug finally entering tumor cells is limited, so PDT effect is poor, and then the tumor grows rapidly. CINP can deliver IR820 to tumor parts through passive targeting, and the existence of CAT can increase local oxygen concentration, so that the yield of singlet oxygen is improved, the tumor inhibition effect is stronger than that of free IR820, but the tumor cells cannot be completely killed due to the effect, so that the later-stage tumor has a growing trend. The HINP nanoparticles can enter tumor cells through active targeting and passive targeting, improve the concentration of drugs in tumors, have strong anti-tumor effect, but can not completely kill the tumor cells due to the lack of CAT effect, and show a trend similar to that of a CINP + NIR group in the later period. HCINP + NIR tumors disappeared almost completely on day 14 due to active and passive tumor targeting with drug accumulation locally in the tumor and efficient production of singlet oxygen due to self-oxygenation. Mouse weight monitoring, blood biochemistry and major histopathology analysis showed no apparent systemic toxicity following HCINP + NIR treatment. These lines of evidence suggest that the use of the HCINP drug delivery system is a safe and effective anti-tumor strategy.

Claims

1. The self-oxygen-producing nanoparticles for mediating tumor photodynamic therapy are characterized in that the self-oxygen-producing nanoparticles are of a core-shell spherical structure, IR820 and CAT are distributed inside the nanoparticles, PLGA forms an inner nanoparticle shell, and HA is coated on the outermost layer to serve as an outer shell, wherein HA is hyaluronic acid, PLGA is polylactic acid-glycolic acid copolymer, IR820 is novel indocyanine green, and CAT is catalase.

2. The self-oxygen generating nanoparticle for mediating tumor photodynamic therapy according to claim 1, wherein the PLGA is a PLGA 50:50 block copolymer.

3. A method for preparing self-oxygen-generating nanoparticles for mediating tumor photodynamic therapy is characterized by comprising the following steps:

4. The method for preparing the self-oxygen-generating nanoparticles for mediating the photodynamic therapy of tumors according to claim 3, wherein the amounts of IR820, CAT, PLGA and HA are respectively as follows:

5. the method for preparing the self-oxygen-generating nanoparticles for mediating tumor photodynamic therapy according to claim 4, wherein the amounts of IR820, CAT, PLGA and HA are respectively as follows:

6. the method for preparing the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy according to claim 5, wherein the amounts of IR820, CAT, PLGA and HA are respectively as follows:

7. the method for preparing self-oxygen generating nanoparticles for mediating tumor photodynamic therapy according to claim 3, wherein the organic solvent in the step (2) is selected from one of the following: dichloromethane, trichloromethane, tetrahydrofuran, ethyl acetate and acetone.

8. The method for preparing the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy according to claim 3, wherein the technical parameters of the ultrasonic emulsification in the step (3) are as follows: 400-500W ultrasonic treatment for 1.5-2.5 min.

9. The method for preparing the self-oxygen generating nanoparticles for mediating tumor photodynamic therapy according to claim 3, wherein the technical parameters of the ultrasonic emulsification in the step (4) are as follows: and (3) carrying out ultrasonic treatment at 380W for 4-6min by using 320-.

10. The use of the self-oxygenating nanoparticle of claim 1in the preparation of a medicament for mediating photodynamic therapy of tumors.