Targeting artificial protein crown modified < 19 > F magnetic resonance nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a < 19 > F MRI (Magnetic Resonance Imaging) composite nano contrast agent based on a targeting artificial protein crown as well as a preparation method and application thereof. According to the invention, folic acid is modified on plasma protein, and the modified plasma protein has folic acid receptor mediated specific targeting and selective tumor cell uptake by utilizing the overexpression characteristic of a folic acid receptor (FR) on the surface of a cancer cell. Meanwhile, by taking 20-fluoro-15-crown-5-ether as a magnetic resonance active molecule, packaging by using polylactic acid-glycolic acid, and synthesizing PLGA-PFCE nano-particles as the < 19 > F MRI nano contrast agent. The PLGA-PFCE nanoparticles and the modified plasma protein are co-incubated in vitro, an artificial protein crown is formed on the surface of the PLGA-PFCE nanoparticles, and the obtained 1PLGA-PFCE nanoparticle-targeting artificial protein crown compound directly realizes targeting to tumors through targeting small molecules on the surface of the protein crown; moreover, the characteristic that the targeting effect is reduced due to the fact that a common nano material adsorbs a protein crown after entering a living body can be overcome, and the signal intensity of < 19 > F MRI tumor in-situ imaging can be improved after the nano material is enriched to the tumor through intravenous injection.

Description

Technical Field

[0001] The present invention belongs to the technical field of magnetic resonance contrast agents, and in particular relates to a composite nanoparticle based on a targeted artificial protein corona, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide. In the later stages of cancer, the effects of existing treatments are quite limited. Therefore, strengthening early diagnosis of cancer and developing non-invasive diagnostic tools can effectively improve the survival rate of patients and are of great significance to the treatment and prognosis of cancer. In the diagnosis of cancer, magnetic resonance imaging (MRI) technology is widely used in clinical practice due to its advantages such as non-invasiveness, no ionizing radiation, and no tissue depth limitation. However, traditional proton-based imaging technology is still not widely used in the clinic. 1 H magnetic resonance imaging has problems such as background signal interference and contrast agent accumulation toxicity. 19 F atoms have the advantages of natural abundance up to 100%, gyromagnetic ratio is very close to that of hydrogen atoms, and the content in the human body is extremely low so there is no endogenous background signal interference. 19 F atoms as imaging nuclei 19 F magnetic resonance imaging technology has now gained wide recognition in the field of cancer diagnosis.

[0003] However, 19 F MRI contrast agents themselves do not have the ability to actively target tumor sites and cannot be enriched in tumor sites. At the same time, they are limited by the dosage and concentration in actual clinical applications, which makes them less sensitive in in situ tumor imaging. This problem greatly limits their clinical application prospects. One of the key ideas to solve this problem is to develop F MRI contrast agents that can target tumor sites. 19 F MRI nanocontrast agent, which has the function of targeting tumors, thereby promoting 19 After entering the body, F MRI nanocontrast agents are enriched at the tumor site to improve the sensitivity and signal intensity of in situ tumor imaging.

[0004] After entering the biological environment and coming into contact with biological media (such as serum or plasma), nanomaterials will rapidly adsorb macromolecules, mainly proteins, and form a protein corona (PC) on their surface. The formation of the protein corona has a complex impact on the biological properties and pharmacokinetics of nanomaterials in vivo. At the cellular level, it is known that nanomaterials enter cells specifically or non-specifically through receptor-mediated endocytosis or endocytosis. After the formation of the protein corona, the nanomaterials actually interact with the cell surface through the proteins adsorbed on the surface. Therefore, the formation of the protein corona reshapes the behavior of nanomaterials in the body, and then dominates their interaction with biological macromolecules, cells and tissues in the body. To a certain extent, the protein corona gives nanomaterials a new identity, and its function determines the biological properties of nanomaterials in the body.

[0005] For nanomaterials with targeting functions, the formation of a protein corona will shield the original surface properties of the nanomaterial, which may interfere with and inhibit the targeting functional molecules on the surface of the nanomaterial, causing the nanomaterial to move in the body in a manner inconsistent with the design, resulting in reduced targeting effect or even off-target effects. At the same time, at the systemic level, the binding of opsonin proteins (such as immunoglobulins, fibrinogen, and complement proteins) increases the chances of nanomaterials being recognized by the mononuclear phagocytic system (MPS) through receptor-mediated internalization, thereby promoting faster clearance of nanomaterials from the body.

[0006] Therefore, the formation of protein corona is considered to be one of the main reasons for the inconsistent behavior of nanomaterials in vivo and in vitro. Most nanomaterials still have the problems of premature clearance by the mononuclear phagocyte system (MPS) and unsatisfactory targeting effect, which makes it difficult for nanomaterials to achieve clinical transformation. Summary of the Invention

[0007] Based on the above prior art, the present invention provides a method based on a targeted artificial protein corona. 19 F MRI composite nano contrast agent and its preparation method and application, the present invention is to carry out tumor targeting functionalization of plasma protein and then combine it with PLGA-PFCE nanoparticles ( 19 F MRI nanocontrast agents) are preformed into composite nanoparticles with targeted functionalized protein crowns, which enable them to accumulate at the tumor site to achieve tumor-specific targeting. 19F MRI imaging. In addition, due to the property of nanoparticles to spontaneously adsorb proteins to form a protein corona after entering the organism, compared with direct targeted modification on the surface of PLGA-PFCE nanoparticles, composite nanoparticles with artificial protein coronas with targeted functionalization will no longer form a protein corona after entering the organism, so their targeting function is not affected after entering the organism; while directly targeting functionalization on the surface of PLGA-PFCE nanoparticles will cause the formation of a protein corona after entering the organism, which will cause the targeting functional groups on its surface to be shielded by the protein corona, thereby reducing its targeting effect. Therefore,

[0008] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0009] A composite nanoparticle based on a targeted artificial protein corona, the composite nanoparticle having a three-layer core-shell structure, wherein the inner core is 20-fluoro-15-crown-5-ether, the middle layer is a polylactic acid-glycolic acid copolymer layer, and the outermost layer is a protein corona with targeted functionalization by chemically modifying folic acid.

[0010] Furthermore, the outermost protein corona with targeted functionalization is obtained by spontaneous adsorption of targeted functionalized proteins by nanoparticles.

[0011] Furthermore, the protein with targeted functionalization is a plasma protein modified by folic acid through covalent coupling.

[0012] A method for preparing composite nanoparticles based on a targeted artificial protein corona comprises the following steps:

[0013] S1, dissolving poly(lactic acid-co-glycolic acid) copolymer in dichloromethane, adding eicosine-15-crown-5-ether and polyvinyl alcohol aqueous solution thereto, and dispersing them uniformly to obtain an emulsion;

[0014] S2. stirring the emulsion to volatilize the dichloromethane, centrifuging the residue, discarding the supernatant, washing the resulting precipitate with ultrapure water, and then dispersing it in ultrapure water to obtain a PLGA-PFCE nanoparticle solution;

[0015] S3. Dissolving folic acid in dimethyl sulfoxide, and adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain a mixed solution, stirring the mixed solution at room temperature for 8-12 hours in the dark to obtain a folic acid active intermediate solution;

[0016] S4. Adjust the pH value of the folate active intermediate solution to 7.2-7.6 with PBS buffer, then add plasma, and stir for 8-12 hours in a dark environment to allow the folate active intermediate to fully react with the plasma protein. After the reaction is completed, dialyze the resulting mixed product, and concentrate the dialyzed solution to obtain a folate-modified protein solution;

[0017] S5. Mix the folic acid-modified protein solution and the PLGA-PFCE nanoparticle solution, shake and incubate for 0.5-2 hours to allow the folic acid-modified protein to be fully adsorbed and wrapped on the surface of the PLGA-PFCE nanoparticles. After the reaction is completed, centrifuge the resulting suspension, discard the supernatant, and resuspend the resulting precipitate in ultrapure water for repeated washing to remove unbound free plasma proteins or plasma proteins with weak binding force, thereby obtaining composite nanoparticles based on targeted artificial protein corona.

[0018] Furthermore, the mass concentration of the polyvinyl alcohol aqueous solution is 40-50 mg / mL, the mass ratio of polylactic acid-glycolic acid copolymer, eicosine-15-crown-5-ether and polyvinyl alcohol is 1:16.8:200-:250, and the mass concentration of eicosine-15-crown-5-ether in the emulsion is 28-32 mg / mL.

[0019] Furthermore, in step S3, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and folic acid is 1.2-1.5:1:1, and the mass concentration of folic acid in the mixed solution is 3-5 mg / mL.

[0020] Furthermore, in the folic acid active intermediate solution, the mass concentration of the folic acid active intermediate is 3.66-6.1 mg / mL

[0021] Furthermore, in the mixed solution of the folic acid active intermediate and plasma, the mass ratio of the folic acid active intermediate to the plasma protein is 11:120-144.

[0022] Furthermore, the mass concentration of the folic acid-modified protein solution is 48-50 mg / mL, the mass concentration of the PLGA-PFCE nanoparticle solution is 220-278 mg / mL, and the volume ratio of the folic acid-modified protein solution to the PLGA-PFCE nanoparticle solution during mixing is 1:1-5:1.

[0023] Preparation of a composite nanoparticle based on a targeted artificial protein corona 19 Application of F MRI nanocontrast agents.

[0024] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0025] 1. The present invention selects folic acid as a targeted functional small molecule, modifies folic acid onto plasma proteins, and utilizes the characteristics of overexpression of folate receptors (FR) on the surface of cancer cells to enable the modified plasma proteins to have folate receptor-mediated specific targeting and selective tumor cell uptake. At the same time, 20-eighty-five-crown-5-ether is used as a magnetic resonance active molecule, polylactic acid-glycolic acid is used for encapsulation, and polyvinyl alcohol is used as a surfactant to synthesize PLGA-PFCE nanoparticles as 19 F MRI nanocontrast agent. Folic acid-modified plasma proteins were co-incubated with PLGA-PFCE nanoparticles in vitro to form a tumor-targeting protein corona on the surface of PLGA-PFCE nanoparticles. 1 The PLGA-PFCE nanoparticle-protein corona complex is injected intravenously into the body. After the folic acid is enriched in the tumor through the targeting effect of folic acid, the tumor can be targeted to increase its enrichment in the tumor site, thereby improving the efficacy of the drug. 19 F MRI tumor in situ imaging signal intensity, improve 19 The problems of low signal intensity and low sensitivity of F magnetic resonance imaging are solved, providing a new method for accurate imaging diagnosis of cancer.

[0026] 2. Since the behavior and biological characteristics of nanomaterials in the body are mainly determined by the protein corona, the formation of the protein corona will mediate the body's clearance of nanomaterials and shield and interfere with the original functional groups on the surface of the nanomaterials. Therefore, the present invention directly functionalizes the protein corona, which will have a higher targeting efficiency than functionalizing the surface of the nanomaterial.

[0027] 3. The present invention utilizes the property of nanomaterials to spontaneously adsorb proteins to form protein corona in physiological environment, and simulates the process of protein corona formation in vitro. 19 The surface of the PLGA-PFCE nanoparticles of the F MRI nanocontrast agent is pre-formed with an artificial protein corona with targeting, which can reduce the adsorption of proteins by the resulting protein corona-nanoparticle complex after intravenous injection into the body, and reduce the body's resistance to the protein corona caused by the formation of the protein corona. 19 Clearance of F MRI nanocontrast agents and interference with their surface targeting function. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are UV-visible absorption spectra of the PLGA-PFCE@FA-PC nanoparticles prepared in Example 1 and the PLGA-PFCE@PC nanoparticles prepared in Comparative Example 1.

[0029] Figure 2 This is the hydrated particle size distribution diagram of the PLGA-PFCE nanoparticles prepared in Example 1.

[0030] Figure 3This is the hydrated particle size distribution diagram of PLGA-PFCE@FA-PC nanoparticles prepared in Example 1.

[0031] Figure 4 This is a PdI comparison chart of PLGA-PFCE@FA-PC nanoparticles prepared in Examples 1-6.

[0032] Figure 5 This is the TEM image of PLGA-PFCE nanoparticles prepared in Example 1.

[0033] Figure 6 TEM image of PLGA-PFCE@FA-PC nanoparticles prepared in Example 1.

[0034] Figure 7 The PLGA-PFCE nanoparticles prepared in Example 1 19 F NMR spectrum.

[0035] Figure 8 The PLGA-PFCE@FA-PC nanoparticles prepared in Example 1 19 F NMR spectrum.

[0036] Figure 9 The PLGA-PFCE@FA-PC nanoparticles and PLGA-PFCE nanoparticles prepared in Example 1 19 A graph showing the effect of F on magnetic resonance imaging as its concentration changes.

[0037] Figure 10 The PLGA-PFCE@FA-PC nanoparticles and PLGA-PFCE nanoparticles prepared in Example 1 19 Linear fitting curve of F magnetic resonance imaging effect changing with its concentration.

[0038] Figure 11 Cells containing PLGA-PFCE@FA-PC nanoparticles, PLGA-PFCE nanoparticles, and FA-PLGA-PFCE nanoparticles prepared in Example 1 19 FComparison chart of magnetic resonance imaging effects. DETAILED DESCRIPTION

[0039] In order to facilitate those skilled in the art to understand and implement the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0040] Example 1

[0041] 1. Dissolve 10 mg of PLGA in 500 μL of dichloromethane, then add 100 μL of 20-eighthfluoro-15-crown-5-ether (density 1.682 g / mL) and 5 mL of a 40 mg / mL PVA aqueous solution. Mix well, and then use a 360 W ultrasonic probe with a 1 second on-2 second off cycle for 40 minutes to obtain a uniformly dispersed emulsion.

[0042] 2. The emulsion was transferred to a flask and stirred open at room temperature for more than 24 hours to evaporate excess dichloromethane. The residue was centrifuged at 14,000 rpm and 4°C for 15 minutes, and the supernatant was discarded. The resulting precipitate was washed three times with ultrapure water to remove dichloromethane and then dispersed in 1.5 mL of ultrapure water to obtain a PLGA-PFCE nanoparticle solution with a mass concentration of 278 mg / mL.

[0043] 3. Collect fresh blood from Balb / c mice using the orbital venous plexus blood sampling method. Use a 1.5 mL EP tube pre-filled with 1.6 mg / mL anticoagulant EDTA to collect the Balb / c mouse blood. Centrifuge the collected blood at 4°C and 1500 g for 15 min. Aspirate the supernatant, i.e., mouse plasma, and store the plasma at -20°C until use.

[0044] 4. Weigh 72 mg of folic acid, 22.5 mg of NHS, and 31.5 mg of EDC and dissolve them in 24 mL of DMSO to obtain a mixed solution with a folic acid concentration of 3 mg / mL. Stir the mixed solution in the dark at room temperature for 12 hours to obtain a solution of the active folic acid intermediate with a concentration of 3.66 mg / mL.

[0045] 5. Take 6 mL of folic acid active intermediate solution, adjust the pH value of the folic acid active intermediate solution to 7.4 using PBS buffer, then add 5 mL of Balb / c mouse plasma, and gently stir for 12 hours in the dark to allow the folic acid active intermediate to fully react with the plasma protein. After the reaction is completed, the resulting mixed product is taken out and placed in a dialysis bag (MWCO: 2500) and dialyzed in deionized water for 2 days. The dialyzed solution is ultrafiltered and concentrated to a volume of 5 mL to obtain a folic acid-modified protein solution with a mass concentration of 50 mg / mL, which is stored at 4°C.

[0046] 6. Mix 100 μL of folic acid-modified protein solution and 50 μL of PLGA-PFCE nanoparticle solution, shake and incubate for 2 hours to allow the folic acid-modified protein to be fully adsorbed and wrapped on the surface of the PLGA-PFCE nanoparticles. After the reaction is completed, the resulting suspension is centrifuged in a low-temperature centrifuge at 4°C and 15,000 rpm for 15 minutes. The supernatant is discarded, and the resulting precipitate is added to 1 mL of ultrapure water and resuspended by ultrasonication on ice (a cycle of ultrasonication for 5 seconds and ice bath for 5 seconds) to remove unbound free plasma proteins or proteins with weak binding force. Repeat the above centrifugation and resuspension steps three times to obtain composite nanoparticles based on a targeted artificial protein corona, recorded as PLGA-PFCE@FA-PC nanoparticles. The composite nanoparticles based on the targeted artificial protein corona are dispersed in ultrapure water to obtain a PLGA-PFCE@FA-PC solution for standby use.

[0047] Example 2

[0048] The operation and steps were basically the same as those in Example 1, except that in step 6, the volume of the folic acid-modified protein solution was 50 μL.

[0049] Example 3

[0050] The operation and steps were basically the same as those in Example 1, except that in step 6, the volume of the folic acid-modified protein solution was 75 μL.

[0051] Example 4

[0052] The operation and steps were basically the same as those in Example 1, except that in step 6, the volume of the folic acid-modified protein solution was 150 μL.

[0053] Example 5

[0054] The operation and steps were basically the same as those in Example 1, except that in step 6, the volume of the folic acid-modified protein solution was 200 μL.

[0055] Example 6

[0056] The operation and steps were basically the same as those in Example 1, except that in step 6, the volume of the folic acid-modified protein solution was 250 μL.

[0057] Comparative Example 1

[0058] The difference from Example 1 is that steps 4-6 were not performed, and 100 μL of unmodified Balb / c mouse plasma was directly incubated with 50 μL of PLGA-PFCE nanoparticle solution. The other steps were the same as in Example 1 to obtain nanoparticles PLGA-PFCE@PC.

[0059] The nanoparticles PLGA-PFCE@FA-PC prepared in Example 1 and the nanoparticles PLGA-PFCE@PC prepared in Comparative Example 1 were subjected to UV-visible absorption spectrum measurement (absorption wavelength 190-700 nm). The obtained UV-visible absorption spectrum is shown in the figure below: Figure 1 As shown by Figure 1 It can be seen that the characteristic ultraviolet absorption peaks of pure folic acid are around 256 nm, 283 nm, and 365 nm. Pure folic acid has maximum ultraviolet absorption at 283 nm. The nanoparticles PLGA-PFCE@PC prepared in Comparative Example 1 have almost no characteristic absorption peaks at 256 nm, 283 nm, and 365 nm, while the peak intensity of PLGA-PFCE@FA-PC prepared in Example 1 at 283 nm is significantly enhanced, indicating that in the nanoparticles PLGA-PFCE@FA-PC, folic acid is successfully modified to plasma proteins through covalent coupling.

[0060] Comparative Example 2

[0061] The difference from Example 1 is that when performing step 1, 1.2 mg of folic acid is dissolved in 5 mL of a 40 mg / mL PVA aqueous solution to obtain a folic acid solution. The folic acid solution, 10 mg of PLGA, and 100 μL of 20-eicosyl-15-crown-5-ether (density of 1.682 g / mL) are respectively added to 500 μL of dichloromethane, mixed evenly, and then sonicated for 40 minutes using a 360 W ultrasonic probe with a cycle of on for 1 second and off for 2 seconds to obtain a uniformly dispersed emulsion. Steps 2 and 3 are the same as in Example 1, and steps 4-6 are not performed to obtain nanoparticles FA-PLGA-PFCE with targeted small molecule folic acid directly loaded on their surface.

[0062] Experiment 1: Dynamic light scattering potential measurement method of composite nanoparticles based on targeted artificial protein corona of the present invention:

[0063] The average size, polydispersity index (PDI), and zeta potential of the PLGA-PFCE nanoparticles prepared in Example 1 and the PLGA-PFCE@FA-PC nanoparticles prepared in Examples 1-6 were measured using a Malvern particle size analyzer. Specifically, 50 μL of the PLGA-PFCE@FA-PC solution or PLGA-PFCE nanoparticle solution prepared in each example was dispersed in 1 mL of room temperature deionized water and then measured using a Malvern particle size analyzer.

[0064] Experimental results:

[0065] The hydrated particle size distribution of the PLGA-PFCE nanoparticles prepared in Example 1 is shown in FIG. Figure 2As shown by Figure 2 It can be seen that the hydrated particle size distribution range of PLGA-PFCE nanoparticles is about 220 nm, with uniform size and uniform distribution.

[0066] The hydrated particle size distribution of the PLGA-PFCE@FA-PC nanoparticles prepared in Example 1 is shown in FIG. Figure 3 As shown by Figure 3 It can be seen that compared with PLGA-PFCE nanoparticles, the average hydrated particle size of PLGA-PFCE@FA-PC increased by about 60 nm, and the size uniformity decreased.

[0067] The polydispersity coefficients of the PLGA-PFCE@FA-PC nanoparticles prepared in Examples 1-6 are as follows: Figure 4 As shown by Figure 4 It can be seen that when the volume ratio of folic acid-modified protein solution to PLGA-PFCE nanoparticle solution is between 50:75 and 50:100, the PdI value of PLGA-PFCE@FA-PC nanoparticles mutates, indicating that when the volume ratio of folic acid-modified protein FA-PC solution to PLGA-PFCE nanoparticle solution is 50:100, the protein required to form the protein corona is saturated. Therefore, when the volume ratio of folic acid-modified protein FA-PC solution to PLGA-PFCE nanoparticle solution is 50:100, it is the most suitable volume ratio for the preparation of PLGA-PFCE@FA-PC nanoparticles.

[0068] Experiment 2: Experimental method for measuring the dynamic light scattering potential of composite nanoparticles based on the targeted artificial protein corona of the present invention:

[0069] 10 μL of each of the PLGA-PFCE nanoparticle solution and the PLGA-PFCE@FA-PC solution prepared in Example 1 was dropwise added to the carbon support film and allowed to air-dry at room temperature. The morphology, particle size, and dispersion of the nanoparticles were observed using a transmission electron microscope at 200 kV.

[0070] Experimental results:

[0071] The TEM image of the PLGA-PFCE nanoparticles prepared in Example 1 is as follows: Figure 5 As shown, the TEM image of the PLGA-PFCE@FA-PC nanoparticles prepared in Example 1 is as follows Figure 6 As shown by Figure 5 and Figure 6As can be seen, the PLGA-PFCE nanoparticles and PLGA-PFCE@FA-PC nanoparticles prepared in Example 1 are both spherical with a particle size of approximately 120 nm. The differences between the transmission electron microscopy and dynamic light scattering (DLS) measurements are due to the fact that the latter method measures the particle size of the nanoparticles in their hydrated state, and the solvent effect causes the nanoparticles to appear larger in hydrated form.

[0072] Experiment 3: Composite nanoparticles based on targeted artificial protein corona of the present invention 19 F NMR spectrum and fluorine atom content analysis

[0073] Experimental methods:

[0074] 1. Take 10 μL of the PLGA-PFCE@FA-PC solution prepared in Example 1 and add it to a mixed solution of 500 μL of D2O (10 vol%) and H2O (90 vol%), and add 5 μL of 200 mM sodium trifluoromethanesulfonate (CF3NaO3S) as an internal reference to obtain a sample solution. The sample solution was placed in a nuclear magnetic resonance tube and the PLGA-PFCE@FA-PC was tested using a 500 MHz nuclear magnetic resonance spectrometer. 19 F NMR spectrum.

[0075] In addition, through 19 The concentration of PFCE can also be calculated from the F NMR spectrum using the following formula:

[0076]

[0077] Where C PFCE is the molar concentration of PFCE, C CF3NaO3S is the molar concentration of CF3NaO3S, m is the molar concentration of PFCE in 19 The integral value of CF3NaO3S in the F NMR spectrum is fixed at 1, where n is the number of equivalent fluorine atoms, nCF3NaO3S = 3, and nPFCE = 20.

[0078] 2. The PLGA-PFCE nanoparticle solution prepared in Example 1 was treated according to the method of step 1.

[0079] Experimental results

[0080] PLGA-PFCE nanoparticles prepared in Example 1 19 F NMR spectrum Figure 7 As shown in Example 1, the PLGA-PFCE@FA-PC nanoparticles prepared 19 F NMR spectrum Figure 8 As shown, by comparison Figure 7 and Figure 8In addition to the sodium trifluoromethanesulfonate peak at -79 ppm, both PLGA-PFCE nanoparticles and PLGA-PFCE@FA-PC nanoparticles exhibit a distinct PFCE peak at -92 ppm. Calculated T2 relaxation time for PLGA-PFCE nanoparticles is 584.6 ms, T1 relaxation time is 766.3 ms, PFCE concentration is 88.5 mM, and fluorine atomic concentration is 1.77 M. PLGA-PFCE@FA-PC nanoparticles have a T2 relaxation time of 579.9 ms, T1 relaxation time is 769.0 ms, PFCE concentration is 79.7 mM, and fluorine atomic concentration is 1.59 M.

[0081] The results showed that PLGA-PFCE was successfully encapsulated in the protein corona modified with folic acid, and the formation of the protein corona had no obvious effect on the relaxation time.

[0082] Experiment 4: In vitro testing of the composite nanoparticles based on the targeted artificial protein corona of the present invention 19 F MRI experimental method:

[0083] 1. The PLGA-PFCE@FA-PC solution prepared in Example 1 was diluted in a gradient manner to obtain a series of sample solutions with PFCE concentrations of 0, 9.0, 18.0, 27.0, 36.0, and 45.0 mM (the corresponding fluorine atomic concentrations were 0, 180.0, 360.0, 540.0, 720.0, and 900.0 mM, respectively). Each sample solution was placed in a nuclear magnetic resonance tube and imaged using a 400 MHz nuclear magnetic resonance imaging instrument. 19 F MRI RARE sequence imaging, characterization 19 F magnetic resonance imaging effect and quantitative analysis of its 19 F MRI imaging signal intensity changes with PLGA-PFCE@FA-PC concentration.

[0084] 2. The PLGA-PFCE nanoparticle solution prepared in Example 1 was treated according to the method of step 1.

[0085] Experimental results:

[0086] PLGA-PFCE@FA-PC nanoparticles and PLGA-PFCE nanoparticles prepared in Example 1 19 Fluid imaging with varying F MRI effects depending on its concentration 19 F MRI images such as Figure 9 As shown in Example 1, the PLGA-PFCE@FA-PC nanoparticles and PLGA-PFCE nanoparticles prepared 19 The linear fitting curve of F MRI signal intensity versus concentration is shown in Figure 10As shown by Figure 9 and Figure 10 It can be seen that for PLGA-PFCE@FA-PC nanoparticles and PLGA-PFCE nanoparticles, as the PFCE concentration increases, 19 F magnetic resonance imaging images all became brighter, 19 The FMRI signals continued to increase, and the lowest imaging concentration of PFCE was 9.0 mM.

[0087] The results showed that PLGA-PFCE@FA-PC nanoparticles have good 19 F MRI imaging ability, the folic acid-modified protein corona does not significantly affect the imaging ability of PLGA-PFCE@FA-PC.

[0088] Experiment 5: Cells of the composite nanoparticles based on the targeted artificial protein corona of the present invention 19 F MRI experimental method:

[0089] 1. Experimental group: 500 μL of the PLGA-PFCE@FA-PC solution prepared in Example 1 was diluted to 3 mL using 1640 basal culture medium to obtain an experimental culture medium. The fluorine atomic concentration in the experimental culture medium was 300 mM, and 4T1 cells were cultured using the experimental culture medium for 4 hours.

[0090] 2. Control group A: 4T1 cells were cultured in 1640 basal medium containing folic acid for 2 hours to saturate the folate receptors on the surface of the 4T1 cells. The 4T1 cells with saturated folate receptors were then cultured in experimental medium for 4 hours.

[0091] 3. Control group B: 500 μL of the PLGA-PFCE nanoparticle solution prepared in Example 1 was diluted to 3 mL using 1640 basal culture medium to obtain control culture medium B. The fluorine atom concentration in control culture medium B was 300 mM. 4T1 cells were cultured using the control culture medium for 4 hours.

[0092] 4. Control group C: 500 μL of the FA-PLGA-PFCE nanoparticle solution prepared in Comparative Example 2 was mixed with 1000 μL of plasma protein, shaken and incubated for 2 hours to allow the protein to fully adsorb and wrap on the surface of the FA-PLGA-PFCE nanoparticles to simulate the ordinary protein corona without targeted functionalization formed after the nanoparticles enter the organism, and obtain PC@FA-PLGA-PFCE nanoparticles. The PC@FA-PLGA-PFCE nanoparticles were diluted to 3 mL using 1640 basal medium to obtain control medium C. The fluorine atom concentration in control medium C was grouped into 300 mM, and 4T1 cells were cultured using control medium for 4 hours.

[0093] 5. The 4T1 cells treated in steps 1-4 were treated as follows: After the culture, the 4T1 cells were centrifuged and resuspended in 500 μL PBS to obtain a 4T1 cell solution. The 4T1 cell solution was placed in a nuclear magnetic resonance tube and imaged using a 400 MHz nuclear magnetic resonance imaging instrument. 19 F MRI RARE sequence imaging, characterization 19 F MRI effect.

[0094] Experimental results:

[0095] 4T1 cells treated with different 19 F magnetic resonance imaging Figure 11 As shown by Figure 11 It can be seen that the 4T1 cells treated with the experimental method 19 The signal intensity of the F MRI image was very good, while the 4T1 cells treated with method B in the control group showed almost no signal. This indicates that the formation of the folic acid-modified protein corona (FA-PC) significantly enhanced the uptake of PLGA-PFCE by 4T1 cells.

[0096] In addition, the 4T1 cells treated with the experimental method 19 The signal intensity of the F magnetic resonance imaging image was significantly higher than that of the 4T1 cells treated with the control group A method, which proved that the improvement of PLGA-PFCE uptake by 4T1 cells was achieved through the targeting effect mediated by folic acid and folic acid receptors. 19 The signal intensity of the F magnetic resonance imaging image was significantly higher than that of the 4T1 cells treated with the C method in the control group, indicating that the targeted functionalized artificial protein corona obtained by modifying folic acid onto plasma proteins has a higher targeting efficiency than directly loading folic acid on the surface of nanoparticles.

Claims

1. A composite nanoparticle based on a targeted artificial protein corona, characterized by: The composite nanoparticles are a three-layer core-shell structure, wherein the core is 20-fluoro-15-crown-5-ether, the middle layer is a polylactic acid-glycolic acid copolymer layer, and the outermost layer is a protein corona with targeted functionalization by chemically modifying folic acid.

2. The composite nanoparticles based on the targeted artificial protein corona according to claim 1, characterized in that: The outermost protein corona with targeted functionalization is obtained by spontaneous adsorption of targeted functionalized proteins by nanoparticles.

3. The composite nanoparticles based on the targeted artificial protein corona according to claim 2, characterized in that: The protein with targeted functionalization is a plasma protein modified by folic acid through covalent coupling.

4. A method for preparing composite nanoparticles based on a targeted artificial protein corona according to any one of claims 1 to 3, characterized in that The steps include: S1, dissolving poly(lactic acid-co-glycolic acid) copolymer in dichloromethane, adding eicosine-15-crown-5-ether and polyvinyl alcohol aqueous solution thereto, and dispersing them uniformly to obtain an emulsion; S2. stirring the emulsion to volatilize the dichloromethane, centrifuging the residue, discarding the supernatant, washing the resulting precipitate with ultrapure water, and then dispersing it in ultrapure water to obtain a PLGA-PFCE nanoparticle solution; S3. Dissolving folic acid in dimethyl sulfoxide, and adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain a mixed solution, stirring the mixed solution at room temperature for 8-12 hours in the dark to obtain a folic acid active intermediate solution; S4. Adjust the pH value of the folate active intermediate solution to 7.2-7.6 with PBS buffer, then add plasma, and stir for 8-12 hours in a dark environment to allow the folate active intermediate to fully react with the plasma protein. After the reaction is completed, dialyze the resulting mixed product, and concentrate the dialyzed solution to obtain a folate-modified protein solution; S5. Mix the folic acid-modified protein solution and the PLGA-PFCE nanoparticle solution, shake and incubate for 0.5-2 hours to allow the folic acid-modified protein to be fully adsorbed and wrapped on the surface of the PLGA-PFCE nanoparticles. After the reaction is completed, centrifuge the resulting suspension, discard the supernatant, and resuspend the resulting precipitate in ultrapure water for repeated washing to remove unbound free plasma proteins or plasma proteins with weak binding force, thereby obtaining composite nanoparticles based on a targeted artificial protein corona.

5. The method for preparing composite nanoparticles based on a targeted artificial protein corona according to claim 4, characterized in that: The mass concentration of the polyvinyl alcohol aqueous solution is 40-50 mg / mL, the mass ratio of polylactic acid-glycolic acid copolymer, eicosine-15-crown-5-ether and polyvinyl alcohol is 1:16.8:200-:250, and the mass concentration of eicosine-15-crown-5-ether in the emulsion is 28-32 mg / mL.

6. The method for preparing composite nanoparticles based on a targeted artificial protein corona according to claim 4, characterized in that: In step S3, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and folic acid is 1.2-1.5:1:1, and the mass concentration of folic acid in the mixed solution is 3-5 mg / mL.

7. The method for preparing composite nanoparticles based on a targeted artificial protein corona according to claim 4, characterized in that: In the folic acid active intermediate solution, the mass concentration of the folic acid active intermediate is 3.66-6.1 mg / mL.

8. The method for preparing composite nanoparticles based on a targeted artificial protein corona according to claim 4, characterized in that: In the mixed solution of the folic acid active intermediate and plasma, the mass ratio of the folic acid active intermediate to the plasma protein is 11:120-144.

9. The method for preparing composite nanoparticles based on a targeted artificial protein corona according to claim 4, characterized in that: The mass concentration of the folic acid modified protein solution is 48-50 mg / mL, the mass concentration of the PLGA-PFCE nanoparticle solution is 220-278 mg / mL, and the volume ratio of the folic acid modified protein solution to the PLGA-PFCE nanoparticle solution during mixing is 1:1-5:

1.

10. A composite nanoparticle based on a targeted artificial protein corona according to any one of claims 1 to 3 in the preparation 19 Application of nanocontrast agents in fMRI.

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