CN114588279B

CN114588279B - Multifunctional nano molecular probe, preparation method thereof and application of multifunctional nano molecular probe as retinoblastoma diagnosis and treatment preparation

Info

Publication number: CN114588279B
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: 2023-10-20
Anticipated expiration: 2042-03-31
Also published as: CN114588279A

Abstract

The application 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 of the multifunctional nano molecular probe as a retinoblastoma diagnosis and treatment preparation. A multifunctional nano molecular probe comprises a lipid shell membrane and a core, wherein Fe is inlaid in the lipid shell membrane ₃ O ₄ The outer side of the lipid shell membrane is loaded with folic acid. The technical scheme can solve the technical problem of single targeting mode of the diagnosis and treatment integrated nano molecular probe in the prior art. In the technical scheme, the specificity of molecular targeting is combined with the high efficiency of magnetic targeting, and a molecular probe for dual targeting retinoblastoma is constructed, so that the targeting efficiency is improved, and a new thought is provided for constructing a safe, efficient 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 application 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 of the multifunctional nano molecular probe as a retinoblastoma diagnosis and treatment preparation.

Background

Retinoblastoma (RB) is the most common eye malignancy of infants, the malignancy degree is high, early detection is difficult, RB growth of most infants is large when the infants are treated due to 'white pupil sign', the eyes possibly cannot be held in the period of seriously damaging eyesight or even life, and treatment is mainly carried out by removing the eyeballs of the eyes, and radiotherapy and chemotherapy are auxiliary. The eyeball removing operation is the most damaging treatment of the ophthalmology, and the children suffering from the operation lose the vision function forever, so that the children suffering from the operation have great trauma to the physical and mental health. Thus, there is an urgent need to explore new directions, new targets and new measures for the treatment of RB. In view of the characteristics of extremely high harm to infants and troublesome treatment of the current RB, early diagnosis and non-invasive treatment implementation of the RB are always targets of clinical pursuit.

The conventional clinical RB inspection means comprise conventional fundus inspection, eye B-ultrasonic, CT and MRI imaging, and the traditional imaging technologies are difficult to achieve early qualitative positioning diagnosis of the RB, and the early diagnosis of the RB is difficult to be matched with the early diagnosis of the infant, so that the household economic burden of the infant is certainly increased by multiple inspections, and the early diagnosis of the RB is once in bottleneck due to the difficulty. Along with the rapid development of molecular imaging and nanotechnology, the molecular imaging and therapeutic means are integrated into a nano platform, and the diagnosis and treatment integrated nano molecular probe with diagnostic capability and therapeutic efficacy is designed and constructed, so that the method has remarkable significance.

Chinese patent CN108014349a discloses a preparation method and application of a multifunctional gene-carrying contrast agent, which can be used in the practice of RB diagnosis and treatment. The multifunctional contrast agent prepared by the patent comprises a lipid shell membrane, folic acid is modified on the lipid shell membrane, positive charges are carried on the surface of the lipid shell membrane, genes are carried on the surface of the lipid shell membrane, and liquid fluorocarbon and indocyanine green are wrapped in the lipid shell membrane. In the technical scheme, the liquid fluorocarbon PFP has good photoinduced phase change property, the ICG has the property of photoacoustic imaging, the PFP and the ICG are simultaneously loaded in a nano carrier, folic acid is modified on a lipid shell in a combined mode, a therapeutic gene is carried on the surface of the carrier, and the contrast agent can meet the requirements of photoacoustic/ultrasonic dual-mode and gene transfection therapy under the action of laser excitation. However, the contrast agent is only provided with one targeting molecule folic acid, and is limited by factors such as receptor expression difference among individuals and receptor-antibody specific binding, and the effect of folic acid on targeting tumors is limited. Moreover, the contrast agent can only realize bimodal imaging, and can not meet the requirements of multi-modal imaging of photoacoustic, ultrasound and nuclear magnetic resonance of the contrast agent in clinical application.

Disclosure of Invention

The application aims to provide a multifunctional nano molecular probe so as to solve the technical problem of single targeting mode of the diagnosis and treatment integrated nano molecular probe in the prior art.

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

a multifunctional nano molecular probe comprises a lipid shell membrane, wherein Fe is loaded on the lipid shell membrane ₃ O ₄ And folic acid.

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

s1: adding DPPC, DSPE-PEG 2000-fonate, DC-chol and oleic acid modified SPIONS into solvent, mixing, and steaming to obtain lipid membrane;

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

s3: and (3) dropwise adding liquid fluorocarbon and indocyanine green into the emulsion, and performing sound vibration emulsification to obtain FCNPIFEs.

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

the scheme simultaneously introduces SPION (namely Fe ₃ O ₄ ) And Folic Acid (FA) are respectively used as a magnetic targeting agent and a molecular targeting agent, so that the targeting efficiency of the nanoparticle probe is improved. The folic acid targeted nano molecular probe constructed in advance by the inventor can be specifically gathered around RB cells through being combined with folic acid receptors on the surfaces of the RB cells. However, the targeting ability of ligand-modified active targeting probes is limited due to saturation effect of specific receptors and obstruction of tumor microenvironment, and how to maximize the number of molecular probes in tumor regions is a big problem. In the technical scheme, the inventor combines folic acid targeting and magnetic targeting, namely, combines the specificity of molecular targeting with the high efficiency of magnetic targeting, and constructs a molecular probe of dual targeting RB, thereby improving targeting efficiency, and realizing the synergistic increase of retinoblastoma by the magnetic targeting and folic acid targetingPotent targeting (see example 3, fig. 11 and table 1). The magnetic targeting is a novel active targeting mode based on interaction between magnetic substances, and under the action of an externally applied magnetic field, a magnetic field is applied to the surface of an eyeball, so that enrichment and retention of magnetic particles on retina can be realized. By Fe ₃ O ₄ Superparamagnetic iron oxide nanoparticles (SPIONs) as cores have high targeted aggregation capability in an externally applied magnetic field. In addition, fe is added to the molecular probe ₃ O ₄ The defect that the ultrasonic imaging and the photoacoustic imaging can not provide anatomical positions due to limited penetration capability is overcome, so that the molecular probe has the function of MR imaging, and the MR imaging is important for early diagnosis of RB in clinic.

Further, the components of the lipid shell membrane include DPPC, DSPE-PEG2000-Folate and DC-chol. The addition of DC-chol to the lipid shell membrane can make the nanoparticle surface positively charged. Folic acid targeting, magnetic targeting and positive charge are synergistic, achieving more excellent tumor targeting and therapeutic effect (see example 3, fig. 11 and table 1, fig. 13, 14 and table 2).

Further, the lipid shell membrane is internally wrapped with liquid fluorocarbon.

According to the scheme, liquid fluorocarbon (PFH, perfluorohexane, with the boiling point of 56 ℃) is selected as a kernel to prepare the phase-change nano molecular probe, and the liquid fluorocarbon is an ideal ultrasonic contrast agent due to the moderate boiling point and strong operability; meanwhile, the device has the characteristics of strong oxygen dissolving capability and high stability, and can ensure the continuous supply of oxygen, the maintenance of the effective activity of active oxygen and the like in the photodynamic therapy (PDT) process. PFH use also reduced HSP70 levels during treatment, reduced stress response due to temperature elevation, and increased therapeutic efficacy (example 6, fig. 29).

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

Photodynamic therapy (PDT) can be achieved using the above described protocol. PDT is a novel treatment mode with no wound, good specificity and small toxicity to normal tissues. Under the irradiation of laser, the photosensitizer is used for converting light energy into toxic Reactive Oxygen Species (ROS), important cell organs such as mitochondria are damaged by oxidizing cell membranes, so that cell death is caused, PDT has a certain damage effect on RB, and the effect on RB cell lines with poor chemotherapy and gene therapy effects is particularly remarkable. Indocyanine green (ICG) is one of the photosensitizers commonly used in PDT treatment, and after laser irradiation, ICG transitions from a low-level ground state to a high-level singlet excited state due to strong absorption peak at 780nm, and electrons in the excited state are unstable and easily release energy through various ways to return to the ground state, including radiation transition, non-radiation transition (photothermal effect), intersystem crossing to a triplet excited state (photodynamic effect), and when electrons fall back from a singlet excited state to the ground state, the energy is released in the form of heat energy or other harmful substances (such as ROS). The ICG is excited by laser to generate energy level transition, so that a synergistic effect of photothermal treatment and photodynamic treatment can be generated, and 'double striking' of tumors can be realized. ICG is often used as the only human available dye approved by the FDA in the united states for ocular fundus choroidal angiography, and for staining of the lens capsule and inner retinal limiting membrane in ophthalmic surgery, and is the most desirable choice for ophthalmic photosensitizers.

Further, 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 nano particles of the scheme have moderate particle size, are suitable for intravenous administration, and can be effectively phagocytized by tumor cells. The surface of the nanoparticle has positive charges, and has positive promotion effect on the 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 was 6mg:2mg:2mg: the concentration of oleic acid modified SPIONs was 10mg/ml at 25. Mu.L.

Further, the solvent includes chloroform and methanol. The solvent can fully dissolve and disperse the lipid such as DPPC, DSPE-PEG 2000-form, DC-chol, etc., and the lipid membrane can be obtained after the solvent is evaporated.

Further, in S3, the ratio of the amount of liquid fluorocarbon to indocyanine green used was 2mg:0.2mL. The dosage 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 preparing a diagnosis or treatment preparation of retinoblastoma.

Retinoblastoma (RB) is one of the most common ocular malignancies in infants. The conventional clinical RB inspection means comprise conventional fundus inspection, eye B-ultrasonic, CT and MRI imaging, and the traditional imaging technologies are difficult to achieve early qualitative positioning diagnosis of the RB, and the early diagnosis of the RB is difficult to be matched with the early diagnosis of the infant, so that the household economic burden of the infant is certainly increased by multiple inspections, and the early diagnosis of the RB is once in bottleneck due to the difficulty. In the aspect of clinical treatment of RB, most infants have larger tumor growth when seeing pathological changes for diagnosis, and eyeball excision is still the main treatment mode adopted. As an invasive and irreversible treatment, eyeball excision is a very destructive treatment in ophthalmic diseases, and brings great pain and mental trauma to the child and family members.

The scheme provides the multi-mode contrast agent integrating the advantages of ultrasound, optoacoustic and magnetic resonance, has the advantage of carrying out multi-mode in-vivo real-time imaging on tumor tissues at the anatomical structure, function and molecular level, and can provide more complete and accurate biological information for clinic. In addition, the nanoprobe provided by the scheme also has Photodynamic (PDT) and Photothermal (PTT) treatment. The nano probe is subjected to targeted enrichment around RB cells under the mediation of folic acid-magnetic double targeting, and phase change in a tumor area is promoted by pulse laser irradiation, so that microbubbles are generated, and photoacoustic effect is enhanced to enhance photoacoustic/ultrasonic/nuclear magnetic resonance imaging; meanwhile, the ICG converts the absorbed light energy into heat energy and generates active oxygen, so that photo-thermal synergistic photodynamic therapy under the monitoring of the trimodal molecular imaging of the RB is realized. The RB multi-mode imaging early diagnosis and the noninvasive treatment are integrated through the nano molecular probe, so that a new idea is developed for establishing an efficient, safe and visual treatment system under the imaging monitoring. Therefore, the multifunctional nano-molecular probe provided by the scheme can be applied to medical practice operation for preparing preparations for diagnosis or treatment of retinoblastoma.

In conclusion, the nano molecular probe has attractive prospect in tumor imaging and treatment. But still existThe targeting is insufficient, and the imaging and the treatment are difficult to integrate synchronously, so that the research and development of a novel multifunctional nano molecular probe realizes the integrated diagnosis and treatment of tumors and is a hot spot of current research. In view of the urgent need of Retinoblastoma (RB) in early diagnosis and noninvasive treatment, the proposal provides a folic acid-magnetic dual-targeting, liquid fluorocarbon (PFH) and indocyanine green (ICG) coated and Fe-loaded ₃ O ₄ Phase-change multifunctional nano molecular probe (Folate-CN-PFH-ICG-Fe) ₃ O ₄ FCNPIFEs, the individual performance parameters of the nanoparticles are detailed in example 2). Under the action of a magnetic field, a large amount of RB cells are enriched (see in-vivo and in-vitro targeting experiments in the embodiment 3 for details), and ultrasonic, photoacoustic and nuclear magnetic resonance images of the RB cells are obtained through photoinduced phase transition under the action of laser (see in-vivo and in-vitro tri-modal imaging experiments in the embodiment 3 for details), so that the accurate positioning of tumors is realized; at the same time, ICG converts the absorbed light energy into heat energy and generates a large amount of active oxygen to exert the synergistic therapeutic effect of light and heat and photodynamic therapy (see the experimental data related to the in-vivo and in-vitro therapeutic effects of example 4, example 5 and example 6 for details). 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 diagram of the structure of FCNPIFEs prepared in example 1 of this application.

FIG. 2 is a schematic representation of the use of FCNPIFEs prepared in example 1 of this application.

FIG. 3 is an electron micrograph of FCNPIFEs of example 2 of this application.

FIG. 4 is a graph showing the particle size distribution of FCNPIFEs of example 2 of this application.

FIG. 5 is a graph showing the potential distribution of FCNPIFEs of example 2 of this application.

FIG. 6 is a graph showing the ultraviolet absorption spectrum and standard curve of FCNPIFEs of example 2 of the present application.

FIG. 7 shows the standard curve and hysteresis curve of Fe for FCNPIFEs of example 2 of this application.

FIG. 8 shows the results of the experiments of the magnetic property detection, the in vitro phase change property detection and the oxygen carrying capacity detection of FCNPIFEs of example 2 of this application.

FIG. 9 shows the confocal microscopy observations (3 h) of the in vitro targeting experiment of example 3 (1) of the present application.

FIG. 10 shows the results of a typical flow cytometry test (3 h) of the in vitro targeting experiment of example 3 (1) of the present application.

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

Fig. 12 is a signal intensity-nanoparticle concentration curve for in vitro ultrasound/photoacoustic/magnetic resonance trimodal imaging of example 3 (2) of the present application.

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

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

FIG. 15 shows the results of in vivo multi-modality imaging study of FCNPIFEs molecular probes of example 3 (4) of the present application.

FIG. 16 shows the experimental results of in vitro photothermal effect studies of FCNPIFEs molecular probes of example 4 of the present application.

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

FIG. 18 shows the results of in vitro ROS production effects of FCNPIFEs molecular probes of example 4 of the present application (laser confocal experiment).

FIG. 19 shows the results of in vitro ROS production assay (flow cytometry assay) of FCNPIFEs molecular probes of example 4 of this application.

FIG. 20 shows the in vitro biosafety study result (CCK-8) of the FCNPIFEs molecular probe of example 4 of the present application.

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

FIG. 22 is a graph showing the temperature change at a tumor site treated in vivo according to example 5 of the present application.

FIG. 23 is a photograph of a tumor of a mouse treated in vivo according to example 5 of the present application.

FIG. 24 is a graph showing the tumor volume change, tumor inhibition rate and mouse weight change of the in vivo treatment according to example 5 of the present application.

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

FIG. 26 is a graph showing the blood oxygen saturation of tumor tissue before and after irradiation of different experimental groups under magnetic field in example 6 of the present application.

FIG. 27 is a HIF-1. Alpha. Fluorescence staining micrograph of tumor tissue of example 6 of the present application.

FIG. 28 is a microscopic image of HSP 70. Alpha. Fluorescence staining of tumor tissue of example 6 of the present application.

FIG. 29 shows the results of quantitative analysis of HSP70 expression level in example 6 of the present application.

FIG. 30 shows the biosafety evaluation result (tissue staining) of example 7 of the present application.

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

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. Unless otherwise indicated, 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 are all commercially available.

Wherein, main reagent includes:

DPPC is known as 1, 2-dioxadecanoyl-rac-glyco-3-phoshocoloine;

DSPE-PEG (2000) -plate (DSPE-PEG 2000-FA) is fully known as:

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

the SPIONs are all called: oleic acid modified Fe ₃ O ₄ Nanoparticle (OA@Fe) ₃ O ₄ )；

The above reagents were all purchased from sienna ruixi (china).

Indocyanine green (ICG), perfluorohexane (PFH) and Dc-cholesterol (DC-chol) were all purchased from Sigma (USA).

Example 1: preparation of FCNPIFEs molecular probes

Weighing a certain amount of DPPC (6 mg), DSPE-PEG2000-FA (2 mg), DC-chol (2 mg), oleic acid modified SPIONS 25 μL (main component is Fe) ₃ O ₄ 10 mg/mL) was placed in a round bottom flask, 5mL of chloroform was added and the solution was stirred with a magnetic stirrer until it became clear. A brown lipid film was formed by rotary evaporation using a rotary evaporator (50 ℃ C., 2 h), and then hydrated with 2mL PBS. Simultaneously, PFH (0.2 mL) and ICG (2 mg) were mixed and emulsified (2 min, 35%. Times.130W, on 5s, off 5s,Heat System Inc,USA) on ice with an ultrasonic sonicator to obtain a first emulsion. Adding the hydrated brown lipid film into the first emulsion, and performing a second ultrasonic treatment (6 min,40% multiplied by 130W, on 5s and off 5 s) to obtain a second emulsion. Finally, centrifuging the second emulsion at 8000rpm for 5min for three times to obtain Folate-CN-PFH-ICG-Fe ₃ O ₄ (FCNPIFEs) and stored in a refrigerator at 4 ℃ for standby.

Besides FCNPIFEs, FNNFEs (Folate-NN-PFH-ICG-Fe) are also prepared ₃ O ₄ ) I.e., the equivalent amount of DC-chol is replaced by common cholesterol (the surface potential of the nanoparticle is-25.325 mV, i.e., the surface is negatively charged). FCNPIs (Folate-CN-PFH-ICG, without SPIONS) and CNPIFEs (NN-PFH-ICG-Fe) are also prepared ₃ O ₄ Substitution of DSPE-PEG2000 with DSPE-PEG 2000-FA), CNPIs (NN-PFH-ICG, substitution of DSPE-PEG2000-FA with DSPE-PEG2000 without SPIONS), FCNPFEs (Folate-CN-PFH-Fe) ₃ O ₄ Without ICG) and FCNIFEs (Folate-CN-ICG-Fe ₃ O ₄ No PFH was added). And incubating the fluorescent dye DiI and the nanoparticles together to construct a fluorescent labeling molecular probe for in-vivo and in-vitro targeting detection and in-vivo distribution detection.

The schematic structure of the FCNPIFEs prepared in this example is shown in FIG. 1, and the efficacy and use of the FCNPIFEs are shown in FIG. 2. FCNPIFEs of the present protocol can be administered intravenously into the blood circulation, reach the tumor tissue, and enter the tumor tissue via EPR effect. In addition, the FCNPIFEs in the technical scheme are multifunctional phase-change nano molecular probes with double targeting advantages, can penetrate through tumor vascular endothelial gaps, target and gather to extravascular tumor cells, liquid fluorocarbon generates liquid-gas phase change under the action of laser with certain energy, so that the multi-mode imaging effect is enhanced, and the excited indocyanine green converts light energy into heat energy and generates active oxygen, plays a photo-thermal synergistic photodynamic treatment effect, improves the diagnosis and treatment effect, reduces systemic toxic and side effects, and provides a new platform for realizing the imaging and noninvasive treatment of RB at the molecular level. The FCNPIFEs of the scheme combine the specificity of molecular targeting with the high efficiency of magnetic targeting, and are molecular probes of double-targeting RB, so that the targeting efficiency is improved.

Example 2: characterization of FCNPIFEs molecular probes

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

Encapsulation efficiency and drug loading detection: detection of ICG and Fe in molecular probe by ultraviolet spectrophotometer ₃ O ₄ Encapsulation efficiency and drug loading rate. See fig. 6 for ICG measurements. FCNPIFE shows a peak at 778 nm. The standard curve regression equation of the ICG is measured by ultraviolet spectrophotometry, and the (93.09+/-0.63)%, and the drug loading (15.51+/-0.11) mg is calculated. Wherein, ICG encapsulation efficiency (%) = (total ICG mass-free ICG mass)/total ICG mass×100%; ICG drug loading (%) = (total ICG mass-free ICG mass)/nanoparticle total mass x 100%.

For Fe ₃ O ₄ See fig. 7 left and hysteresis curves see fig. 7 right. And (3) preparing an Fe standard curve, and calculating to obtain the Fe encapsulation rate of (4.37+/-0.80)%, and the drug loading rate of (0.0026+/-0.05)%. Wherein Fe is ₃ O ₄ Encapsulation efficiency (%) =fe in nanoparticles ₃ O ₄ mass/Fe of (2) ₃ O ₄ Total input x 100%; fe (Fe) ₃ O ₄ Drug loading (%) =fe in nanoparticles ₃ O ₄ Is multiplied by 100% of the mass per nanoparticle total mass.

And (3) magnetic performance detection: t magnetic properties of FCNPIFEs at 300K were studied using a vibrating sample magnetometer (vibrating sample magnetometer, VSM). The FCNPIFEs solution is placed in a glass bottle, and the aggregation condition of nanoparticles under a magnetic field (4T) for 6 hours is observed, and the experimental result is shown in the left part of FIG. 8.

In vitro phase change performance detection: using confocal microscopy, FCNPIFEs were observed at 808nm laser (1.5W/cm ² 5 min), the experimental results are shown in fig. 8, which illustrates that the nanoparticles of the present solution can undergo phase transition under the excitation of laser.

Oxygen carrying capacity detection: after the oxygen carrying capacity of the nanoparticle is detected, FCNPIFEs, FCNIFEs and PBS are excited to generate oxygen for 10min, 1ml of the substance to be detected is added into deaerated water, water bath is carried out at 47 ℃ for 10min, a portable dissolved oxygen monitor is used for detecting the oxygen content in the water, and the experimental result shows that PFH can effectively store oxygen, and the detailed view is shown in the right of fig. 8. At 48℃ambient temperature, the concentration of dissolved oxygen in the FCNPIFEs experimental set increased rapidly from 3.21 to 13.20mg/ml over 60s and maintained higher levels over 10min, compared to FCNIFE and PBS. By heating to burst FCNPIFEs, the oxygen stored in PFH overflows in large amounts, rapidly increasing dissolved oxygen levels occur, and over time, the dissolved oxygen levels decrease and maintain equilibrium levels. The above data demonstrates that PFH has desirable oxygen carrying capacity, can provide oxygen for PDT and enhance PDT action.

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

(1) FCNPIFEs molecular probe in-vitro targeting performance detection

In vitro culture of RB cells: human RB Y79 cell line in 1640 culture solution containing 10% fetal bovine serum, 5% CO ₂ Conventional culture passaging at 37 ℃.

In vitro targeting experiments: according to the previous method, CNPIFE, FCNPIFECNPI, CNPI and FNNFFE were Dil stained and seeded with Y79 cells into 24-well plates (2X 10 per well, respectively ⁵ Individual cells) into 7 groups: control group, CNPI, FCNPI+folic Acid (FA),Cnpipe+magnetic field (M.F), FCNPI, FNNPIFE +magnetic field, fcnpife+magnetic field. Nanoparticle concentrations were all 0.4mg/ml, the magnetic field was completed by a 4T magnet attached to the side of the well and the cells were incubated for additional 1, 3 and 6 hours. After the incubation with nanoparticles was completed, cells were fixed with paraformaldehyde for 15 minutes, and then incubated with DAPI for 15 minutes to stain nuclei, and then incubated with DiO for 10 minutes to stain cell membranes. Intracellular uptake behavior of each group of nanoparticles was observed by confocal, and each group was quantitatively analyzed by flow cytometry. Confocal observations are seen in fig. 9 (scale bar 25 μm), flow cytometry detection is seen in fig. 10, phagocytosis statistics are seen in fig. 11 and table 1 (phagocytosis is the percentage of Y79 cells phagocytosed with nanoparticles to total cell number, and is the quantitative measurement result using flow cytometry, n=3, ×p < 0.01, ×p < 0.0001). In vitro targeting experiments prove that the effect of the three targeting groups (folic acid targeting, magnetic targeting and cations) is strongest at 3h, and the signal is strongest (6 h is needed for other groups) as compared with other groups, and the phagocytosis rate is better than that of other groups. In fig. 11, it can be observed that at 3h, the phagocytosis rate (percentage of Y79 cells detecting fluorescent signal) of nanoparticles was significantly lower for the fnnppe+magnetic field group (nanoparticle surface non-positive charge), the FCNPI group (no magnetic targeting) and the cnpipe+magnetic field group (no leaf acid targeting) for the three experimental groups. This suggests that three factors, folic acid targeting, magnetic targeting and DC-chol (positive charge), are indispensable for increasing the targeting of nanoparticles, and there is a synergistic effect between the three. Again, with detailed analysis of the data in table 1, the phagocytosis rate of fcnpife+ M.F group had reached a peak of 95.08±0.88% at 3h, and the peak was consistently maintained to 6h (96.69 ±0.32%) while the phagocytosis rate of the other experimental groups at 3h was much lower than that of fcnpife+ M.F group, and even after 6h was much lower than that of fcnpife+ M.F group. Nanoparticles in the CNPIs group lack folic acid targeting and magnetic targeting, but the surface charge is positive, and the phagocytic rate is 25.76+/-0.15% when the surface charge is 3 hours; nanoparticles in the FNNFEs+ 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% at 3 hours; the sum of phagocytosis rates of CNPIs group (surface positive charge single targeting) and FNNFEs+ M.F group (folic acid and magnetic double targeting) (about 80)36%) is still smaller than the phagocytic rate of the fcnpifes+ M.F group (three targeting), the effect of the three targeting is not a mere superposition of the respective effects of the three factors, further confirming the synergistic effect between the three targeting. At 3h, the phagocytosis rate of the CNPIFE+ 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 rate of the CNPIFE+ M.F group (folic acid targeting+surface cations) is far lower than that of the FCNPIFEs+ M.F group (three targeting), which also shows that the folic acid targeting and the magnetic targeting have a synergistic effect. And compared with the FNNFEs+ M.F group (the phagocytic rate is 54.60 +/-9.65%), the synergistic effect between the folic acid targeting and the magnetic targeting is not obvious under the condition of no positive ions on the surfaces of the nano particles although the synergistic effect exists between the folic acid targeting and the magnetic targeting. The analysis of the data in table 1 further illustrates that the nanoparticles of the present approach use a three-targeting combined approach, allowing the nanoparticles to be rapidly enriched in tumor cells; and the effect of three targets is not simply added, but a synergistic effect of 1+1+1 > 3 is generated.

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

(2) FCNPIFEs in vitro multimodal imaging study

In vitro imaging: to assess the in vitro PA function of FCNPIFEs, samples were diluted (0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) and added to agar gel molds for PA imaging, recorded and analyzed by the VEVO laser PA imaging system, according to ICG concentration. In vitro ultrasound function was investigated by MyLab90, and FCNPIFEs liposomes were diluted (0, 0.4, 0.8, 1.2 and 2.4 mg/ml) and exposed to 808nm laser (2 w/cm) ² 5 min), the signal intensity after laser excitation was analyzed using DFY (institute of ultrasound imaging at university of Chongqing medical science, chongqing in 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 0.24 mM) and placed in EP tubes using Philips Achieva 3.A 0T TX MR scanner (philips medical system, netherlands) acquires T2WI images and analyzes the Signal Intensity (SI) of a region of interest (ROI) by Sante DICOM, the T2 parameters are as follows: tr=161ms, te=14.60 ms, field=3t, dfov= 250mm,slice hickness =3.0 mm. From in vitro imaging experiments, FCNPIFE can perform ultrasound/photoacoustic/magnetic resonance three-mode imaging simultaneously, and the signal intensity has obvious linear correlation with the nanoparticle concentration (fig. 12).

(3) Detection of in vivo distribution and targeting ability of FCNPIFEs molecular probes

Establishing a nude mouse RB subcutaneous planting tumor model: the cell concentration was adjusted to 5X 10 using RB Y79 cells in logarithmic growth phase ⁷ Per ml, 0.2 ml/nude mice inoculated subcutaneously on the dorsum of buttocks of 3-4 week old BALB/c nude mice. Two weeks or so, the back of the buttocks of the nude mice grow tumor to about 1cm multiplied by 1cm for standby.

In vivo targeting experiments: y79 tumor-bearing mice were randomly divided into 6 groups (the amount of tail vein injected nanoparticles was 200 μl,0.4mg/mL, n=3): CNPIs (i), cnpifs (ii), cnpifs+magnetic field (iii), FCNPIs (iv), fnnpis+magnetic field (v), fcnpifes+magnetic field (vi), fluorescent images (0, 3,6, 24 h) at different time points after nanoparticle injection were obtained using a small animal in vivo fluorescence assay (Fx 7 IR Spectra, vilber Lourmat, france) by applying a magnetic field to a stationary magnet (4T) above the tumor. Finally, tumors and major organs of mice were collected for fluorescence imaging and corresponding fluorescence signals were analyzed.

Fluorescence imaging of Y79 tumor-bearing mice (fig. 13 and 14) further validated the targeting advantage of fcnpifes+ magnetic fields. Fig. 14 is a graph showing fluorescence intensity statistics (n=3, ×p < 0.0001) of nanoparticles in different organs and tumor tissues 24h after injection, and table 2 shows specific fluorescence intensity statistics. Referring to the left panel of fig. 13, the fcnpifes+ magnetic field set began to fluoresce at the tumor region (circled in the figure) 3h after injection, peaking at 6h and exhibiting higher fluorescence energy within 24 h. The FNNFFEs+ magnetic field is inferior in appearance, weak fluorescent signals are observed in tumor areas at 6h, the signals reach the maximum at 24h, and the rest groups detect weak signals or even no signals at the tumor parts at 24 h. Experimental results show that the three-targeting nanoparticles can be accumulated at the tumor part more quickly, and the targeting treatment effect is realized. The biodistribution of the relevant nanoliposomes was shown by imaging the major organs and tumors (right in fig. 13) and each fluorescence intensity was quantified using a fluorescence analysis system (fig. 14 and table 2). Except for the FCNPIFEs+ magnetic field group spleens, each group has obvious accumulation of liver and spleen. This is due to the general metabolic pathways by which the uptake behavior of the reticuloendothelial system proceeds in vivo. As shown by experimental results, the FCNPIFEs nanoparticle has optimal safety and minimal accumulation in non-tumor parts. Referring to fig. 13, 14 and table 2, since the fcnpifes+magnetic field group had a signal peak at 6h, the signal was significantly reduced at 24h, and the fnnpifes+magnetic field group reached maximum intensity at 24 h. This demonstrates that the nanoparticles FCNPIFEs of the present approach can rapidly achieve therapeutic effects in vivo, and that the nanoparticles can be metabolized and cleared in a shorter time, with higher biosafety relative to FNNPIFEs. In summary, FCNPIFEs not only have minimal damage to major organs, but also accumulate most efficiently in tumors.

Table 2: in vivo fluorescence intensity statistics (mean±sd, n=3)

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

Y79 nude mice were subjected to in vivo photoacoustic imaging, and were divided into four groups (fcnpifes+magnetic field, CNPIs, fcnifes+magnetic field, fcnpfes+magnetic field), tail intravenous nanoparticles (200 μl,0.4mg/mL, n=3), and the test results were shown in the lower two figures of fig. 15 by capturing images at different time points (0, 3,6, 24 h) using the VEVO laser PA imaging system by applying a magnetic field to a stationary magnet (4T) above the tumor. Wherein, the lower left is a typical PA image of tumor sites of tumor-bearing mice, and the lower right is a PA intensity statistical graph of tumor sites at different time points (n=3, ×p < 0.0001).

In vivo ultrasonic imaging is carried out on the nano particles, and the nano particles are divided into the following groups: fcnpifes+magnetic field, CNPIs, cnifes+magnetic field (200 μl,0.4mg/mL, n=3), and sound intensity values were analyzed using DFY software. The experimental results are shown in the upper two graphs of FIG. 15. Wherein, the upper left is a typical ultrasonic image of tumor site of tumor-bearing mice, the upper right is an echo intensity statistical graph before and after 808nm laser irradiation (n=3, ×p < 0.001, ×p < 0.0001).

To examine the in vivo MR imaging effect of nanoparticles, nude mice were divided into 2 groups: fcnpifes+magnetic field, FCNPIs (200 μl,0.4mg/mL, n=3), T2WI images were recorded at different time points (0, 3,6, 24 h) using a philips Achieva 3.0T TX MR scanner, and ROI signals were analyzed using Sante DICOM. The T2 weighted image is obtained using the following parameters: tr=5560 ms, te=85 ms, field=3t, dfov= 80mm,slice thickness =0.7 mm. The experimental results are shown in the middle two graphs of FIG. 15. Wherein, the middle left is a typical T2 image of tumor sites of tumor-bearing mice, the middle right is a statistical graph of signal intensity of tumor sites at different time points (n=3, p < 0.05, p < 0.0001).

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

Example 4: research on in vitro photo-thermal effect (PTT), photodynamic effect (PDT) and cytotoxicity of FCNPIFEs molecular probes

PBS, FCNPFEs (0.4 mg/mL) and FCNPIFEs (0.05, 0.1, 0.2, 0.4 mg/mL) at different concentrations were irradiated with 808nm laser for 5min (1.5W/cm) ² ) The experimental results are shown in fig. 16, wherein the upper left is a thermal energy image recorded per minute and the upper right is a temperature profile over time. And laser beams (0.5,1,1.5,2.0W/cm) ² ) The FCNPIFEs solution (0.4 mg/ml) was irradiated for 5min. 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 profile over time. The thermal infrared imaging camera was used to record the temperature change per second of the solution to be tested and to take images per minute.

Next, photo-thermal stability studies on FCNPIFEs (0.4 mg/mL) were performed using 808nm (1.5W/cm) ² ) The experimental results, see left of fig. 17, of 5 cycles of laser irradiation, demonstrate that FCNPIFEs are good in photo-thermal stability.UV-vis-NIR spectra (5 min, 1.5W/cm) of FCNIFEs (0.4 mg/mL) and ICG (0.4 mg/mL) were measured for FCNPIFEs (0.4 mg/mL) ² ) The experimental results are shown on the right of fig. 17.

The DCFH-DA method was used to study the condition of the cells producing ROS. Y79 cells (2X 10) ⁵ Individual cells per well) were incubated with FCNPIFEs (0.4 mg/ml), FCNIFEs (0.4 mg/ml) and ICG (0.4 mg/ml) in 24-well plates for 3h and a 4T magnetic field was applied, followed by 808nm laser (1.5W/cm) ² ) Irradiating for 5min. DCFH-DA was then added to the cells and incubated for 30min, and finally the ROS production was observed using confocal microscopy and quantitated using a flow cytometer. Wherein the anoxic group was placed in an anaerobic box throughout the experiment. Fluorescence confocal images (scale bar 100 μm) are seen in fig. 18, flow cytometry analysis results are seen on the left of fig. 19, and quantitative analysis results are seen on the right of fig. 19. The fluorescence confocal experiment result shows that: FCNPIFEs produce similar amounts of ROS by PDT action in anaerobic and aerobic environments; whereas for FCNIFEs and free ICGs, there is a difference in the amount of ROS produced by PDT action, both in anaerobic and aerobic environments. PFH is a good oxygen carrying substance, and flow cytometry experimental results also confirm the laser confocal experimental results.

In vitro safety of FCNPIFEs was tested using CCK-8 method, Y79 and ARPE-19 cells were seeded in 96-well plates (1X 10) ⁴ Individual cells per well), after 24h of culture, 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, CCK-8 assays were performed and absorbance at 450nm was measured for each well with a microplate reader. The experimental results are shown in fig. 20, and the cell viability is greater than 90%, which indicates that the biosafety of the nanoparticles is high without laser irradiation. The Y79 cells are subjected to laser irradiation for 5min, and then CCK-8 experiments are carried out, and the experimental results are shown on the right side of FIG. 20, so that the nanoparticles have ideal cell killing effect by applying the laser irradiation.

And carrying out in vitro treatment effect experiments on the nanoparticles. Y79 cells were seeded in 96-well plates (5X 10) ⁴ Individual cells per well), after 24h of incubation, the cells were divided into 8 groups and subjected to irradiation treatment (806 nm,2w,5 min): blank controls (normoxic and anoxic), PTT group (normoxic and anoxic), PDT group (normoxic and anoxic), ptt+pdt group (normoxic and anoxic). In PDT treatment, culture plate is ice-bath to avoid photo-thermal effect, naN is added in PTT treatment ₃ (100 mM) to avoid photodynamic effects. Quantitative flow cytometry analysis results referring to fig. 21, demonstrate that ptt+pdt treatment can increase the cell killing capacity of nanoparticles.

Example 5: PDT and PTT in vivo synergistic treatment effect experiment

When the tumor of the Y79 tumor-bearing mice grows to 100mm ³ Mice 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), (IX) FCNPIFEs+ M.F +laser group. The laser conditions of group (VIII) are: 808nm, 1.5W/cm ² On 30s/off 30s, 10min to ensure that the temperature is at 41 ℃ to avoid excessive temperature, is considered a single PDT treatment. The laser conditions adopted in the group (II), the group (IV), the group (V), the group (VI), the group (VII) and the group (VIII) are as follows: 808nm, 1.5W/cm ² And (5) 10min. Nanoparticles were administered by tail vein injection (200 μl,0.4mg/mL, n=3), and after 6h the tumor sites were irradiated with laser light. Thermal imaging and temperature change were recorded by a thermal infrared imager, tumor volume and weight of mice were recorded every two days, and tumor suppression rate was calculated. Hematoxylin and Eosin (HE) of tumors and major organs were collected the third day after treatment, and Proliferating Cell Nuclear Antigen (PCNA) and terminal deoxynucleotidyl transferase mediated dUTP-biotin notch end marker (TUNEL) staining to assess histological changes and apoptosis levels.

The temperature change at the tumor sites of the different treatment groups is shown on the left of fig. 22, and the temperature change at the tumor sites of the (viii) group is shown on the right of fig. 22. In the free icg+ laser treated group, only a modest temperature rise was observed. Whereas in the group for photodynamic therapy the temperature was kept below 41 ℃ to avoid photothermal effects. For mice irradiated with continuous irradiation a significant local increase in temperature was expected, with maximum temperatures reaching approximately 47℃and 47.5℃respectively. These two similar temperature curves mean that there is no significant difference in the photo-thermal efficiencies of FCNPIFE and FCNIFE. In the fcnpifes+laser group, the tumor gradually decreased. In other groups, either the treatment is poor or the tumor recurs. After different treatments, tumors were extracted. The tumor volume group of fcnpifes+lasers gradually decreased throughout the observation period and the treatment effect was significantly better than the other groups. Consistent with previous results, the tumor weight of FCNPIFEs+lasers was significantly lower than that of other groups, and mice varied little in weight during the study (FIG. 23), suggesting that FCNPIFEs+lasers combination was safe and effective. In fig. 3, the left graph shows the tumor volume (V/V0) versus time, the middle graph shows the tumor inhibition ratio (n=3, ×p < 0.0001, ×p < 0.001), and the right graph shows the body weight change of 16 days mice. HE. Microscopic images of tumor tissue sections after PCNA and TUNNEL staining are shown in FIG. 24. Furthermore, the tumor weight inhibition was further verified for the anti-tumor effect of the different treatments. Consistent with expectations, the tumor weight inhibition rate of the fcnpifes+laser group was highest among all groups.

Example 6: PDT and PTT synergy mechanism research 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): normal saline, fcnifes+magnetic field (M.F), fcnpifes+magnetic field. After injecting the nanoparticles (200. Mu.L, 0.4 mg/mL) for 6 hours, the nanoparticles were irradiated with 808nm laser for 10min (1.5W/cm) ² ) Before and after injecting each group of nano particles for 24 hours, detecting the change of blood oxygen saturation of the tumor part by using a blood oxygen mode of a Vevo Laser photoacoustic imager, taking tumor tissues to carry out HIF-1 alpha staining, and detecting the improvement condition of a molecular probe on the hypoxia state of the tumor part. Fig. 25 shows the blood oxygen saturation images of tumor tissues before and after irradiation of different experimental groups under a 4T magnetic field on the left, and fig. 26 shows the quantitative measurement result of the ratio of oxyhemoglobin (λ=850 nm) to deoxyhemoglobin (λ=750 nm) at the tumor tissues on the right. FIG. 27 shows HIF-1. Alpha. Fluorescent staining microscopy images of tumor tissue (day after irradiation treatment). The experimental results show that in the FCNPIFEs+ magnetic field group, the content of oxyhemoglobin in tumor tissues is increased from 33.34+/-2.91% to 74.21+/-7.08% before treatment, and further show that PFH has good oxygen extracting capacity in an anoxic environment.

In order to investigate the effect of photodynamic effects on photothermal effects, the inventors have further investigated the expression level of HSP70 in tumor tissue. Tumor-bearing mice were divided into 5 groups (n=3): normal saline group, fcnpis+magnetic field, fcnpifes+magnetic field, fcnifes+magnetic field+laser, and fcnpifes+magnetic field+laser. The processing mode of the laser processing group is the same as that of the previous section. And detecting the expression condition of heat shock protein HSP70 at the tumor part by an immunofluorescence staining method and a western immunoblotting method, and further confirming whether the enhanced photodynamic effect has a promoting effect on the photothermal effect. Fig. 28 shows HSP70 a fluorescent staining microscopic image of tumor tissue (day after irradiation treatment), and fig. 29 shows the quantitative analysis result of HSP70 expression level (proportion of HSP 70-containing region). The test results show that: the increased level of HSP70 will cause the cells to develop resistance to heat, the proportion of HSP 70-containing regions of the fcnpifes+magnetic field+laser group is down-regulated by approximately 19.33% (relative to fcnifes+magnetic field+laser), suggesting that nanoparticles using this approach may reduce HSP70 expression by irreversibly destroying mitochondrial metabolism related substances by ROS generated by photodynamic action, thereby promoting killing of cancer cells, further explaining the synergistic principle of PDT and PTT.

Example 7: biosafety study of FCNPIFEs molecular probes

The mice tested in vivo in example 5 were taken for tissue sections and safety assessment. To assess the in vivo safety of FCNPIFE, 12 female nude mice were subjected to blood routine and serum biochemical tests by tail vein injection of FCNPIFEs (200 μl,0.4mg/mL, n=3) followed by collection of blood samples (n=3) at different time points (control, 1d, 7d, 14 d).

According to the in vivo safety results, no obvious abnormality appears in the blood biochemical indexes of the mice before and after the FCNPIFEs are injected (figure 31), which shows that the FCNPIFEs have good biological safety. After the end of in vivo treatment, H & E staining of major organs including heart, liver, spleen, lung, kidney (fig. 30) showed no apparent physiological abnormalities, indicating negligible toxicity before and after treatment.

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content 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 membrane ₃ O ₄ And folic acid; the components of the lipid shell membrane comprise DPPC, DSPE-PEG 2000-form and DC-chol; the lipid shell membrane is internally wrapped with perfluorohexane and indocyanine green;

the multifunctional nano molecular probe is prepared by the following method:

s1: adding DPPC, DSPE-PEG 2000-focus, DC-chol and oleic acid modified SPIONs into a solvent, uniformly mixing, and evaporating the solvent to obtain a lipid membrane;

s3: and dropwise adding perfluorohexane and indocyanine green into the emulsion, and performing sound vibration emulsification to obtain the multifunctional nano molecular probe.

2. The multifunctional nanomolecular probe of claim 1, wherein: the particle size is 338.63 + -10.90 nm, and the potential is 31.86+ -3.49 mV.

3. The method for preparing a multifunctional nano-molecular probe according to any one of claims 1 or 2, wherein: the method comprises the following steps of:

s3: and dropwise adding perfluorohexane and indocyanine green into the emulsion, and performing sound vibration emulsification to obtain the multifunctional nano molecular probe FCNPIFEs.

4. A method for preparing a multifunctional nano-molecular probe according to claim 3, wherein: in S1, the dosage ratio of DPPC, DSPE-PEG2000-Folate, DC-chol and oleic acid modified SPIONs was 6mg:2mg:2mg: the concentration of oleic acid modified SPIONs was 10mg/ml at 25. Mu.L.

5. A method for preparing a multifunctional nano-molecular probe according to claim 3, wherein: in S1, the solvent includes chloroform and methanol.

6. A method for preparing a multifunctional nano-molecular probe according to claim 3, wherein: in S3, the dosage ratio of perfluorohexane to indocyanine green is 2mg:0.2mL.

7. Use of a multifunctional nanomolecular probe according to any one of claims 1 or 2 for the preparation of a diagnostic or therapeutic formulation for retinoblastoma.