CN114588279A

CN114588279A - Multifunctional nano molecular probe, preparation method thereof and application of multifunctional nano molecular probe as retinoblastoma diagnosis and treatment preparation

Info

Publication number: CN114588279A
Application number: CN202210344008.4A
Authority: CN
Inventors: 武明星; 刘丹宁; 郑政; 周希瑗
Original assignee: Second Affiliated Hospital of Chongqing Medical University
Current assignee: Second Affiliated Hospital of Chongqing Medical University
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-06-07
Anticipated expiration: 2042-03-31
Also published as: CN114588279B

Abstract

The invention relates to the technical field of diagnosis and treatment integrated nano preparations, in particular to a multifunctional nano molecular probe, a preparation method thereof and application thereof as a retinoblastoma diagnosis and treatment preparation. A multifunctional nano molecular probe comprises a lipid shell membrane and a core, wherein Fe is embedded in the lipid shell membrane₃O₄And folic acid is loaded outside the lipid shell membrane. The technical scheme can solve the technical problem that the diagnosis and treatment integrated nano molecular probe in the prior art is single in targeting mode. In the technical scheme, the specificity of molecular targeting and the high efficiency of magnetic targeting are combined to construct a molecular probe of dual-targeting retinoblastoma, so that the targeting efficiency is improved, and a new thought is provided for constructing a safe, high-efficiency and noninvasive visual diagnosis and treatment system integrating early diagnosis, treatment and curative effect evaluation of retinoblastoma.

Description

Multifunctional nano molecular probe, preparation method thereof and application of multifunctional nano molecular probe as retinoblastoma diagnosis and treatment preparation

Technical Field

The invention relates to the technical field of diagnosis and treatment integrated nano preparations, in particular to a multifunctional nano molecular probe, a preparation method thereof and application thereof as a retinoblastoma diagnosis and treatment preparation.

Background

Retinoblastoma (RB) is the most common eye malignant tumor of infants, has high malignancy degree and difficult early detection, and most of children patients have large RB growth when seeing a doctor due to 'white pupil syndrome', which indicates that the children may not be able to keep the eyeballs when entering a period seriously damaging eyesight and even life, and the treatment mainly comprises the removal of the eyeballs of the patients and the radiotherapy and chemotherapy as assistance. The eye ball extirpation operation is the most destructive treatment of ophthalmology, and the infant after the operation loses the visual function forever, causes the very big wound to infant's physical and mental health. Therefore, there is an urgent need to explore new directions, new targets and new measures for treating RB. In view of the characteristics of extremely high harm and troublesome treatment of RB to infants at present, early diagnosis and implementation of noninvasive treatment are always targets of clinical pursuit.

The common clinical RB examination means comprise conventional fundus examination, eye B-ultrasound, CT, MRI imaging and the like, the traditional imaging technology is difficult to achieve early qualitative positioning diagnosis of RB, and moreover, children with diseases are young and difficult to match, and the economic burden of families of the children with diseases is undoubtedly increased by multiple examinations, so that the early diagnosis of RB is once in a bottleneck. With the rapid development of molecular imaging and nanotechnology, molecular imaging and treatment means are integrated into a nano platform, and the design and construction of diagnosis and treatment integrated nano molecular probes with diagnosis capability and treatment efficacy have significant significance.

Chinese patent CN108014349A discloses a preparation method and application of a gene-loaded multifunctional contrast agent, and the contrast agent can be used in the practical operation of RB diagnosis and treatment. The multifunctional contrast agent prepared by the patent comprises a lipid shell membrane, wherein folic acid is modified on the lipid shell membrane, the surface of the lipid shell membrane is positively charged and carries genes, and liquid fluorocarbon and indocyanine green are wrapped inside the lipid shell membrane. In the technical scheme, the liquid fluorocarbon PFP has good photoinduced phase transition characteristic, the ICG has the characteristic of photoacoustic imaging, the PFP and the ICG are simultaneously loaded in a nano carrier, folic acid is modified on a lipid shell, therapeutic genes are carried on the surface of the carrier, and the contrast agent can meet the requirements of photoacoustic/ultrasonic bimodal and gene transfection treatment under the excitation action of laser. However, the contrast agent only has one targeting molecular folic acid, and is limited by factors such as the difference of receptor expression between individuals, receptor-antibody specific binding and the like, and the tumor targeting effect of folic acid is limited. Moreover, the contrast agent can only realize bimodal imaging, and cannot meet the requirements of photoacoustic imaging, ultrasonic imaging and nuclear magnetic resonance multimodal imaging of the contrast agent in clinical application.

Disclosure of Invention

The invention aims to provide a multifunctional nano molecular probe, which aims to solve the technical problem that the diagnosis and treatment integrated nano molecular probe in the prior art is single in targeting mode.

In order to achieve the purpose, the invention adopts the following technical scheme:

a multifunctional nano molecular probe comprises a lipid shell membrane, wherein Fe is loaded on the lipid shell membrane₃O₄And folic acid.

The scheme also provides a preparation method of the multifunctional nano molecular probe, which comprises the following steps of:

s1: adding DPPC, DSPE-PEG 2000-foil, DC-chol and oleic acid modified SPIONs into a solvent, uniformly mixing, and performing rotary evaporation to obtain a lipid membrane;

s2: adding water into the lipid membrane, and homogenizing and dispersing under an ice bath condition to obtain emulsion;

s3: and (3) dropwise adding liquid fluorocarbon and indocyanine green into the emulsion, and then emulsifying by sonic vibration to obtain FCNPIFEs.

The principle and the advantages of the scheme are as follows:

the scheme introduces SPION (namely Fe) at the same time₃O₄) And Folic Acid (FA)) The magnetic targeting agent and the molecular targeting agent are respectively used, so that the targeting efficiency of the nanoparticle probe is improved. The folic acid targeted nano molecular probe constructed by the inventor in the early stage can be specifically gathered around RB cells by combining with a folic acid receptor on the surface of the RB cells. However, since the specific receptor has saturation effect and the obstruction of tumor microenvironment, the targeting ability of the ligand-modified active targeting probe is limited, and how to increase the number of molecular probes in the tumor region to the maximum extent is a big problem. In the technical scheme, the inventor combines folate targeting and magnetic targeting, namely the specificity of molecular targeting and the high efficiency of magnetic targeting, and constructs a dual RB targeting molecular probe, so that the targeting efficiency is improved, and the magnetic targeting and the folate targeting realize synergistic targeting effect on retinoblastoma (see example 3, fig. 11 and table 1). The magnetic targeting is a novel active targeting mode based on the interaction between magnetic substances, and under the action of an external magnetic field, a magnetic field is applied to the surface of an eyeball, so that the enrichment and retention of magnetic particles on a retina can be realized. With Fe₃O₄Superparamagnetic iron oxide nanoparticles (SPIONs) as cores have high targeted aggregation capability in an applied magnetic field. In addition, Fe is added into the molecular probe₃O₄The defect that the ultrasonic and photoacoustic imaging cannot provide anatomical positions due to limited penetration capacity is overcome, so that the molecular probe has the function of MR imaging, and the MR imaging is important for clinically early diagnosis of RB.

Further, the components of the lipid shell membrane include DPPC, DSPE-PEG2000-Folate and DC-chol. The DC-chol is added into the lipid shell membrane, so that the surface of the nanoparticle can be positively charged. Folate targeting, magnetic targeting, and positively charged triproteins synergize, achieving superior tumor targeting and therapeutic effects (see example 3, fig. 11 and table 1, fig. 13, 14, and table 2).

Further, liquid fluorocarbon is wrapped in the lipid shell membrane.

According to the scheme, liquid fluorocarbon (PFH, perfluorohexane, boiling point 56 ℃) is selected as an inner core to prepare the phase-change type nano molecular probe, and the phase-change type nano molecular probe is an ideal ultrasonic contrast agent due to moderate boiling point and strong operability; meanwhile, the composite material has the characteristics of strong oxygen dissolving capacity and high stability, and can ensure the continuous supply of oxygen in the process of photodynamic therapy (PDT), maintain the effective activity of active oxygen and the like. The use of PFH also decreased HSP70 levels during treatment, decreased stress response due to elevated temperatures, and increased therapeutic efficacy (example 6, figure 29).

Furthermore, indocyanine green is wrapped in the lipid shell membrane.

Photodynamic therapy (PDT) can be achieved using the above described protocols. PDT is a novel treatment mode which is noninvasive, good in specificity and low in toxicity to normal tissues. Under laser irradiation, photosensitizer is used to convert light energy into toxic Reactive Oxygen Species (ROS), cell death is caused by oxidizing cell membranes and damaging important organelles such as mitochondria, PDT also has certain damage effect on RB, and especially has obvious effect on RB cell lines with poor chemotherapy and gene therapy resisting effects. Indocyanine green (ICG) is one of the photosensitizers commonly used in PDT treatment, and due to its strong absorption peak at 780nm, after laser irradiation, ICG transits from a low-energy ground state to a high-energy singlet excited state, electrons in the excited state are unstable and easily return to the ground state by releasing energy through various pathways, including radiative transition, non-radiative transition (photothermal effect), intersystem crossing to a triplet excited state (photodynamic effect), and the like, and when electrons fall from the singlet excited state to the ground state, energy is released in the form of thermal energy or other harmful substances (such as ROS and the like). ICG is excited by laser to generate energy level transition, which can generate the synergistic effect of photo-thermal treatment and photodynamic treatment, and realize 'double-strike' to tumor. ICG, the only FDA approved human-available dye in the united states, is commonly used for fundus choroidal angiography, and for staining of the lens capsule and the retinal limiting membrane in ophthalmic surgery, and is the most desirable choice for ophthalmic photosensitizers.

Furthermore, the particle size of the multifunctional nano molecular probe is 338.63 +/-10.90 nm, and the potential is 31.86 +/-3.49 mV.

The nanoparticle of the scheme has moderate particle size, is suitable for intravenous administration, and can be effectively phagocytized by tumor cells. The surface of the nanoparticle of the scheme is positively charged, and the nanoparticle has a positive promoting effect on phagocytosis of the nanoparticle by tumor cells.

Further, in S1, the mass ratio of DPPC, DSPE-PEG2000-Folate, DC-chol and oleic acid modified SPIONs is 6 mg: 2 mg: 2 mg: 25 μ L, oleic acid-modified SPIONs at a concentration of 10 mg/ml.

Further, the solvent includes chloroform and methanol. The solvent can be used for dissolving and dispersing lipid such as DPPC, DSPE-PEG 2000-foil, and DC-chol, and evaporating to dryness to obtain lipid membrane.

Further, in S3, the ratio of the liquid fluorocarbon to the indocyanine green is 2 mg: 0.2 mL. The use amount of the liquid fluorocarbon and indocyanine green can fully exert PTT and PDT effects under the condition of tumor hypoxia.

The scheme also provides application of the multifunctional nano molecular probe in preparation of a diagnostic or therapeutic preparation of the retinoblastoma.

Retinoblastoma (RB) is one of the most common ocular malignancies in infants. The common clinical RB examination means comprise conventional fundus examination, eye B-ultrasound, CT, MRI imaging and the like, the traditional imaging technology is difficult to achieve early qualitative positioning diagnosis of RB, and moreover, children with diseases are young and difficult to match, and the economic burden of families of the children with diseases is undoubtedly increased by multiple examinations, so that the early diagnosis of RB is once in a bottleneck. In the clinical treatment of RB, most children with diseases have large tumor growth when being treated with pathological changes, and the eye ball removal is still the main treatment method. As an invasive and irreversible treatment, enucleation is a very destructive treatment for ophthalmic diseases, causing great pain and mental trauma to children and family members.

The scheme provides the multi-mode contrast agent integrating three imaging advantages of ultrasound, optoacoustic and magnetic resonance, has the advantage of performing multi-mode in-vivo real-time imaging on tumor tissues at the levels of anatomical structures, functions and molecules, and can provide more complete and accurate biological information for clinic. In addition, the nano-probe provided by the scheme has Photodynamic (PDT) and Photothermal (PTT) treatment. The nano probe in the scheme is targeted and enriched around RB cells under the mediation of folic acid-magnetism dual-target, and the phase change of the nano probe in a tumor region is promoted through the irradiation of pulse laser, so that micro bubbles are generated, and the photoacoustic effect, the ultrasonic effect and the nuclear magnetic resonance imaging are enhanced; meanwhile, the ICG converts absorbed light energy into heat energy and generates active oxygen, and photo-thermal synergetic photodynamic therapy under the monitoring of RB trimodal molecular imaging is realized. RB multi-modal imaging early diagnosis and noninvasive treatment are integrated through a nano molecular probe, and a new thought is developed for establishing a high-efficiency, safe and visual treatment system under the condition of imaging monitoring. Therefore, the multifunctional nano molecular probe provided by the scheme can be applied to the medical practice operation of preparing a preparation for diagnosing or treating retinoblastoma.

In conclusion, the nano molecular probe shows attractive prospects in the aspects of tumor imaging and treatment. However, the method still has the serious defects that the target is not enough and the imaging and the treatment are difficult to be synchronously integrated, so that the research and development of a novel multifunctional nano molecular probe and the realization of the integrated diagnosis and treatment of tumors are the hot spots of the current research. In view of the urgent need for early diagnosis and non-invasive treatment of Retinoblastoma (RB), the present protocol proposes a folate-magnetic dual-targeting, encapsulated liquid fluorocarbon (PFH) and indocyanine green (ICG) with Fe loading₃O₄The phase change type multifunctional nano molecular probe (follow-CN-PFH-ICG-Fe)₃O₄FCNPIFEs, the nanoparticles performance parameters are detailed in example 2). Under the action of a magnetic field, a large amount of RB cells are enriched in the RB cells (see in-vivo and in-vitro targeting experiments in example 3 for details), and ultrasonic, photoacoustic and nuclear magnetic resonance images of the RB cells are obtained through photoinduced phase change under the action of laser (see in-vivo and in-vitro three-modal imaging experiments in example 3 for details), so that accurate positioning of tumors is realized; meanwhile, the ICG converts absorbed light energy into heat energy and generates a large amount of active oxygen to exert photothermal and photodynamic synergistic treatment effects (see experimental data related to in vivo and in vitro curative effects of example 4, example 5 and example 6). The nanoparticles of this protocol also showed good biosafety, as detailed in example 7. The scheme provides a new idea for constructing a safe, efficient and noninvasive visual diagnosis and treatment system integrating RB early diagnosis, treatment and curative effect evaluation.

Drawings

FIG. 1 is a schematic structural diagram of FCNPIFEs prepared in example 1 of the present invention.

FIG. 2 is a schematic representation of the use of FCNPIFEs prepared in example 1 of the present invention.

FIG. 3 is an electron micrograph of FCNPIFEs of example 2 of the present invention.

FIG. 4 is a graph showing the particle size distribution of FCNPIFEs in example 2 of the present invention.

FIG. 5 is a graph showing the potential distribution of FCNPIFEs in example 2 of the present invention.

FIG. 6 is a UV absorption spectrum and a calibration curve of FCNPIFEs of example 2 of the present invention.

Fig. 7 shows the Fe calibration curve and hysteresis curve of FCNPIFEs of example 2 of the present invention.

FIG. 8 shows the results of the magnetic property test, the in vitro phase transition property test and the oxygen carrying capacity test of FCNPIFEs in example 2 of the present invention.

FIG. 9 shows the observation result (3h) of the confocal microscope in the in vitro targeting experiment of example 3(1) of the present invention.

FIG. 10 shows typical flow cytometry results (3h) of in vitro targeting experiments according to example 3(1) of the present invention.

FIG. 11 shows the statistics of phagocytosis rate of quantitative flow cytometry analysis of in vitro targeting experiments of example 3(1) of the present invention.

Fig. 12 is a signal intensity-nanoparticle concentration curve of the in vitro ultrasound/photoacoustic/magnetic resonance three-mode imaging according to example 3(2) of the present invention.

Fig. 13 is a fluorescence image (left) of the distribution in vivo of the nanoparticles of example 3(3) of the present invention and a distribution image (right) of DIR-labeled nanoparticles in the major organ after 24h injection.

Fig. 14 is a fluorescence intensity statistical graph (quantitative fluorescence intensity analysis) of nanoparticles in different organs and tumor tissues after 24h injection according to example 3(3) of the present invention.

FIG. 15 shows the results of in vivo multi-modal imaging studies of FCNPIFEs molecular probes of example 3(4) of the present invention.

FIG. 16 shows the results of in vitro photothermal effects of FCNPIFEs molecular probes of example 4 of the present invention.

FIG. 17 shows the photo-thermal stability and UV NIR spectra of the FCNPIFEs molecular probe of example 4 of the present invention.

FIG. 18 shows the results of the in vitro ROS production effect experiment (laser confocal experiment) of the FCNPIFEs molecular probe of example 4 of the present invention.

FIG. 19 shows the results of the in vitro ROS production assay (flow cytometry assay) using the FCNPIFEs molecular probe of example 4 of the present invention.

FIG. 20 shows the results of in vitro biosafety studies of the FCNPIFEs molecular probe of example 4 of the present invention (CCK-8).

FIG. 21 shows the experimental results of PTT and PDT in vitro combination therapy of FCNPIFEs molecular probes of the invention example 4.

FIG. 22 is a temperature profile of a tumor site treated in vivo in accordance with example 5 of the present invention.

FIG. 23 is a photograph of a tumor in mice treated in vivo in example 5 of the present invention.

FIG. 24 is a graph of the change in tumor volume, tumor inhibition and mouse body weight for the in vivo treatment of example 5 of the present invention.

FIG. 25 is a stained section of tumor tissue after in vivo treatment according to example 5 of the present invention.

Fig. 26 is an image of blood oxygen saturation levels of tumor tissues before and after irradiation of different experimental groups in a magnetic field according to embodiment 6 of the present invention.

FIG. 27 is a fluorescent staining micrograph of HIF-1. alpha. of tumor tissue according to example 6 of the present invention.

FIG. 28 is a fluorescent staining micrograph of HSP70 alpha of tumor tissue of example 6 of the present invention.

FIG. 29 is the result of quantitative analysis of the expression level of HSP70 in example 6 of the invention.

FIG. 30 shows the results of biosafety evaluation (tissue staining) in example 7 of the present invention.

FIG. 31 shows the results of biosafety evaluation (blood biochemical index) in example 7 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available.

Wherein, the main reagent comprises:

DPPC is called 1, 2-dihexanoyl-rac-Glycero-3-Phospholine;

the DSPE-PEG (2000) -folate (DSPE-PEG2000-FA) is called as follows:

1,2-distearoyl-sn-glyc-ero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-2000]；

SPIONs are all referred to as: oleic acid modified Fe₃O₄Nanoparticles (OA @ Fe)₃O₄)；

The above reagents were purchased from west anruix (china).

Indocyanine green (ICG), Perfluorohexane (PFH) and Dc-cholesterol (Dc-cholesterol) were purchased from Sigma (usa).

Example 1: preparation of FCNPIFEs molecular probe

Weighing a certain amount of DPPC (6mg), DSPE-PEG2000-FA (2mg), DC-chol (2mg) and oleic acid modified SPIONs (the main component is Fe)₃O₄10mg/mL) was placed in a round bottom flask, 5mL of chloroform was added, and the solution was stirred with a magnetic stirrer until it was clear. A brown lipid film (50 ℃ C., 2h) was formed by rotary evaporation using a rotary evaporator and hydrated by adding 2mL of PBS. At the same time, PFH (0.2mL) and ICG (2mg) were mixed and emulsified on ice with an ultrasonic vibrometer (2min, 35% X130W; on 5s, off 5s, Heat System Inc, USA) to obtain a first emulsion. The hydrated brown lipid film was added to the first emulsion and subjected to a second sonication (40% x 130W for 6 min; on 5s, off 5s) to obtain a second emulsion. Finally, the second emulsion was centrifuged three times at 8000rpm and 5min to obtain Folate-CN-PFH-ICG-Fe₃O₄(FCNPIFEs) and stored in a refrigerator at 4 ℃ for later use.

In addition to FCNPIFEs, FNNPIFEs (Folate-NN-PFH-ICG-Fe) were prepared₃O₄) I.e., equivalent replacement of DC-chol with ordinary cholesterol (this)The surface potential of the nanoparticles was-25.325 mV, i.e., the surface was negatively charged). FCNPIs (foil-CN-PFH-ICG, without SPIONs), CNPIFEs (NN-PFH-ICG-Fe) were also prepared₃O₄Using DSPE-PEG2000 instead of DSPE-PEG2000-FA), CNPIs (NN-PFH-ICG, using DSPE-PEG2000 instead of DSPE-PEG2000-FA, and no SPIONs), FCNPFEs (foil-CN-PFH-Fe)₃O₄Without ICG) and FCNIFEs (Folate-CN-ICG-Fe)₃O₄Without PFH). Incubating the fluorescent dye DiI and the nanoparticles together to construct a fluorescent labeled molecular probe for in vivo and in vitro targeted detection and in vivo distribution condition detection.

The schematic structure of FCNPIFEs prepared in this example is shown in fig. 1, and the efficacy and use of FCNPIFEs are shown in fig. 2. The FCNPIFEs of the scheme can enter blood circulation in a mode of intravenous administration, reach tumor tissues and enter the tumor tissues through an EPR effect. In addition, FCNPIFEs of this technical scheme is one kind has dual targeting advantage, multi-functional nanometer molecular probe of looks transition type, can pass tumour blood vessel endothelium clearance, the targeting gathers to extravascular tumour cell, liquid fluorocarbon takes place the liquid gas phase transition under the laser effect of certain energy, thereby strengthen the multimodality imaging effect, indocyanine green after arousing simultaneously turns into light energy and generates active oxygen with heat energy, exert light and heat synergy photodynamic therapy effect, when improving diagnosis treatment effect, reduce whole body toxic and side effect, provide new platform for realizing RB at the picture of molecular level and noninvasive treatment. The FCNPIFEs combine the specificity of molecular targeting with the high efficiency of magnetic targeting, and are a molecular probe of double-targeting RB, so that the targeting efficiency is improved.

Example 2: characterization of FCNPIFEs molecular probes

Particle size and potential detection: the electron micrograph of FCNPIFE prepared in example 1 is shown in figure 3. The particle size distribution and the surface potential of the molecular probe were measured by a laser particle size analyzer/Zeta potentiometer, and the results are shown in FIGS. 4 and 5, 338.63 + -10.90 nm and Zeta potential of 31.86 + -3.49 mV.

And (3) detecting the encapsulation efficiency and the drug loading rate: detection of ICG and Fe in molecular probe by ultraviolet spectrophotometer₃O₄Encapsulation efficiency and drug loading. Measurement of ICGThe results are shown in FIG. 6. FCNPIFE showed the highest peak at 778 nm. The regression equation of the standard curve of ICG measured by ultraviolet spectrophotometry can calculate to obtain (93.09 +/-0.63)%, and the drug-loading rate (15.51 +/-0.11) mg. Wherein the ICG encapsulation ratio (%) (total ICG mass-free ICG mass)/total ICG mass × 100%; ICG drug loading (%) (total ICG mass-free ICG mass)/total nanoparticle mass × 100%.

For Fe₃O₄See fig. 7 left and the hysteresis curve see fig. 7 right. An Fe standard curve is prepared, and the Fe encapsulation rate is (4.37 +/-0.80)%, and the drug loading rate is (0.0026 +/-0.05)%, which can be obtained by calculation. Wherein, Fe₃O₄Encapsulation efficiency (%) - (Fe in nanoparticle)₃O₄Mass of (1)/Fe₃O₄The total input amount is multiplied by 100 percent; fe₃O₄The drug loading capacity (%) ═ Fe in the nanoparticles₃O₄The mass of (2)/the total mass of the nanoparticles is multiplied by 100%.

And (3) magnetic property detection: magnetic properties of FCNPIFEs at 300K were studied using a Vibrating Sample Magnetometer (VSM). The FCNPIFEs solution was placed in a glass vial and the nanoparticles were observed for 6h aggregation in a magnetic field (4T) and the experimental results are shown in fig. 8.

In-vitro phase change performance detection: confocal microscopy was used to observe FCNPIFEs at 808nm laser (1.5W/cm)²5min), and the experimental result is shown in fig. 8, which illustrates that the nanoparticle of the present scheme can undergo phase change under the excitation of laser.

Detecting the oxygen carrying capacity: detecting the oxygen carrying capacity of the nanoparticles, after exciting FCNPIFEs, FCNIFEs and PBS to generate oxygen for 10min, adding 1ml of the substance to be detected into degassed water, carrying out water bath at 47 ℃ for 10min, and detecting the oxygen content in the water by using a portable dissolved oxygen monitor, wherein the experimental result shows that PFH can effectively store oxygen, and the detailed result is shown on the right of FIG. 8. At 48 ℃ ambient temperature, the concentration of dissolved oxygen in the FCNPIFEs increased rapidly from 3.21 to 13.20mg/ml within 60s and remained high for 10min, compared to FCNIFE and PBS. The FCNPIFEs explode by heating, the oxygen stored in the PFH overflows in a large amount, a phenomenon occurs in which the dissolved oxygen content rapidly rises, and as time goes on, the dissolved oxygen content falls and maintains an equilibrium level. The above data demonstrate that PFH has ideal oxygen carrying capacity to provide oxygen for PDT and enhance PDT.

Example 3: FCNPIFEs in vivo and in vitro targeting performance and three-mode imaging research

(1) In-vitro targeting performance detection of FCNPIFEs molecular probe

In vitro culture of RB cells: human RB Y79 cell line in 1640 culture medium containing 10% fetal bovine serum, 5% CO₂And conventional culture passage at 37 ℃.

In vitro targeting experiments: according to the previous method, CNPIFE, FCNPIFECNPI, CNPI and FNNPIFE were Dil stained and seeded separately with Y79 cells into 24-well plates (2X 10 cells per well)⁵Individual cells), divided into 7 groups in total: control, CNPI, FCNPI + Folic Acid (FA), CNPIFE + magnetic field (M.F), FCNPI, FNNPIFE + magnetic field, FCNPIFE + magnetic field. The nanoparticle concentrations were all 0.4mg/ml, the magnetic field was completed by 4T magnets attached to the sides of the wells, and the cells were incubated for additional 1, 3 and 6 hours. After incubation with nanoparticles was complete, cells were fixed with paraformaldehyde for 15 minutes, incubated with DAPI for another 15 minutes to stain nuclei, and then incubated with DiO for 10 minutes to stain cell membranes. Intracellular absorption behavior of each group of nanoparticles was observed by confocal and each group was quantitatively analyzed by flow cytometry. Confocal observations are shown in fig. 9 (scale bar 25 μm), flow cytometry results are shown in fig. 10, and phagocytosis statistics are shown in fig. 11 and table 1 (phagocytosis, i.e., the percentage of Y79 cells that have phagocytosed nanoparticles to the total number of cells, is a quantitative measurement using flow cytometry, n is 3, p is < 0.01, p is < 0.0001). In vitro targeting experiments prove that the effects of three targeting groups (folic acid targeting, magnetic targeting and cation) are strongest already at 3h, and compared with other groups, the signals are strongest at the fastest speed (other groups need 6h), the phagocytosis rate is also superior to that of other groups. In fig. 11, it can be observed that at 3h, FNNPIFE + magnetic field set (nanoparticles surface non-positive charge), FCNPI set (no magnetic target) and CNPIFE + magnetic field set (no folate target), the nanoparticle phagocytosis rate (percentage of Y79 cells where fluorescence signal was detected) was significantly lower for the three experimental groups than for the FCNPIFE + magnetic field set. This demonstrates that folic acid targeting, magnetic targeting, to increase targeting of nanoparticlesAnd DC-chol (positive charge) are all the three factors, and a synergistic effect exists among the three factors. By analyzing the data in table 1 in detail, the FCNPIFEs + M.F group reached a peak of 95.08 ± 0.88% at 3h and the peak was consistently maintained at 6h (96.69 ± 0.32%), while the other experimental groups had a phagocytosis rate at 3h much lower than that of the FCNPIFEs + M.F group, and even after 6h, the other experimental groups had a phagocytosis rate much lower than that of the FCNPIFEs + M.F group. The nanoparticles in the CNPIs group lack folic acid targeting and magnetic targeting, but the surface charge is positive, and the phagocytosis rate is 25.76 +/-0.15% in 3 h; the nanoparticles in FNNPIFEs + M.F group have magnetic targeting and folic acid targeting, but the surface charge is negative, and the phagocytosis rate is 54.60 +/-9.65% in 3 h; the sum (about 80.36%) of the phagocytosis rate of the CNPIs group (surface positive charge single targeting) and the FNNPIFEs + M.F group (folic acid and magnetic double targeting) is still less than that of the FCNPIFEs + M.F group (triple targeting), the action effect of triple targeting is not the simple superposition of the action effects of the three factors, and the synergistic effect between the triple targeting is further confirmed. At 3h, the phagocytosis rate of the CNPIFEs + M.F group (magnetic targeting + surface cations) is 41.71 +/-2.30%, the phagocytosis rate of the FCNPIs group (folic acid targeting + surface cations) is 45.91 +/-3.32%, and the sum of the phagocytosis rates (about 87.62%) is far lower than that of the FCNPIFEs + M.F group (triple targeting), which also indicates that the folic acid targeting and the magnetic targeting have a synergistic effect. Comparing with FNNPIFEs + M.F group (phagocytosis rate 54.60 +/-9.65%), it is shown that the synergistic effect of folic acid targeting and magnetic targeting is not significant under the condition of no surface cations of the nanoparticles, although the synergistic phenomenon exists between folic acid targeting and magnetic targeting. The analysis of the data in table 1 further shows that the nanoparticles in the scheme use a technical scheme of combining three targets, so that the nanoparticles can be rapidly enriched in tumor cells; and the effect of the triple targeting is not a simple effect superposition, but a synergistic effect of 1+1+1 > 3.

Table 1: phagocytosis rate statistics for different treatment groups (mean ± SD, n ═ 3)

(2) FCNPIFEs in vitro multi-modal imaging study

In vitro imaging: to evaluate FCNPIFEs for in vitro PA function, samples were diluted (0.025, 0.05, 0.1, 0.2 and 0.4mg/mL) according to ICG concentration and added to agar gel phantom for PA imaging, recorded and analyzed by a VEVO laser PA imaging system. In vitro ultrasound function was investigated by MyLab90, FCNPIFEs liposomes diluted (0, 0.4, 0.8, 1.2 and 2.4mg/ml) and exposed to 808nm laser (2 w/cm)²5min), the signal intensity after laser excitation was analyzed using DFY (ultrasonic imaging institute of university of Chongqing, China). To assess the function of in vitro MR imaging, samples were diluted (Fe concentrations: 0.015, 0.03, 0.06, 0.12, 0.18 and 0.24mM) and placed in EP tubes, T2WI images were obtained using a philips Achieva 3.0T TX MR scanner (philips medical system, netherlands), and the Signal Intensity (SI) of a region of interest (ROI) was analyzed by Sante DICOM, with the following T2 parameters: TR 1610ms, TE 14.60ms, Field 3T, DFOV 250mm, and slice hickness 3.0 mm. According to in vitro imaging experiments, the FCNPIFE can simultaneously perform ultrasonic/photoacoustic/magnetic resonance three-mode imaging, and the signal intensity has an obvious linear correlation with the nanoparticle concentration (fig. 12).

(3) Detection of in vivo distribution and targeting capability of FCNPIFEs molecular probe

Establishing a nude mouse RB subcutaneous tumor model: RB Y79 cells in logarithmic growth phase were used to adjust the cell concentration to 5X 10⁷0.2ml of each of the mice was inoculated under the skin of the hip back of a 3-4 week-old BALB/c nude mouse. About two weeks, the nude mice developed tumors on the dorsal and gluteal sides to about 1cm by 1cm in size for use.

In vivo targeting experiments: y79 tumor-bearing mice were randomly divided into 6 groups (tail vein injected nanoparticles amount of 200 μ L,0.4mg/mL, n ═ 3): CNPIs (I), CNPIFEs (II), CNPIFEs + magnetic field (III), FCNPIs (IV), FNNPIFEs + magnetic field (V), FCNPIFEs + magnetic field (VI), applying magnetic field by fixing magnet (4T) above tumor, and obtaining fluorescence images (0, 3, 6, 24h) of different time points after nanoparticle injection by using small animal living body fluorescence detection (Fx7 IR Spectra, Vilber Lourmat, France). Finally, tumors and major organs of the mice were collected for fluorescence imaging and the corresponding fluorescence signals were analyzed.

Fluorescence imaging of Y79 tumor-bearing mice (fig. 13 and 14) further validated the targeting advantage of FCNPIFEs + magnetic field. Fig. 14 is a statistical graph of the fluorescence intensity of nanoparticles in different organs and tumor tissues after 24h injection (n-3, p < 0.0001), and table 2 is a specific statistical data of fluorescence intensity. Referring to the left panel of fig. 13, FCNPIFEs + field set started to fluoresce in the tumor region (circled marks in the figure) 3h after injection, peaking at 6h and exhibiting higher fluorescence energy within 24 h. The FNNPIFEs + magnetic field showed the second time, weak fluorescence signals were observed in the tumor region at 6h, the fluorescence signals reached the maximum at 24h, and weak signals or even no signals were detected in the tumor region at 24h in the remaining groups. The experimental result shows that the tri-targeting nanoparticles can be rapidly accumulated at the tumor part, and the targeting treatment effect is realized. Biodistribution of the relevant nanoliposomes was shown by imaging the major organs and tumors (fig. 13 right), and each fluorescence intensity was quantified using a fluorescence analysis system (fig. 14 and table 2). Except for the FCNPIFEs + magnetic field group spleen, there was significant accumulation in the liver and spleen of each group. This is due to the regular metabolic pathways that take place in vivo by the uptake behavior of the reticuloendothelial system. According to experimental results, the FCNPIFEs nanoparticle has the most ideal safety and the smallest accumulation amount at non-tumor parts. Referring to the experimental results of fig. 13, fig. 14 and table 2, the FCNPIFEs + field set has a significantly reduced signal at 24h due to the fact that a peak signal has occurred at 6h, while the FNNPIFEs + field set reaches a maximum intensity at 24 h. This shows that the nanoparticles FCNPIFEs of the present protocol can rapidly achieve therapeutic effects in vivo, and the nanoparticles can be metabolized and cleared in a shorter time, which is safer than FNNPIFEs. In conclusion, FCNPIFEs not only minimally damage major organs, but also accumulate most efficiently in tumors.

Table 2: statistical data of fluorescence intensity of living body (mean. + -. SD, n ═ 3)

(4) FCNPIFEs molecular probe in vivo multi-modal imaging research

Y79 nude mice were subjected to in vivo photoacoustic imaging and divided into four groups (FCNPIFEs + field, CNPIs, FCNIFEs + field, FCNPFEs + field), nanoparticles (200 μ L,0.4mg/mL, n ═ 3) were injected into the tail vein, images (0, 3, 6, 24h) at different time points were captured using the VEVO laser PA imaging system by applying a magnetic field to the magnet (4T) fixed above the tumor, and the experimental results are shown in the two lower panels of fig. 15. The lower left is a typical PA image of the tumor site of tumor-bearing mice, and the lower right is a PA intensity histogram of the tumor site at different time points (n-3, p < 0.0001).

Carrying out in vivo ultrasonic imaging on the nanoparticles, and grouping the nanoparticles into: FCNPIFEs + field, CNPIs, CNIFEs + field (200 μ L,0.4mg/mL, n 3) and analyzed for intensity values using DFY software. The experimental results are shown in detail in the upper two panels of FIG. 15. The top left is a typical ultrasonic image of the tumor site of the tumor-bearing mouse, and the top right is a statistical plot of the echo intensities before and after 808nm laser irradiation (n is 3, p is less than 0.001, p is less than 0.0001).

To examine the in vivo MR imaging effect of nanoparticles, nude mice were divided into 2 groups: FCNPIFEs + magnetic field, FCNPIs (200 μ L,0.4mg/mL, n ═ 3), T2WI images were recorded at different time points (0, 3, 6, 24h) using a philips Achieva 3.0T TX MR scanner, and ROI signals were analyzed using Sante DICOM. The T2 weighted image was obtained using the following parameters: TR 5560ms, TE 85ms, Field 3T, DFOV 80mm, and slice thickness 0.7 mm. The experimental results are shown in the middle two panels of FIG. 15. Of these, the central left is a typical T2 image of the tumor site of tumor-bearing mice, and the central right is a statistical plot of the signal intensity of the tumor site at different time points (n-3, p < 0.05, p < 0.0001).

In-vivo imaging results prove that FCNPIFEs are efficiently and effectively accumulated in tumor tissues, ultrasonic/photoacoustic/magnetic resonance three-mode imaging can be effectively carried out, and in addition, in consideration of the imaging results, the optimal time window of future laser irradiation treatment experiments is 6 hours after different nano-carriers are injected.

Example 4: research on FCNPIFEs molecular probe in vitro photothermal effect (PTT), photodynamic effect (PDT) and cytotoxicity

PBS, FCNPFEs (0.4mg/mL) and FCNPIFEs (C: (B) ((C))0.05, 0.1, 0.2, 0.4mg/ml) was irradiated under 808nm laser for 5min (1.5W/cm)²) The experimental results are shown in fig. 16, wherein the upper left is the thermal energy image recorded per minute, and the upper right is the temperature change curve with time. And selecting lasers (0.5,1,1.5, 2.0W/cm) with different irradiation intensities²) FCNPIFEs solution (0.4mg/ml) was irradiated for 5 min. The experimental results are shown in fig. 16, wherein the lower left is the thermal energy image recorded per minute, and the lower right is the temperature change curve with time. And recording the temperature change amount of the solution to be measured per second and shooting images per minute by using a thermal infrared imaging camera.

Next, photothermal stability studies of FCNPIFEs (0.4mg/mL) were performed using 808nm (1.5W/cm)²) The laser irradiation is carried out for 5 cycles, and the experimental result is shown in the left part of FIG. 17, which shows that the FCNPIFEs have good photo-thermal stability. UV-vis-NIR spectra (5min, 1.5W/cm) of FCNPIFEs (0.4mg/mL), FCNIFEs (0.4mg/mL) and ICG (0.4mg/mL) were measured²) See fig. 17, right for experimental results.

The DCFH-DA method was used to study ROS production by cells. Y79 cell (2X 10)⁵Each cell per well) were incubated with FCNPIFEs (0.4mg/ml), FCNIFEs (0.4mg/ml) and ICG (0.4mg/ml) in 24-well plates for 3h and a 4T magnetic field was applied, followed by a 808nm laser (1.5W/cm)²) Irradiating for 5 min. DCFH-DA was added to the cells and incubated for 30min, and ROS production was observed using confocal microscopy and quantified using flow cytometry. Wherein the hypoxic group was placed in an anaerobic cassette throughout the experiment. Fluorescence confocal images (scale bar 100 μm) are shown in fig. 18, flow cytometry analysis is shown on the left in fig. 19, and quantitation is shown on the right in fig. 19. The fluorescence confocal experiment result shows that: FCNPIFEs generate similar amount of ROS through PDT in anaerobic and aerobic environments; however, for FCNIFEs and free ICGs, there is a difference in the amount of ROS produced by PDT in anaerobic as well as aerobic environments. PFH is a good oxygen carrying substance, and the flow cytometry experimental result also confirms the laser confocal experimental result.

In vitro safety of FCNPIFEs Using CCK-8 method, Y79 and ARPE-19 cells were seeded in 96-well plates (1X 10)⁴Each cell per well), culturingAfter 24h of incubation, the medium was replaced with fresh medium containing FCNPIFEs (FCNPIFEs concentrations 0.1, 0.2, 0.4, 0.8 and 1.6 mg/mL). After 24h incubation, the CCK-8 assay was performed and the absorbance at 450nm was measured for each well using a microplate reader. The experimental results are shown in fig. 20, and the cell survival rates are all higher than 90%, which shows that the biological safety of the nanoparticles is high under the condition of no laser irradiation. The Y79 cells are subjected to laser irradiation for 5min, and then a CCK-8 experiment is carried out, and the experimental result is shown on the right side of the figure 20, which shows that the nanoparticles have ideal cell killing effect by applying the laser irradiation.

The nanoparticles were tested for in vitro therapeutic effect. Y79 cells were seeded in 96-well plates (5X 10)⁴Each cell per well), after 24h of culture, divided into 8 groups, and subjected to irradiation treatment (808nm, 2w, 5 min): blank control (normoxia and hypoxia), PTT group (normoxia and hypoxia), PDT group (normoxia and hypoxia), PTT + PDT group (normoxia and hypoxia). In PDT treatment, the plates were incubated in ice to avoid photothermal effects, and NaN was added in PTT treatment₃(100mM) to avoid photodynamic effects. Results of quantitative flow cytometry analysis see fig. 21, which illustrates that PTT + PDT treatment can increase the cell killing ability of the nanoparticles.

Example 5: experiment of in vivo synergistic therapeutic effects of PDT and PTT

When the tumor of the Y79 tumor-bearing mouse grows to 100mm³Mice were divided into 9 groups: PBS group, (II) laser treatment group, (III) FCNPIFEs + M.F group, (IV) ICG + laser group, (V) FCNIFEs + M.F + laser group, (VI) FCNPFEs + M.F + laser group, (VII) CNPIs + laser group, (VIII) FCNPIFEs + M.F + laser group (intermittent laser treatment), and (IX) FCNPIFEs + M.F + laser group. Group (VIII) laser conditions were: 808nm, 1.5W/cm²On 30s/off 30s, 10min to ensure temperature at 41 ℃ to avoid excessive temperature, as a single PDT treatment. The laser conditions adopted by the groups (II), (IV), (V), (VI), (VII) and (VIII) are as follows: 808nm, 1.5W/cm²And 10 min. The nanoparticles were administered by tail vein injection (200 μ L,0.4mg/mL, n ═ 3), and the tumor site was irradiated with laser after 6 h. And recording thermal imaging and temperature change by using a thermal infrared imager, recording the tumor volume and the tumor weight of the mouse once every two days, and calculating the tumor inhibition rate. Tumor and main device collected on the third day after treatmentFunctional Hematoxylin and Eosin (HE), Proliferating Cell Nuclear Antigen (PCNA) and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end marker (TUNEL) staining to assess histological changes and levels of apoptosis.

The temperature changes of the tumor sites of the different treatment groups are shown in the left of FIG. 22, and the temperature changes of the tumor sites of the group (VIII) are shown in the right of FIG. 22. In the free ICG + laser treated group, only a modest temperature increase was observed. Whereas in the group used for photodynamic therapy the temperature was kept below 41 ℃ to avoid photothermal effects. A significant increase in local temperature was noted for the mice irradiated with continuous irradiation, with the maximum temperatures reaching approximately 47 ℃ and 47.5 ℃ respectively. These two similar temperature profiles imply no significant difference in photothermal efficiency for FCNPIFE and FCNIFE. In the FCNPIFEs + laser group, tumors gradually shrunk. In other groups, either the treatment was less effective or the tumor had recurred. After various treatments, tumors were extracted. The tumor volume groups of FCNPIFEs + laser gradually decreased throughout the observation period and the treatment was significantly better than the other groups. Consistent with previous results, the tumor weight of FCNPIFEs + laser was significantly lower than that of the other groups, and the body weight of mice did not change much during the study (FIG. 23), suggesting that the FCNPIFEs + laser combination was safe and effective. In fig. 3, the left graph is the change of tumor volume (V/V0) with time, the middle graph is the tumor inhibition rate (n-3, p < 0.0001, p < 0.001), and the right graph is the change of mouse body weight for 16 days. HE. Microscopic images of tumor tissue sections after PCNA and TUNNEL staining are shown in FIG. 24. In addition, tumor weight inhibition rate further verifies the antitumor effect of the different treatments. Consistent with the expectation, the tumor weight inhibition rate was highest in all groups for the FCNPIFEs + laser group.

Example 6: research on PDT and PTT synergistic action mechanism of FCNPIFEs molecular probe

To study the saturation level of oxyhemoglobin in tumor tissue, tumor-bearing mice were randomly divided into 3 groups (n ═ 3): saline, FCNIFEs + magnetic field (M.F), FCNPIFEs + magnetic field. After injection of nanoparticles (200. mu.L, 0.4mg/mL) for 6h, laser irradiation at 808nm for 10min (1.5W/cm)²) 24h before and after each group of nanoparticles is injected, Vevo Laser photoacoustic imager is used for detecting the blood oxygen saturation of the tumor part in the blood oxygen modeAnd taking tumor tissue to perform HIF-1 alpha staining, and detecting the improvement condition of the molecular probe on the hypoxia state of the tumor part. Fig. 25 shows the blood oxygen saturation images of the tumor tissue before and after irradiation of different experimental groups under a 4T magnetic field on the left, and fig. 26 shows the quantitative detection result of the proportion of oxyhemoglobin (λ 850nm) and deoxyhemoglobin (λ 750nm) at the tumor tissue on the right. FIG. 27 shows HIF-1. alpha. fluorescent staining micrographs of tumor tissue (one day after irradiation treatment). The experimental results show that in the FCNPIFEs + magnetic field set, the content of oxygenated hemoglobin in the tumor tissues is increased from 33.34% + -2.91% before treatment to 74.21% + -7.08%, and further show that PFH has good oxygen-extracting capacity in an anoxic environment.

In order to study the influence of photodynamic action on photothermal action, the inventors further studied the expression level of HSP70 in tumor tissues. Tumor-bearing mice were divided into 5 groups (n ═ 3): saline group, FCNPIs + magnetic field, FCNPIFEs + magnetic field, FCNIFEs + magnetic field + laser and FCNPIFEs + magnetic field + laser. The processing method of the laser processing group is the same as that of the previous stage. And detecting the expression condition of the heat shock protein HSP70 at the tumor part by an immunofluorescence staining method and a protein immunoblotting method, and further confirming whether the enhanced photodynamic effect has a promotion effect on the photothermal effect. Fig. 28 shows fluorescence-stained microscopic images of HSP70 α of tumor tissues (one day after irradiation treatment), and fig. 29 shows the results of quantitative analysis of the expression level of HSP70 (proportion of HSP 70-containing region). The test result shows that: the increase of the HSP70 level can lead the cells to generate heat resistance, and the proportion of HSP 70-containing area of the FCNPIFEs + magnetic field + laser group is reduced by about 19.33 percent (relative to FCNIFEs + magnetic field + laser), which shows that the nanoparticles using the scheme can destroy mitochondrial metabolism related substances through ROS generated irreversibly through photodynamic action, reduce HSP70 expression, further promote the killing of cancer cells, and further explain the synergistic action principle of PDT and PTT.

Example 7: biological safety research of FCNPIFEs molecular probe

Mice from the in vivo experiment of example 5 were taken for tissue sectioning and safety assessment. To evaluate FCNPIFE safety in vivo, 12 female nude mice were subjected to blood routine and serum biochemical tests by collecting blood samples (n-3) at different time points (control, 1d, 7d, 14d) after tail vein injection of FCNPIFEs (200 μ L,0.4mg/mL, n-3).

According to the evaluation of in vivo safety results, no obvious abnormality appears in the blood biochemical indexes of the mice before and after FCNPIFEs injection (figure 31), and the FCNPIFEs have good biological safety. H & E staining of major organs including heart, liver, spleen, lung, kidney (fig. 30) did not show significant physiological abnormalities at the end of in vivo treatment, indicating negligible toxicity before and after treatment.

The above description is only an example of the present invention, and the general knowledge of the known specific technical solutions and/or characteristics and the like in the solutions is not described herein too much. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, and these should also be considered as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. A multifunctional nano molecular probe is characterized in that: comprises a lipid shell membrane, wherein Fe is loaded on the lipid shell membrane₃O₄And folic acid.

2. The multifunctional nanomolecular probe according to claim 1, wherein: the lipid shell membrane comprises DPPC, DSPE-PEG2000-Folate and DC-chol.

3. The multifunctional nanomolecular probe according to claim 2, wherein: liquid fluorocarbon is wrapped in the lipid shell membrane.

4. The multifunctional nanomolecular probe according to claim 3, wherein: indocyanine green is further wrapped in the lipid shell film.

5. The multifunctional nanomolecular probe according to claim 4, wherein: the particle diameter is 338.63 + -10.90 nm, and the potential is 31.86 + -3.49 mV.

6. The method for preparing a multifunctional nano molecular probe according to any one of claims 1 to 5, wherein the method comprises the following steps: comprises the following steps of:

s1: adding DPPC, DSPE-PEG2000-Folate, DC-chol and oleic acid modified SPIONs into a solvent, uniformly mixing, and evaporating the solvent to obtain a lipid membrane;

7. The method for preparing a multifunctional nano molecular probe according to claim 6, wherein the method comprises the following steps: in S1, the dosage ratio of DPPC, DSPE-PEG2000-Folate, DC-chol and oleic acid modified SPIONs is 6 mg: 2 mg: 2 mg: 25 μ L, oleic acid-modified SPIONs at a concentration of 10 mg/ml.

8. The method for preparing a multifunctional nano molecular probe according to claim 4, wherein the method comprises the following steps: in S1, the solvent includes chloroform and methanol.

9. The method for preparing a multifunctional nano molecular probe according to claim 4, wherein the method comprises the following steps: in S3, the dosage ratio of the liquid fluorocarbon to the indocyanine green is 2 mg: 0.2 mL.

10. Use of the multifunctional nano-molecular probe according to any one of claims 1 to 5 for preparing a diagnostic or therapeutic preparation for retinoblastoma.