FILM-FORMING AGENT COMPOSITION FOR CONTRAST AGENT, FILM-FORMING LIPID SOLUTION FOR CONTRAST AGENT, CONTRAST AGENT AND PREPARATION METHOD THEREOF

Abstract

Disclosed are a film-forming agent composition for contrast agent, a film-forming lipid solution including the film-forming agent composition, a contrast agent including the film-forming lipid solution, and a preparation method thereof. The film-forming agent composition for contrast agent includes a lipid, an emulsifier and a surface charge modifier; relative to 100 parts by weight of the lipid, the content of the emulsifier is 20-50 parts by weight, and the content of the surface charge modifier is 10-35 parts by weight; and the lipid is a carboxylated phospholipid, and the surface charge modifier is a polyelectrolyte. Based on the composition, the nanodroplets of the resulting contrast agent have more uniform particle size, higher stability and better controllability, as well as a lower threshold value for ultrasonic gasification.

Claims

1. A film-forming agent composition for contrast agent, comprising a lipid, an emulsifier and a surface charge modifier, wherein: relative to 100 parts by weight of the lipid, a content of the emulsifier is 20-50 parts by weight, and a content of the surface charge modifier is 10-35 parts by weight; and the lipid is a carboxylated phospholipid, and the surface charge modifier is a polyelectrolyte.

2. The film-forming agent composition for contrast agent according to claim 1, wherein relative to 100 parts by weight of the lipid, the content of the emulsifier is 25-45 parts by weight, and the content of the surface charge modifier is 15-30 parts by weight.

3. The film-forming agent composition for contrast agent according to claim 1, wherein the lipid is a carboxylated phospholipid, which is any one or more selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, 1,2-bis(diphenylphosphine)ethane, and distearoylphosphatidylethanolamine-polyethylene glycol.

4. The film-forming agent composition for contrast agent according to claim 1, wherein the emulsifier is any one or more selected from the group consisting of: polyethylene glycol 4000, polyethylene glycol 40s, polyoxypropylene polyoxyethylene block polyether, polyethylene glycol 1400, and polysorbate-80.

5. The film-forming agent composition for contrast agent according to claim 1, wherein the surface charge modifier is any one or more selected from the group consisting of: hyaluronic acid, chitosan, sodium hydroxymethyl cellulose, carbomer, sodium alginate, polyamine, and hard amine.

6. The film-forming agent composition for contrast agent according to claim 1, further comprising: a photosensitizer, relative to 100 parts by weight of the lipid, the content of the photosensitizer is 10-35 parts by weight.

7. The film-forming agent composition for contrast agent according to claim 6, wherein relative to 100 parts by weight of the lipid, the content of the photosensitizer is 15-30 parts by weight.

8. The film-forming agent composition for contrast agent according to claim 6, wherein the photosensitizer is any one or more selected from: methylene blue, porphyrin, hematoporphyrin, photoporphyrin, mesoporphyrin, sodium porphyrin, gallium porphyrin, hydrophilic chlorin derivative, protoporphyrin and copper protoporphyrin.

9. The film-forming agent composition for contrast agent according to claim 1, further comprising: gold particles modified with amino groups on the surface, relative to the total weight of the lipid, emulsifier, surface charge modifier and photosensitizer, the content of the gold particles is 20-50 parts by weight.

10. The film-forming agent composition for contrast agent according to claim 9, wherein the particle size of the gold particles is 1-30 nm.

11. A film-forming lipid solution for contrast agent, wherein the film-forming lipid solution for contrast agent comprises, or is prepared from, the film-forming agent composition for contrast agent according to claim 1.

12. A contrast agent, comprising a nanodroplet consisting of a shell and the content wrapped by the shell, the shell being produced by the film-forming lipid solution for contrast agent according to claim 11.

13. The contrast agent according to claim 12, wherein the content is a biocompatible substance which comprises drug or does not comprise drug and is a gaseous state or a phase-changeable liquid.

14. The contrast agent according to claim 13, wherein the biocompatible substance is any one or more selected from the group consisting of: air, nitrogen, carbon dioxide, oxygen, hydrogen, nitrogen oxide, inert gas, halosilane, halosilane, haloalkane, and sulphur halide.

15. The contrast agent according to claim 13, wherein the content is a perfluorocarbon liquid.

16. A method for preparing a contrast agent, comprising: (1) mixing the film-forming agent composition for contrast agent according to claim 1 with a first organic solvent to obtain a film-forming lipid solution for contrast agent; (2) mixing the film-forming lipid solution for contrast agent with perfluorocarbon liquid to obtain solution A; (3) mixing the solution A and the hydration solution to produce the Ouzo effect, leaving the resulting material to stand for stratification, and separating the lipid phase to obtain material B; (4) resuspending the material B in a buffer solution to obtain an initial nanodroplet solution; (5) contacting the initial nanodroplet solution with gold particles modified with amino groups on the surface and an initiator.

17. The method according to claim 16, wherein in step (2), the saturability of perfluorocarbon in the solution A is 30-100%.

18. The method according to claim 16, wherein in step (3), the hydration solution is a mixture of glycerol, propylene glycol and phosphate in a volume ratio of 1:(0.5-3):(5-12).

19. The method according to claim 16, wherein in step (3), the volume ratio of the solution A to the hydration solution is 1:(0.3-2).

20. The method according to claim 16, wherein in step (5), the weight ratio of the amount of the initiator to that of the gold particles modified with amino groups on the surface is (0.5-4):1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0070] FIG. 1 is an image of contrast agent I1 obtained in Example 1 under a transmission electron microscope.

[0071] FIG. 2 is an image of the contrast agent I1 obtained in Example 1 before activation (a) and after activation (b).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0072] The present application will be described in detail below by the Examples. The described Examples of the present application are only a part of the examples of the present application, but not all of the examples. Based on the Examples of the present application, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0073] Unless otherwise specified, the materials used in the following Examples are all commercially available analytical grades.

[0074] The PBS buffer solution used in the following Examples is prepared by the following method: weighing 0.097 g of KCl, 4.005 g of NaCl, 1.145 g of Na.sub.2HPO.sub.4.H.sub.2O and 0.096 g of KH.sub.2PO.sub.4 to a beaker of 1 L, adding deionized water and making up to 500 ml to prepare phosphate buffered saline (PBS solution) for use (if it is not enough, it may be prepared multiple times).

EXAMPLE 1

[0075] (1) Carboxylated dipalmitoyl phosphatidyl choline (DPPC) (lipid; 1 carboxyl group per molecule, CAS: 63-89-8, purchased from SigmaAldrich), polyethylene glycol 40s (PEG40s) (emulsifier), sodium alginate (surface charge modifier, CAS: 9005-38-3) and methylene blue (photosensitizer) are mixed in a weight ratio of 100:35:25:25 to fully dissolve in the organic solvent ethanol (the amount of organic solvent is such that the concentration of lipid is 2 mg/mL), thereby obtaining a film-forming lipid solution for contrast agent;

[0076] (2) Part of the film-forming lipid solution for contrast agent is taken out, and excess perfluorohexane liquid is added to obtain a saturated perfluorocarbon solution; the saturated perfluorocarbon solution is mixed with the remaining film-forming lipid solution for contrast agent to prepare solution A with 80% saturability;

[0077] (3) The solution A is mixed with a hydration solution (a mixture of glycerol, propylene glycol and PBS buffer in a volume ratio of 1:1.5:8.5) in a volume ratio of 1:0.8, allowing to stand for 20 min; the solution is divided into two layers, sucking off the upper layer solution, and centrifuging the lower layer liquid for 10 min at a speed of 4000 rpm; then the upper layer solution is sucked off again to obtain material B;

[0078] (4) The material B is resuspended in PBS buffer solution, centrifuged, and the upper layer solution is sucked off, then the obtained mixture is added into PBS buffer solution again to be resuspended and centrifuged; this process is repeated for three times; the finally obtained lower layer material (referred to as material C) is mixed with PBS buffer solution to make the lipid concentration in the mixed solution to be 4 mg/mL, and to be resuspended to obtain the initial nanodroplet solution;

[0079] (5) Gold particles (whose surface modified with amino groups, each gold particle includes 3 amino groups, and the average particle size of the gold particles is about 10 nm; manufacturer: SigmaAldrich; product number: 765309) and an initiator (consisting of EDC and NHS in a weight ratio of 1:0.75) are added to the initial nanodroplet solution according to material C:gold particles:initiator in a weight ratio of 10:3:4, the obtained mixture being incubated at room temperature 20° C. for 24 h to obtain a contrast agent, denoted as I1.

[0080] The image of the contrast agent I1 under the transmission electron microscope is shown in FIG. 1. It can be seen from FIG. 1 that the nanodroplets are spheres with a core-shell structure.

EXAMPLE 2

[0081] (1) Carboxylated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (lipid; 1 carboxyl group per molecule, CAS: 816-94-4, purchased from SigmaAldrich), polyethylene glycol 4000 (PEG4000) (emulsifier), polyethyleneimine (surface charge modifier, CAS: 9002-98-6) and methylene blue (photosensitizer) are mixed in a weight ratio of 100:30:28:22 to fully dissolved in the organic solvent isopropanol (the amount of organic solvent is such that the concentration of lipid is 4 mg/mL), thereby obtaining a film-forming lipid solution for contrast agent;

[0082] (2) Part of the film-forming lipid solution for contrast agent is taken out, and excess perfluorohexane liquid is added to obtain a saturated perfluorocarbon solution; the saturated perfluorocarbon solution is mixed with the remaining film-forming lipid solution for contrast agent to prepare solution A with 90% saturability;

[0083] (3) The solution A is mixed with a hydration solution (a mixture of glycerol, propylene glycol and PBS buffer in a volume ratio of 1:1:10) in a volume ratio of 1:1, allowing to stand for 20 min; the solution is divided into two layers, sucking off the upper layer solution, and centrifuging the lower layer liquid for 10 min at a speed of 4000 rpm; then the upper layer solution is sucked off again to obtain material B;

[0084] (4) The material B is resuspended in PBS buffer solution, centrifuged, and the upper layer solution is sucked off, then the obtained mixture is added into PBS buffer solution again to be resuspended and centrifuged; this process is repeated for three times; the finally obtained lower layer material (referred to as material C) is mixed with PBS buffer solution to make the lipid concentration in the mixed solution to be 3 mg/mL, and to be resuspended to obtain the initial nanodroplet solution;

[0085] (5) Gold particles (whose surface modified with amino groups, each gold particle includes 3 amino groups, and the average particle size of the gold particles is about 20 nm; SigmaAldrich product number: 765341) and an initiator (consisting of EDC and NHS in a weight ratio of 1:0.85) are added to the initial nanodroplet solution according to material C:gold particles:initiator in a weight ratio of 10:4:6, the obtained mixture being incubated at room temperature 20° C. for 24 h to obtain a contrast agent, denoted as I2.

EXAMPLE 3

[0086] (1) Carboxylated distearoylphosphatidylethanolamine (DSPE) (lipid; 1 carboxyl group per molecule, CAS: 1069-79-0, purchased from SigmaAldrich), Pluronic-F68 (emulsifier), polyallylamine hydrochloride (surface charge modifier, CAS: 30551-89-4) and porphyrin (photosensitizer) are mixed in a weight ratio of 100:40:22:28 to fully dissolve in the organic solvent triethanolamine (the amount of organic solvent is such that the concentration of lipid is 4 mg/mL), thereby obtaining a film-forming lipid solution for contrast agent;

[0087] (2) Part of the film-forming lipid solution for contrast agent is taken out, and excess perfluorohexane liquid is added to obtain a saturated perfluorocarbon solution; the saturated perfluorocarbon solution is mixed with the remaining film-forming lipid solution for contrast agent to prepare solution A with 60% saturability;

[0088] (3) The solution A is mixed with a hydration solution (a mixture of glycerol, propylene glycol and PBS buffer in a volume ratio of 1:2:7) in a volume ratio of 1:0.5, allowing to stand for 20 min; the solution is divided into two layers, sucking off the upper layer solution, and centrifuging the lower layer liquid for 10 min at a speed of 4000 rpm; then the upper layer solution is sucked off again to obtain material B;

[0089] (4) The material B is resuspended in PBS buffer solution, centrifuged, and sucking off the upper layer solution is sucked off, then the obtained mixture is added into PBS buffer solution again to be resuspended and centrifuged; this process is repeated for three times; the finally obtained lower layer material (referred to as material C) is mixed with PBS buffer solution to make the lipid concentration in the mixed solution to be 3.5 mg/mL, and to be resuspended to obtain the initial nanodroplet solution;

[0090] (5) Gold particles (whose surface modified with amino groups, each gold particle includes 3 amino groups, and the average particle size of the gold particles is about 20 nm; manufacturer: SigmaAldrich; product number: 765341) and an initiator (consisting of EDC and NHS in a weight ratio of 1:1) are added to the initial nanodroplet solution according to material C:gold particles:initiator in a weight ratio of 10:3:6, the obtained mixture being incubated at room temperature 20° C. for 24 h to obtain a contrast agent, denoted as I3.

EXAMPLE 4

[0091] Referring to the method of Example 1, the difference is that the weight ratio of lipid, emulsifier, surface charge modifier and photosensitizer is changed to 100:23:12:12, and the total weight is kept unchanged.

[0092] Finally, a contrast agent is obtained and denoted as I4.

EXAMPLE 5

[0093] Referring to the method of Example 1, the difference is that the weight ratio of lipid, emulsifier, surface charge modifier and photosensitizer is changed to 100:46:32:32, and the total weight is kept unchanged.

[0094] Finally, a contrast agent is obtained and denoted as I5.

EXAMPLE 6

[0095] Referring to the method of Example 1, the difference is that in step (5), the gold particles modified with amino groups on the surface are replaced with the same weight of silver particles with amino groups modified on the surface (Yoshikura Nano, JCSNP03-0010).

[0096] Finally, a contrast agent is obtained and denoted as I6.

EXAMPLE 7

[0097] Referring to the method of Example 1, the difference is that in step (2), the saturability of the solution A is changed to 40%.

[0098] Finally, a contrast agent is obtained and denoted as I7.

EXAMPLE 8

[0099] Referring to the method of Example 1, the difference is that in step (2), the saturability of the solution A is changed to 100%.

[0100] Finally, a contrast agent is obtained and denoted as I8.

EXAMPLE 9

[0101] Referring to the method of Example 1, the difference is that no photosensitizer is added.

[0102] Finally, a contrast agent is obtained and denoted as I9.

EXAMPLE 10

[0103] Referring to the method of Example 1, the difference is that the amount of the photosensitizer is changed so that the weight ratio of lipid to photosensitizer is 100:20.

[0104] Finally, a contrast agent is obtained and denoted as I10.

EXAMPLE 11

[0105] Referring to the method of Example 1, the difference is that the amount of the photosensitizer is changed so that the weight ratio of lipid to photosensitizer is 100:60.

[0106] Finally, a contrast agent is obtained and denoted as I11.

EXAMPLE 12

[0107] Referring to the method of Example 1, the difference is that no gold particles are added.

[0108] Finally, a contrast agent is obtained and denoted as I12.

COMPARATIVE EXAMPLE 1

[0109] With reference to the method of Example 1, the difference is that no surface charge modifier, emulsifier and photosensitizer are added; particularly, in step (1), the DPPC with a weight equal to the total weight of DPPC, PEG40s, sodium alginate and methylene blue in Example 1 is fully dissolved in the same amount of organic solvent ethanol as in Example 1, thereby obtaining a film-forming lipid solution for contrast agent.

[0110] Finally, a contrast agent is obtained and denoted as D1.

COMPARATIVE EXAMPLE 2

[0111] A contrast agent is prepared by applying mechanical external force, referring to a preparation method described by Geoffrey P. et al., Supporting information, Super-resolution ultrasound imaging in vivo with transient laser-activated nanodroplets, Nano letters, 2016, 16, 4, 2556-2559. Particularly, the method includes:

[0112] (D-1) According to step (1) of Example 1 and referring the above article of Geoffrey P. et al., the lipid DPPC is replaced with the same weight of water, thereby obtaining a film-forming lipid solution for contrast agent;

[0113] (D-2) In a 180 W ultrasonic bath, the film-forming lipid solution for contrast agent is mixed with a perfluorohexane liquid (the perfluorohexane is excessive with reference to the above article of Geoffrey P. et al.), thereby obtaining D-A solution;

[0114] (D-3) The D-A solution is vortexed for 10 seconds, and sonicated in an ultrasonic bath for 5 min to obtain material D-B;

[0115] (D-4) The material D-B is washed by centrifugation at 1000 rcf for 5 min, resuspending in PBS buffer solution, and waiting for 30 min.

[0116] Finally, a contrast agent is obtained and denoted as D2.

TEST EXAMPLE

[0117] (1) Particle Size and Homogeneity of Nanodroplets

[0118] The number-average particle size (Dn, unit: nm) of the nanodroplets is detected by a particle size analyzer (Malvern, Mastersizer 3000) based on the DLS principle, and the results are recorded in Table 1;

[0119] The weight-average particle size (Dw, unit: nm) of the nanodroplets is also detected by the particle size analyzer. The polydispersity coefficient=Dw/Dn is calculated, and the results are recorded in Table 1.

[0120] (2) Stability Test

[0121] The stability of the contrast agent in vivo is reflected by the half-life of the contrast agent, and the longer the half-life, the higher the stability. Particularly, the test method includes: taking a Japanese long-eared white rabbit as the experimental object, injecting the drug-loaded nanodroplets through the ear vein of the rabbit, adjusting the concentration of the drug-loaded nanodroplets to 1×10.sup.11/ml, and the injection dose to 0.1 ml/kg; collecting blood from the long-eared white rabbits at different times, and monitoring the content of phospholipid in the blood by high performance liquid chromatography. The half-life of the contrast agent is a period corresponding to the time when the phospholipid concentration in the blood is half of the initial injection concentration.

[0122] The measured half-life results of the contrast agents from the Examples and Comparative Examples are recorded in Table 1, respectively.

[0123] (3) Detection of Ultrasound Activation Threshold (without Optical Assistance)

[0124] In vitro ultrasound activation experiment is performed, and the results are recorded in Table 1. Particularly, the test method includes: embedding drug-loaded nanodroplets into an agar model for in vitro site-specific activation experiments. Herein, the concentration of drug-loaded nanodroplets is 1×10.sup.9/ml, and the content of agar is 1% (w/v). The ultrasonic probe (center frequency: 7.8 MHz) is placed just above the agar model, and the probe and the agar model are acoustically coupled through ultrasonic couplant, adjusting the mechanical index of the ultrasound probe, and observing the change of grayscale in the ultrasound image. Since there is a weak signal in the ultrasonic image before activation of the droplets, however, after activation there is a strong signal in the ultrasonic image due to the phase change of the droplets into microbubbles. Accordingly, the activation of the droplets may be reflected by the change of the grayscale in the ultrasonic image. In the region of interest, we set the mechanical index corresponding to the ultrasound that may induce 10% nanodroplet activation as the activation threshold of the droplets.

[0125] (4) Detection of Ultrasound Activation Threshold with Optical Assistance

[0126] In vitro site-specific ultrasound activation experiment is performed with optical assistance, and the results are recorded in Table 1. Particularly, the test method includes: embedding drug-loaded nanodroplets into an agar model for in vitro site-specific activation experiments. Herein, the concentration of drug-loaded nanodroplets is 1×10.sup.9/ml, and the content of agar is 1% (w/v). The ultrasonic probe (center frequency: 7.8 MHz) is placed just above the agar model, and the probe and the agar model are acoustically coupled through ultrasonic couplant. The difference from the threshold of ultrasonic activation is that we irradiate the side of the agar probe with a 760 nm laser, adjusting the laser energy to 1 W/cm.sup.2; furthermore, adjusting the mechanical index of the ultrasound probe, and observing the change of grayscale in the ultrasound image. Since there is a weak signal in the ultrasonic image before activation of the droplets, however, after activation there is a strong signal in the ultrasonic image due to the phase change of the droplets into microbubbles. Accordingly, the activation of the droplets may be reflected by the change of the grayscale in the ultrasonic image. In the region of interest, we set the mechanical index corresponding to the ultrasound that may induce 10% nanodroplet activation as the activation threshold of the droplets with optical assistance.

[0127] (5) Determination of Ultrasound Activation Effect

[0128] Take the ultrasound contrast agent I1 prepared in Example 1 as an example for testing. Particularly, taking a Japanese long-eared white rabbit as the experimental object, a peripheral vein channel is established through the marginal ear vein in the left ear of the rabbit, and a three-way tube is connected at the end of the catheter, and one of the channels is used for injecting the drug-loaded ultrasound contrast agent prepared by the present application, and one channel for injecting normal saline solution. The Japanese white rabbit is anesthetized with 3% (40 mg/kg) sodium pentobarbital. After the rabbit is fully anesthetized, the right waist is depilated to facilitate renal imaging. The concentration of the drug-loaded ultrasound contrast agent I1 prepared in Example 1 is adjusted to 1×10.sup.11/ml, and then bolus injection is performed through the marginal ear vein at a dose of 0.1 ml/kg, followed by flushing the pipeline with 1 ml of normal saline solution. The B mode of the Vearsonics ultrasound imaging system is used for observation, the center frequency of the ultrasound probe is 7.8 MHz, the mechanical index (MI) of the imaging ultrasound pulse before activation is 0.4, and the mechanical index of the ultrasound pulse during the activation is 1.5 (the safety threshold value of the human body is 1.9). The observed real-time ultrasound images before and after the ultrasound activation are shown in (a) and (b) of FIG. 2, respectively. It can be seen from FIG. 2 that, before activation there is dark signal in the ultrasound image since the nanodroplets are not vaporized, however, after activation there is bright signal in the ultrasound image since the nanodroplets are vaporized. Therefore, nanodroplets are capable of in vivo controlled imaging.

TABLE-US-00001 TABLE 1 Ultrasound Ultrasound Number activation activation average Half- threshold threshold particle Polydispersity life without optical with optical size (nm) coefficient (h) assistance assistance I1 270 0.14 6 1.4 0.8 I2 250 0.23 5.5 1.3 0.8 I3 320 0.20 6 1.4 0.8 I4 330 0.24 5 1.4 0.9 I5 350 0.26 5.5 1.3 0.8 I6 260 0.14 6 1.4 1.0 I7 220 0.30 4 1.7 1.0 I8 220 0.23 5 1.4 0.8 I9 260 0.14 6 1.4 1.4 I10 270 0.14 6 1.4 1.2 I11 380 0.30 4 1.3 0.7 I12 270 0.14 6 1.4 0.9 D1 420 0.35 3 1.8 1.8 D2 520 0.70 4 2.3 2.1

[0129] It can be seen from Table 1, compared with the prior art, the nanodroplets of contrast agent obtained from the contrast agent composition according to the present application have smaller particle size and higher homogeneity (lower polydispersity coefficient), higher stability (longer half-life), and lower threshold of ultrasound activation under ultrasound conditions. In addition, the contrast agent obtained by the contrast agent composition according to the present application also has optical control properties, and the threshold of ultrasound activation may be reduced to below 1.4 with optical assistance.

[0130] The preferred embodiments of the present application have been described above in detail; however, the present application is not limited thereto. Within the scope of the technical concept of the present application, a variety of simple modifications may be made to the technical solutions of the present application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present application, and they all belong to the protection scope of the present application.