WO2023130948A1 - Preparation method for biosynthetic targeted nano ultrasound contrast agent, and application thereof - Google Patents

Abstract

Provided are a preparation method for a biosynthetic targeted nano ultrasound contrast agent, and an application thereof. The preparation method comprises the following steps: preparing a nano biological balloon and a fluorescent probe modified target object, and connecting the nano biological balloon to the fluorescent probe modified target object to obtain the biosynthetic targeted nano ultrasound contrast agent. The biosynthetic targeted nano ultrasound contrast agent is prepared on the basis of a nano biological balloon generated by a microorganism; the nano biological balloon has a size of 100-300 nm, is completely composed of a protein, and is uniform in particle size and good in stability. The biosynthetic targeted nano ultrasound contrast agent obtained by means of the preparation method can identify a target point specifically binding to a target object on the surface of a cell, has the potential of penetrating a blood vessel to enter a tissue for imaging, and is excellent in ultrasonic imaging performance.

Description

Preparation method and application of a biosynthetic targeted nano-ultrasound contrast agent technical field

The invention belongs to the technical field of ultrasonic molecular imaging, and in particular relates to a preparation method and application of a biosynthesis targeting nano-ultrasonic contrast agent.

Background technique

Ultrasound contrast agent is a specific molecule that displays images at the tissue level, cell level, and subcellular level through imaging means, reflecting changes at the molecular level in the living state. Ultrasound molecular imaging technology is a qualitative and Quantitative Research Techniques. Ultrasound molecular imaging has unique advantages, including non-invasive and radiation-free, good safety, strong tissue penetration, high spatial and temporal resolution, and real-time, dynamic, and repeated observation of target tissues.

Ultrasonic molecular imaging has achieved good imaging results in disease models such as thrombosis, atherosclerosis, and microvascular inflammation, and has extremely high application value in early diagnosis of diseases. In 2017, the team of Professor K. Willmann in the United States applied ultrasonic molecular imaging technology to clinical practice for the first time. The accuracy rate of ultrasonic molecular imaging in clinical trials of breast cancer was as high as 77%, and the accuracy rate in clinical trials of ovarian cancer was as high as 93%. Ultrasound molecular imaging has been demonstrated in a large number of preclinical studies and in a large number of disease models. The introduction of ultrasound molecular imaging into tumor imaging plays an important role in the early detection stages of cancer.

technical problem

The ultrasound contrast agent currently used clinically is a synthetic ultrasound contrast agent, which consists of lipid, polymer or protein shell-wrapped gas microbubbles, with a diameter of 1-8 μm and a relatively large volume. The inability to penetrate the vessel wall to image non-vascular sites limits its use in ultrasonographic molecular imaging. Nano-scale ultrasound contrast agents that have been studied more at this stage mainly include acoustic liposomes, fluorocarbon nano-droplets, and nanobubbles, among which acoustic liposomes are mainly prepared by freeze-drying liposomes, and the gas composition depends on Obtained by means of atmospheric pressure infiltration. However, acoustic liposomes and nanobubbles have different internal gas contents and are easy to leak, which easily leads to poor ultrasound imaging performance; fluorocarbon nanodroplets have better ultrasound imaging performance, but it requires special external stimuli to improve The temperature of the nanodroplet. More importantly, the above-mentioned nano-scale ultrasound contrast agents are all derived from chemical synthesis, and they all have many problems such as uneven particle size, poor stability, large toxic and side effects, and unfriendly environment.

Therefore, the development of a biosynthesized nano-ultrasound contrast agent with excellent ultrasound imaging performance, good stability and targeting effect plays an important role in the field of ultrasound molecular imaging technology.

technical solution

In view of the deficiencies in the prior art, the purpose of the present invention is to provide a preparation method and application of biosynthetic targeted nano-ultrasound contrast agent. The biosynthetic targeted nano-ultrasound contrast agent prepared by the preparation method can recognize the target specifically bound to the target on the cell surface, has the potential to penetrate blood vessels and enter tissue for imaging, and has excellent ultrasonic imaging performance. The biosynthetic targeted nano-ultrasound contrast agent is prepared based on nano-biological airbags produced by microorganisms. The size of the nano-biological airbags is 100-300 nm, and it is completely composed of protein. The particle size of the nano-biological airbags is uniform and stable. Good sex.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a method for preparing a biosynthetic targeted nano-ultrasound contrast agent, the method for preparing a biosynthesized targeted nano-ultrasound contrast agent comprises the following steps:

preparing a nano-biological airbag and a fluorescent probe-modified target, and connecting the nano-biological airbag and the fluorescent probe-modified target to obtain the biosynthetic targeted nano-ultrasound contrast agent;

The nanobiological airbag is produced by natural vesicle-containing microorganisms and/or genetically engineered vesicle-containing microorganisms.

In the present invention, the biosynthetic targeted nano-ultrasound contrast agent is prepared based on nano-biological airbags produced by microorganisms, and the nano-biological airbags are a kind of gas vesicles. GV is an organelle composed of proteins. The shape of GV is a cylindrical or spindle shape with two pointed ends and a round middle. The width is between 45-250 nm, the length can reach 100-300 nm, and the thickness of the capsule wall is 2 nm. In the present invention, the nanobiological airbag is applied to ultrasonic imaging, and the nanobiological airbag is coupled with a targeting substance, so that the nanobiological airbag can recognize the target specifically combined with the targeting substance on the cell surface. The present invention also utilizes the small molecule fluorescent probe to mark the target, occupies the redundant amino groups on the target, and reduces the self-linkage of the target when the nano-biological airbag is coupled to the target.

Preferably, the fluorescent probes include any one or a combination of at least two of coumarin-based probes, fluorescein-based probes, rhodamine-based probes, BODIPY-based probes or Cyanine-based probes.

Preferably, the rhodamine-based probe includes AF488.

Preferably, the preparation method of the nano-bio-airbag comprises: isolating the nano-bio-airbag from natural vesicle-containing microorganisms or isolating the nano-bio-airbag from genetically engineered vesicle-containing microorganisms.

In the present invention, the nano-biological airbags can be isolated from natural vesicle-containing microorganisms, and can also be isolated from genetically engineered vesicular microorganisms. Both nano-biological airbags are completely composed of proteins, and their particle diameters are uniform. In the range of 100-300 nm, such as 100 nm, 200 nm or 300 nm, etc., have good stability and have the potential to penetrate blood vessels and enter tissues for imaging. In addition, the genetically engineered vesicle-containing microorganisms constructed by genetic engineering are convenient to cultivate, easy to obtain, and can obtain a large number of nanobiological air sacs.

Preferably, the natural vesicle-containing microorganisms include any one or a combination of at least two of halophilic archaea, algae or Bacillus megaterium.

Preferably, the genetically engineered vesicle-containing microorganism contains at least one copy of a plasmid carrying a bio-air sac gene cluster.

Preferably, the chassis cells of the genetically engineered vesicle-containing microorganism include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida.

Preferably, said isolating nanobiological air sacs from natural vesicle-containing microorganisms includes:

The lysate is mixed with the natural vesicle-containing microorganism to lyse, and the lysed solution is centrifuged to obtain the nano-biological airbag.

Preferably, the pH of the lysate is 7.3-7.7, such as 7.3, 7.4, 7.5, 7.6 or 7.7.

Preferably, the lysate includes Tris-HCl, MgCl ₂ and CaCl ₂ .

Preferably, the concentration of Tris-HCl in the lysate is 8-12 mM, such as 8 mM, 9 mM, 10 mM, 11 mM or 12 mM etc.

Preferably, the concentration of MgCl ₂ in the lysate is 1.5-2.5 mM, such as 1.5 mM, 1.8 mM, 2 mM, 2.2 mM, 2.4 mM or 2.5 mM.

Preferably, the concentration of CaCl ₂ in the lysate is 1.5-2.5 mM, such as 1.5 mM, 1.8 mM, 2 mM, 2.2 mM, 2.4 mM or 2.5 mM.

Preferably, the volume ratio of the lysate mixed with the natural vesicle-containing microorganism is (0.8-3):1, for example, it can be 0.8:1, 1:1, 2:1 or 3:1, etc.

Preferably, the centrifugal force of the centrifuge is 200-400 g, such as 200 g, 250 g g, 300 g, 350 g or 400 g, etc., the centrifugation time is 3-5 h, such as 3 h, 3.5 h, 4 h, 4.5 h or 5 h, etc.

In the present invention, the buoyancy of the nanobiological airbags produced by natural bubble-containing microorganisms is greater than the buoyancy of the cell thallus, the particle diameter of the nanobiological airbags is 220 ± 20 nm, and the nanobiological airbags are easy to float on the liquid surface. After low-speed centrifugation at -400 g and 4°C, other cell thalli and organelles sink to the lower layer, and the nanobiological airbags float on the liquid surface, and the nanobiological airbags can be obtained by removing the lower layer of liquid. The remaining unlysed natural vesicle-containing microorganisms were sucked out with a syringe, and the lysate was added again for treatment until all the microorganisms were completely lysed, and the obtained nanobiological airbags were transferred to a new centrifuge tube and stored at 4°C for later use.

Preferably, isolating nanobiological air sacs from genetically engineered vesicle-containing microorganisms comprises:

Transforming the plasmid carrying the bio-airbag gene cluster into the disc cells to obtain genetically engineered vesicle-containing microorganisms, culturing and inducing the genetically engineered vesicle-containing microorganisms, mixing the lysate with the induced genetically engineered vesicle-containing microorganisms, adding lysate Enzyme incubation, followed by addition of DNase for incubation, and centrifugation of the lysed solution to obtain the nanobiological airbag.

In the present invention, all microorganisms are completely lysed through the above steps, and the released nano-biological airbags are transferred to a new centrifuge tube and stored at 4°C for future use.

Preferably, the chassis cells include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida.

Preferably, the mixed volume ratio of the lysate and the induced genetically engineered vesicle-containing microorganism is (5-10):(7-8), for example, it can be 5:7, 6:7, 7:7, 8: 7, 9:7, 10:7, 5:8, 6:8, 7:8, 9:8 or 10:8, etc.

Preferably, the final concentration of the lysozyme is 20-1000 μg/mL, for example, can be 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 500 μg/mL or 1000 μg/mL, etc.

Preferably, the temperature for adding lysozyme to incubate is 25-37°C, for example, it can be 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C or 37°C, etc., the adding lysozyme The incubation time is 1-3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h or 3 h, etc.

Preferably, the final concentration of the DNase is 20-1000 μg/mL, such as 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 500 μg/mL or 1000 μg/mL wait.

Preferably, the temperature for adding DNase and incubating is 25-37°C, such as 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C or 37°C, etc., the incubation time It is 1-12 h, for example, it can be 1 h, 2 h, 4 h, 6 h, 8 h, 10 h or 12 h, etc.

Preferably, the centrifugal force of the centrifuge is 300-400 g, such as 300 g, 320 g g, 340 g, 360 g, 380 g or 400 g, etc., the centrifugation time is longer than 1 h, for example, it can be 2 h, 2.5 h, 3 h or 3.5 h, etc.

In the present invention, the particle diameter of the nano-biological airbags separated from the genetically engineered vesicle-containing microorganisms is 110 ± 20 nm. The nanobio-air sacs separate.

Preferably, the preparation method of the fluorescent probe modified target comprises:

The fluorescent probe is esterified with EDC and NHS, the esterified fluorescent probe, target and medium are mixed for reaction, and the fluorescent probe not combined with the target is removed to obtain the fluorescent probe-modified target.

In the present invention, the target itself has an amino group and a carboxyl group. In order to avoid the occurrence of self-linkage between the target and the reduction of labeling efficiency during the esterification of the target, the present invention uses a small molecule fluorescent probe to occupy the amino position of the target. The dots avoid self-ligation, and the amount of target labeling can be calculated from the fluorescence of the fluorescent probe.

Preferably, the target comprises IgG.

The targeting substance in the present invention can specifically bind to the target on the cell surface, and through the specific combination of the targeting substance and the target, the biosynthetic targeted nano-ultrasound contrast agent can reach the corresponding tissue and cell surface The target-specific binding realizes targeted imaging.

Preferably, the mass ratio of the esterified fluorescent probe, target and medium mixed (0.5-1):(1-2):(2000-4000), for example, can be 0.5:1:2000, 0.5: 2:2000, 0.5:1:4000, 0.5:2:4000, 1:1:2000, 1:2:2000 or 1:2:4000 etc.

Preferably, the medium includes any one or a combination of at least two of deionized water, sodium borate solution, sodium bicarbonate solution or PBS buffer.

In the present invention, the concentration of Na ₃ BO ₃ in the sodium borate solution is 50-60 mM, the pH is 8.0-8.5, and the concentration of Na ₃ BO ₃ can be 50 mM, 53 mM, 55 mM, 58 mM or 60 mM, for example. mM, etc., the pH can be, for example, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, etc.

In the present invention, the concentration of _NaHCO in the sodium bicarbonate solution is 0.05-0.2 M, the pH is 8.3-9.0, and the concentration of _NaHCO can be, for example, 0.05 M, 0.08 M, 0.1 M, 0.13 M, 0.15 M, 0.18 M or 0.2 M, etc., the pH can be, for example, 8.3, 8.5, 8.7, 8.9 or 9.0, etc.

In the present invention, the concentration of Na ₃ PO ₄ in the PBS buffer solution is 0.1-0.2 M, the concentration of NaCl is 0.15-0.25 M, and the pH is 7.2-7.5. The concentration of Na ₃ PO ₄ can be, for example, 0.1 M, 0.13 M, 0.15 M, 0.18 M or 0.2 M, etc., the concentration of NaCl, for example, can be 0.15 M, 0.18 M, 0.2 M, 0.23 M, or 0.25 M, etc., and the pH can be, for example, 7.2, 7.3, 7.4 or 7.5, etc.

Preferably, the reaction time is 1-5 h, such as 1 h, 2 h, 3 h, 4 h or 5 h, etc.

Preferably, the removal of the fluorescent probes not bound to the targeting substance is performed through a dialysis bag.

Preferably, the molecular cut-off of the dialysis bag: the molecular weight of the fluorescent probe<the molecular cut-off<the molecular weight of the target object.

Preferably, the steps of linking the nano-biological airbag and the fluorescent probe-modified target include:

(a) mixing the fluorescent probe-modified targeting substance, protective agent, EDC and MES buffer, and stirring to react to obtain a reaction solution;

(b) Mixing and reacting the nano-biological airbag solution with the reaction solution in step (a), and centrifuging to obtain the biosynthesis targeting nano-ultrasound contrast agent.

Preferably, in step (a), the final concentration of the fluorescent probe-modified targeting substance in the mixed solution is 0.05-3 mg/mL.

Preferably, in step (a), the final concentration of the protective agent in the mixed solution is 3-6 mM, such as 3 mM, 4 mM, 5 mM or 6 mM etc.

Preferably, in step (a), the protective agent includes NHS or thio-NHS.

Preferably, in step (a), the final concentration of EDC in the mixed solution is 2-3 mM, for example, can be 2 mM, 2.2 mM, 2.4 mM, 2.6 mM, 2.8 mM or 3 mM, etc.

Preferably, in step (a), the pH of the MES buffer is 5.5-6.5, for example, it may be 5.5, 5.7, 5.9, 6.0, 6.1, 6.3 or 6.5.

Preferably, in step (a), the protective agent includes NHS or thio-NHS.

Preferably, in step (a), the rotational speed of the stirring reaction is 100-400 rpm, such as 100 rpm, 200 rpm rpm, 300 rpm or 400 rpm, etc., the stirring reaction time is 15-30 min, such as 15 min, 18 min, 20 min, 22 min, 25 min, 28 min or 30 min, etc.

Preferably, in step (b), the nano-bio-air vesicle solution is a buffer containing nano-bio-air vesicles.

Preferably, in step (b), the concentration of the nano-bio-airbag solution has an OD ₅₀₀ of 10-120, such as 10, 20, 40, 60, 80, 100 or 120, etc., and the pH of the nano-bio-airbag solution is 7.5-8.5, for example, can be 7.5, 7.7, 7.9, 8.0, 8.1, 8.3 or 8.5, etc.

Preferably, in step (b), the buffer is an amino-free buffer.

Preferably, in step (b), the volume ratio of the nano-bio-airbag solution mixed with the reaction solution in step (a) is (1.5-4):(0.5-10), for example, it can be 1.5:0.5, 1.5: 2. 1.5:4, 1.5:6, 1.5:8, 1.5:10, 3:10, 3.5:10, 4:0.5, 4:1.5 or 4:3 etc.

Preferably, in step (b), the mixing reaction time is 2-3 h, for example, it can be 2 h, 2.5 h h or 3 h etc.

Preferably, in step (b), the centrifugal force of the centrifuge is 200-300 g, such as 200 g, 220 g, 240 g, 260 g, 280 g or 300 g, etc., the centrifugation time is 2-4 h, such as 2 h, 2.5 h, 3 h, 3.5 h or 4 h.

Preferably, in step (b), the method for calculating the amount of target labeling of the biosynthesized targeted nano-ultrasound contrast agent includes:

Labeling amount of target substance = (A _{bubble max} × C _{target substance} ) / (A _{target substance max} × dilution factor × C _bubble )

in,

A _{bubble max} : crush the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

C _{target object} : the molar concentration of the target object;

A _{target object max} : Dilute the target object modified by the small molecule fluorescent probe, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

_Bubble C: molar concentration of biosynthesized targeted nano-ultrasound contrast agents.

In the present invention, after the fluorescent probe-modified target is coupled to the nano-biological airbag, the connected biosynthetic targeted nano-ultrasonic contrast agent is separated from the solution by low-speed centrifugation, and the centrifugal force is The centrifugation time is 2-4 h under the condition of 200-300 g. The biological air bag connected with the target object is suspended in the upper layer of the sample tube, and the unconnected target object is in the solution. Use a syringe to draw the clear solution at the bottom of the sample tube, and then Resuspend in PBS, centrifuge, and repeat this step 2-3 times to obtain biosynthetic targeted nano-ultrasound contrast agent.

As a preferred technical solution of the present invention, the preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:

(1) Preparation of nano-biological airbags:

Isolation of nanobiological air sacs from natural vesicular microorganisms or isolation of nanobiological air sacs from genetically engineered vesicular microorganisms:

Isolation of nanobiological air sacs from natural vesicular microorganisms:

The lysate with a pH of 7.3-7.7 and the natural vesicle-containing microorganism are mixed and lysed at a volume ratio of (0.8-3): 1, and the lysate includes 8-12 mM Tris-HCl, 1.5-2.5 mM MgCl ₂ and 1.5-2.5 mM CaCl ₂ , centrifuge the lysed solution for more than 3-5 h under the condition of a centrifugal force of 200-400 g to obtain the nano-biological airbag.

Isolation of nanobiological air sacs from genetically engineered vesicle-containing microorganisms:

Transforming the plasmid carrying the bio-airbag gene cluster into the disc cells to obtain genetically engineered vesicle-containing microorganisms, cultivating and inducing the genetically engineered vesicle-containing microorganisms, the volume ratio of the lysate to the induced genetically engineered vesicle-containing microorganisms is (5-10): (7-8) mix, add lysozyme, the final concentration of the lysozyme is 20-1000 μg/mL, incubate at 25-37 ℃ for 1-3 h, then add DNase, the The final concentration of DNase is 20-1000 μg/mL, incubate at 25-37°C for 1-12 h, the lysed solution was subjected to a centrifugal force of 300-400 g under the condition of centrifugation for more than 1 h to obtain the nanobiological airbag.

(2) Preparation of fluorescent probe-modified targets:

Use EDC and NHS to esterify the fluorescent probe, and mix the esterified fluorescent probe, target and medium according to the mass ratio (0.5-1):(1-2):(2000-4000) for 1-5 h, Fluorescent probes not bound to the targeting substance are removed through the dialysis bag to obtain the fluorescent probe-modified targeting substance.

(3) Connecting the nano-biological airbags to the target modified by the fluorescent probe:

(a) Mix the fluorescent probe-modified targeting substance, protective agent, EDC, and MES buffer with a pH of 5.5-6.5, and the final concentration of the fluorescent probe-modified targeting substance in the solution after mixing is 0.05 -3 mg/mL, the final concentration of the protective agent is 3-6 mM, the final concentration of EDC is 2-3 mM, the rotation speed is 100-400 rpm and the reaction is stirred for 15-30 min to obtain the reaction solution;

(b) Mixing and reacting the nano-bio-airbag solution with the reaction solution in step (a) in a volume ratio of (1.5-4):(0.5-3) for 2-3 h, and the pH of the nano-bio-airbag solution is 7.5- 8.5. With an OD ₅₀₀ of 10-120, centrifuge for 2-4 h under the condition of a centrifugal force of 200-300 g to obtain the biosynthetic targeted nano-ultrasound contrast agent.

In the present invention, the target marker amount of the biosynthetic targeted nano-ultrasound contrast agent can be calculated by the following formula:

Labeling amount of target substance = (A _{bubble max} × C _{target substance} ) / (A _{target substance max} × dilution factor × C _bubble )

in,

A _{bubble max} : crush the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

C _{target object} : the molar concentration of the target object;

A _{target object max} : Dilute the target object modified by the small molecule fluorescent probe, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

_Bubble C: molar concentration of biosynthesized targeted nano-ultrasound contrast agents.

In the second aspect, the present invention provides an application of the preparation method of biosynthetic targeted nano-ultrasound contrast agent in the first aspect in the field of ultrasonic molecular imaging.

The numerical ranges described in the present invention not only include the above-mentioned point values, but also include any point values between the above-mentioned numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention will not exhaustively list the above-mentioned point values. Specific point values covered by the stated ranges.

It should be noted that the scientific and technical terms and their abbreviations used in the present invention have the meanings commonly understood by those skilled in the art. The following lists some terms and abbreviations used in the present invention:

BODIPY probes: boron fluoride dipyrrole probes.

Cyanine probes: Cyanine probes.

EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

NHS: N-Hydroxy succinimide, N-Hydroxy succinimide.

MES: Morpholinoethanesulfonic acid, 2-Morpholinoethanesulfonic Acid.

DSPC: Distearoylphosphatidylcholine.

PDI: Polymer dispersion index, Polymer dispersion index.

Compared with the prior art, the present invention has the following beneficial effects:

Beneficial effect

Compared with the prior art, the present invention has the following beneficial effects:

(1) The size of the biosynthetic targeted nano-ultrasound contrast agent of the present invention is 100-300 nm, and the size of the nano-bio-airbag is completely composed of protein. The particle size of the nano-bio-airbag is uniform and stable. Synthetic targeted nano-ultrasound contrast agents can recognize the target that specifically binds to the target on the cell surface, have the potential to penetrate blood vessels into tissue imaging, and have excellent ultrasonic imaging performance. The biosynthetic targeted nano-ultrasound contrast agent prepared by the present invention has no significant difference in the stability test and imaging performance test between the unmodified contrast agent, and also has a targeting effect, and can specifically bind to target cells and target tissues , to achieve targeted imaging.

(2) In the preparation method of the biosynthetic targeted nano-ultrasound contrast agent of the present invention, the fluorescent probe is used to occupy the amino site of the target to avoid the self-connection of the target, and the target can be calculated by the fluorescence of the fluorescent probe amount of markers.

Description of drawings

Fig. 1 is a comparison chart of the fluorescence intensity of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 1.

Fig. 2 is a comparison chart of in vitro ultrasound imaging of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 2.

FIG. 3 is a statistical diagram of the signal intensity of the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent in Test Example 2.

Fig. 4 is a comparison chart of the adhesion between biosynthetic targeting nano-ultrasound contrast agent and biosynthetic non-targeting nano-ultrasound contrast agent and human breast cancer MCF-7 cell line with high expression of E-cadherin in Test Example 3.

5 is a particle size distribution diagram of the biosynthesized targeted nano-ultrasound contrast agent in Test Example 4 and the phospholipid microbubble contrast agent obtained in Comparative Example 3.

FIG. 6 is a statistical graph of the PDI dispersion index of the biosynthesized targeted nano-ultrasound contrast agent in Test Example 5 and the phospholipid microbubble contrast agent obtained in Comparative Example 3. FIG.

Embodiments of the present invention

The technical solutions of the present invention will be further described below through specific embodiments. It should be clear to those skilled in the art that the examples are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

In the following examples, the sources of materials, reagents and instruments are as follows:

name	factory	Grade
Haloarchaea	ATCC	Halobacterium sp. NRC-1
lysate	Genlantis	SoluLyse
Lysozyme	Thermo Scientific	Lysozyme
DNase	McLean	DNAse
AF488	polyfluorescein	AF488
IgG	SAB	E-cadherin antibody
NHS	Sigma-Aldrich	N-Hydroxysuccinimide
10 kDa Dialysis Bag	source leaves	10 kDa
MES	McLean	Morpholineethanesulfonic acid
EDC	Merida	Methyl dihydrojasmonate
PBS	McLean	PBS buffer
DSPC	source leaves	DSPC
DSPE-PEG2000	source leaves	DSPE-PEG2000
Ultrasound Imaging System	Mindray	Resona 7
confocal microscope	Nikon	A1 Confocal Laser Microscope System

Example 1

This embodiment provides a method for preparing a biosynthesized targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesized targeted nano-ultrasound contrast agent come from natural bubble-containing microorganisms. The preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:

(1) Preparation of nano-biological airbags:

Isolation of nanobiological air sacs from the halophilic archaea Halobacterium sp. NRC-1 (NRC-1 for short):

The lysate with a pH of 7.5 and the NRC-1 are mixed and lysed at a volume ratio of 2:1, and the lysed solution is centrifuged for 4 h under the condition of a centrifugal force of 300 g to obtain the nanobiological airbag; the lysed The solution included 10 mM Tris-HCl, 2.5 mM MgCl ₂ and 2 mM CaCl ₂ .

(2) Preparation of targeting antibodies modified by fluorescent probes:

AF488 was esterified with EDC and NHS to obtain AF488-NHS ester. AF488-NHS ester, IgG and deionized water were mixed and reacted for 3 h at a mass ratio of 0.5:1:2000. AF488 bound by the targeting antibody was obtained to obtain the targeting antibody modified by the fluorescent probe, and the targeting antibody modified by the fluorescent probe was named AF488-IgG-1.

(3) Linking nanobiological airbags and fluorescent probe-modified targeting antibodies:

(a) Mix the AF488-IgG-1, EDC, NHS and MES buffer with a pH of 6.0, the final concentration of the AF488-IgG-1 in the mixed solution is 2 mg/mL, and the final concentration of EDC is 2 mM, the final concentration of NHS is 5 mM, the MES buffer includes 0.1 M MES and 0.5 M NaCl, 300 rpm stirring reaction for 25 min, to obtain a reaction solution;

(b) Mix the nano-bio-airbag solution with the reaction solution in step (a) at a volume ratio of 1.5:0.5 and react for 2.5 h. The pH of the nano-bio-airbag solution is 8.0, the OD ₅₀₀ is 60, and the centrifugal force is 250 g centrifuged under the same conditions for 3 h to obtain the biosynthesized targeted nano-ultrasound contrast agent.

In this embodiment, the target marker amount of the biosynthesized targeted nano-ultrasound contrast agent can be calculated by the following formula:

Labeling amount of target substance = (A _{bubble max} × C _{target substance} ) / (A _{target substance max} × dilution factor × C _bubble )

in,

A _{bubble max} : After crushing the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, measure the absorption value of the 1 cm optical path at the absorption peak wavelength of the small molecule fluorescent probe;

C _{target object} : the molar concentration of the target object;

A _{target object max} : the target object modified by the small molecule fluorescent probe measures the absorption value of the 1 cm optical path at the absorption peak wavelength of the small molecule fluorescent probe;

_Bubble C: molar concentration of biosynthesized targeted nano-ultrasound contrast agents.

After measurement, the A _{bubble max} was measured as 10000; the C _{target object} was 1.3×10 ^-8 M, and the A _{target object max} was 8000; the C _bubble was 3.5×10 ^-13 M; The absorption value of the target substance modified by the small molecule fluorescent probe was measured, and the dilution factor was 1.

Labeling amount of target substance=(10000×1.3×10 ^-8 )/(8000×1×3.5×10 ^-13 )=4642.8

The calculation results showed that 4642.8 targets were coupled to each bioairbag on average.

Example 2

This embodiment provides a method for preparing a biosynthesis-targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesis-targeted nano-ultrasound contrast agent come from genetically engineered microbes containing bubbles. The preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:

(1) Preparation of nano-biological airbags:

Isolation of nanobiological air sacs from genetically engineered vesicle-containing microorganisms:

Transform the plasmid carrying the bio-airbag gene cluster into Escherichia coli (BL21) to obtain genetically engineered vesicle-containing microorganisms, cultivate and induce the genetically engineered vesicle-containing microorganisms, and mix the lysate with the induced genetically engineered vesicle-containing microorganisms according to Mix at a volume ratio of 5:7, add lysozyme, the final concentration of lysozyme is 20 μg/mL, incubate at 30°C for 2 h, then add DNase, the final concentration of DNase is 10 μg/mL, Incubate at 30° C. for 6 h, and centrifuge the lysed solution for 2 h at a centrifugal force of 350 g to obtain the nanobiological airbag.

(2) Preparation of targeting antibodies modified by fluorescent probes:

AF488 was esterified with EDC and NHS to obtain AF488-NHS ester. AF488-NHS ester, IgG, and deionized water were mixed and reacted for 5 h at a mass ratio of 1:2:4000, and the non-conjugated protein was removed by a dialysis bag with a molecular cutoff of 10 kDa. AF488 bound by the targeting antibody was obtained to obtain the targeting antibody modified by the fluorescent probe, and the targeting antibody modified by the fluorescent probe was named AF488-IgG-2.

(3) Linking nanobiological airbags and fluorescent probe-modified targeting antibodies:

(a) Mix the AF488-IgG-2, EDC, NHS and MES buffer with a pH of 6.5, the final concentration of AF488-IgG-2 in the solution after mixing is 2 mg/mL, and the final concentration of EDC is 3 mM, the final concentration of NHS is 5 mM, the MES buffer includes 0.1 M MES and 0.5 M NaCl, 400 rpm stirring reaction for 15 min, to obtain a reaction solution;

(b) Mix the nano-bio-airbag solution with _the reaction solution in step (a) at a volume ratio of 4:3 and react for 3 h. centrifuged under the same conditions for 2 h to obtain the biosynthesized targeted nano-ultrasound contrast agent.

Comparative example 1

This comparative example provides a method for preparing a biosynthesized non-targeting nano-ultrasound contrast agent, wherein the nano-biological balloon in the biosynthesized nano-ultrasound contrast agent comes from the halophilic archaea NRC-1. The preparation method of the biosynthetic targeted nano-ultrasound contrast agent comprises the following steps:

The lysis solution with a pH of 7.7 and the NRC-1 were mixed and lysed at a volume ratio of 3:1, and the lysis solution included 12 mM Tris-HCl, 2 mM MgCl ₂ and 2.5 mM CaCl ₂ , and the lysed solution was The biosynthetic nano-ultrasonic contrast agent was obtained by centrifuging for 5 h under the condition of a centrifugal force of 200 g.

Comparative example 2

This comparative example provides a method for preparing a biosynthesized non-targeted nano-ultrasound contrast agent, wherein the nano-biological airbags in the biosynthesized targeted nano-ultrasound contrast agent come from genetically engineered vesicle-containing microorganisms. The preparation method of the biosynthetic nano-ultrasound contrast agent comprises the following steps:

Cultivate the genetically engineered vesicle-containing microorganisms described in Example 2, cultivate and induce the genetically engineered vesicle-containing microorganisms, mix the lysate with the induced genetically engineered vesicle-containing microorganisms at a volume ratio of 10:8, add lysozyme, and The final concentration of lysozyme was 25 μg/mL, incubated at 37°C for 3 h, then added DNase, the final concentration of DNase was 15 μg/mL, incubated at 25°C for 12 h, and the lysed solution was The biosynthetic nano-ultrasound contrast agent was obtained by centrifuging for 2 h at a centrifugal force of 400 g.

Comparative example 3

This comparative example provides a method for preparing an ultrasound contrast agent, the ultrasound contrast agent is a phospholipid microbubble contrast agent, and the preparation method of the phospholipid microbubble contrast agent is as follows:

(1) Measure 5.2 mg DSPC and 4.3 mg DSPE-PEG ₂₀₀₀ , add 1 mL chloroform to dissolve, and transfer the solution to a dry and clean test tube.

(2) Blow dry the test tube with N ₂ at room temperature to obtain a phospholipid film, and vacuum it for 4 h to completely remove residual chloroform.

(3) Add pH7.4 buffer solution into the test tube, the buffer solution includes 10 mM Tris-HCl, glycerol, 1,2-propanediol, the volume ratio of Tris-HCl, glycerol and 1,2-propanediol It is 8:1:1.

(4) Place the test tube in a 65°C water-bath ultrasonic cleaner, and sonicate for 10 min until a transparent phospholipid solution is obtained.

(5) Take clean vials and pack them separately, cover and seal, vacuumize for 15 min, and inject C ₃ F ₈ gas for replacement.

(6) Place the vial on a high-frequency oscillator and vibrate mechanically for 30 s to obtain the phospholipid microbubble contrast agent.

test case 1

The fluorescence intensity of the biosynthesized targeted nano-ultrasound contrast agent obtained in Example 1 and the biosynthesized non-targeted nano-ultrasound contrast agent obtained in Comparative Example 1 was tested.

The test method is as follows:

(1) The prepared biosynthetic targeted nano-ultrasound contrast agent and non-targeted nano-ultrasound contrast agent were crushed with ultrasound to transform from a suspension state into a clear solution.

(2) Dilute the above two solutions according to a certain ratio, and use a microplate reader to measure the fluorescence intensity of the two contrast agents at a wavelength of 488/520 nm.

The comparison results of the fluorescence intensity of the ultrasound contrast agents in Example 1 and Comparative Example 1 are shown in Figure 1. It can be seen from Figure 1 that after the biosynthesis targeting nano-ultrasound contrast agent is connected with a fluorescent small molecule, it has a very strong Fluorescent signal, indicating that the targeting antibody is successfully linked to the nanobiological airbag. The comparison results of the fluorescence intensities of the ultrasound contrast agents in Example 2 and Comparative Example 2 are similar to the comparison results of the fluorescence intensities of the ultrasound contrast agents in Example 1 and Comparative Example 1.

test case 2

The biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the biosynthetic non-targeted nano-ultrasound contrast agent obtained in Comparative Example 1 were diluted with PBS to OD ₅₀₀ =10, and the two ultrasonic contrast agents were respectively transferred into the prepared 2 % (mass percentage) in the agarose phantom pipeline, the Mindray Resona 7 high-end color Doppler ultrasound diagnostic system was used to detect the imaging effects of different ultrasound contrast agents to obtain ultrasound contrast signals.

The results are shown in Figures 2 and 3. Figure 2 is a comparison of in vitro ultrasound imaging between biosynthesized targeted nano-ultrasound contrast agents and biosynthesized non-targeted nano-ultrasound contrast agents, and Figure 3 is a comparison of biosynthesized targeted nano-ultrasound contrast agents The signal intensity statistical diagram of the biosynthetic non-targeted nano-ultrasound contrast agent, as can be seen from Figure 2 and Figure 3, the biosynthesized targeted nano-ultrasound contrast agent and the biosynthesized non-targeted nano-ultrasound contrast agent ultrasound contrast agent Consistent imaging performance at the same concentration. The biosynthetic targeted nano-ultrasound contrast agent obtained in Example 2 and the biosynthesized non-targeted nano-ultrasound contrast agent obtained in Comparative Example 2 also have similar imaging properties.

Test case 3

The biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the biosynthetic non-targeted nano-ultrasound contrast agent obtained in Comparative Example 1 were incubated with the cultured MCF-7 cells for 15 min, and the cells were washed with PBS, and repeated 3 times. 5 min each time, placed under a confocal microscope (A1+ laser confocal system) for observation.

The results are shown in Figure 4, the biosynthetic targeted nano-ultrasound contrast agent has a large amount of adhesion on the surface of MCF-7 cells, while the biosynthetic non-targeted nano-ultrasound contrast agent is not seen on the surface of MCF-7 cells The obvious adhesion phenomenon shows that the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 has good targeting. The biosynthetic targeted nano-ultrasound contrast agent obtained in Example 2 also has similar targeting properties and can also adhere to the surface of MCF-7 cells.

Test case 4

The particle diameters of the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the phospholipid microbubble contrast agent obtained in Comparative Example 3 were measured respectively.

The measurement method is as follows:

(1) The prepared biosynthetic targeting nano-ultrasound contrast agent and phospholipid microbubble contrast agent were diluted according to a certain ratio, and the particle size distribution of the two contrast agents was measured with a Malvern particle size analyzer (Zetasizer Nano).

The results are shown in Figure 5. As can be seen from Figure 5, the average particle size of the biosynthetic targeted nano-ultrasound contrast agent in Example 1 is 200 nm, and the average particle diameter of the phospholipid microbubble contrast agent described in Comparative Example 3 is 200 nm. The particle size is 1 μm. Compared with the phospholipid microbubble contrast agent obtained in Comparative Example 3, the biosynthetic targeted nano-ultrasound contrast agent provided in Example 1 has a smaller particle size and has the potential to penetrate blood vessels and enter tissues for imaging.

Test case 5

The PDI dispersion index of the biosynthetic targeted nano-ultrasound contrast agent obtained in Example 1 and the phospholipid microbubble contrast agent obtained in Comparative Example 3 were respectively measured.

The measurement method is as follows:

(1) The prepared biosynthetic targeting nano-ultrasound contrast agent and phospholipid microbubble contrast agent were diluted according to a certain ratio, and the PDI dispersion index of the two contrast agents was measured with a Malvern particle size analyzer (Zetasizer Nano).

The results are shown in Figure 6, as can be seen from Figure 6, the PDI dispersion index of the biosynthetic targeted nano-ultrasound contrast agent in Example 1 is 0.065, and the dispersion index of the phospholipid microbubble contrast agent described in Comparative Example 3 is 0.485. The results show that the biosynthetic targeted nano-ultrasound contrast agent provided in Example 1 has a uniform particle size and better imaging performance.

In summary, the biosynthetic targeted nano-ultrasound contrast agent prepared by the preparation method of the biosynthetic targeted nano-ultrasound contrast agent of the present invention can recognize the target that the cell surface specifically binds to the target substance, has good stability, It has the potential of penetrating blood vessels and entering tissues for imaging. The biosynthetic targeted nano-ultrasound contrast agent in the present invention has excellent ultrasonic imaging performance and has important application prospects in the field of ultrasonic molecular imaging technology.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, and those skilled in the art should understand that any person skilled in the art should be aware of any disclosure in the present invention Within the technical scope, easily conceivable changes or substitutions all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. A method for preparing a biosynthetic targeted nano-ultrasound contrast agent, characterized in that the method for preparing a biosynthesized targeted nano-ultrasound contrast agent comprises the following steps:

   preparing a nano-biological airbag and a fluorescent probe-modified target, and connecting the nano-biological airbag and the fluorescent probe-modified target to obtain the biosynthetic targeted nano-ultrasound contrast agent;

   The nanobiological airbag is produced by natural vesicle-containing microorganisms and/or genetically engineered vesicle-containing microorganisms.
2. The preparation method of biosynthetic targeting nano-ultrasound contrast agent according to claim 1, wherein said fluorescent probes include coumarin probes, fluorescein probes, rhodamine probes, BODIPY probes Any one or a combination of at least two of needles or Cyanine probes;

   Preferably, the rhodamine-based probe includes AF488.
3. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to claim 1 or 2, characterized in that, the preparation method of the nano-biological airbag comprises: isolating the nano-biological airbag from natural vesicle-containing microorganisms or from genetic engineering containing Separation of nano-biological air sacs from bubble microorganisms;

   Preferably, the natural vesicle-containing microorganisms include any one or a combination of at least two of halophilic archaea, algae or Bacillus megaterium;

   Preferably, the genetically engineered vesicle-containing microorganism contains at least one copy of a plasmid carrying the bio-air sac gene cluster;

   Preferably, the chassis cells of the genetically engineered vesicle-containing microorganism include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida.
4. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to claim 3, wherein said isolating nano-biological air sacs from natural bubble-containing microorganisms comprises:

   mixing the lysate with the natural vesicle-containing microorganisms to lyse, and centrifuging the lysed solution to obtain the nanobiological airbags;

   Preferably, the pH of the lysate is 7.3-7.7;

   Preferably, the lysate includes Tris-HCl, MgCl ₂ and CaCl ₂ ;

   Preferably, the concentration of Tris-HCl in the lysate is 8-12 mM;

   Preferably, the concentration of _MgCl in the lysate is 1.5-2.5 mM;

   Preferably, the concentration of _CaCl in the lysate is 1.5-2.5 mM;

   Preferably, the volume ratio of the lysate mixed with the natural vesicle-containing microorganism is (0.8-3):1;

   Preferably, the centrifugal force of the centrifuge is 200-400 g, the centrifugation time is 3-5 h.
5. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to claim 3 or 4, wherein isolating nano-biological airbags from genetically engineered vesicle-containing microorganisms comprises:

   Transforming the plasmid carrying the bio-airbag gene cluster into the disc cells to obtain genetically engineered vesicle-containing microorganisms, culturing and inducing the genetically engineered vesicle-containing microorganisms, mixing the lysate with the induced genetically engineered vesicle-containing microorganisms, adding lysate Enzyme incubation, then adding DNase for incubation, centrifuging the lysed solution to obtain the nanobiological airbag;

   Preferably, the chassis cells include any one or a combination of at least two of Escherichia coli, Streptomyces, yeast, cyanobacteria or Pseudomonas putida;

   Preferably, the mixed volume ratio of the lysate and the induced genetic engineering vesicle-containing microorganism is (5-10):(7-8);

   Preferably, the final concentration of the lysozyme is 20-1000 μg/mL;

   Preferably, the temperature for adding lysozyme for incubation is 25-37°C, and the incubation time is 1-3 h;

   Preferably, the final concentration of the DNase is 20-1000 μg/mL;

   Preferably, the temperature for adding DNase for incubation is 25-37°C, and the incubation time is 1-12 h;

   Preferably, the centrifugal force of the centrifuge is 300-400 g, the centrifugation time is greater than 1 h.
6. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to any one of claims 1-5, characterized in that the preparation method of the fluorescent probe-modified targeting substance comprises:

   Esterify the fluorescent probe with EDC and NHS, mix and react the esterified fluorescent probe, target and medium, remove the fluorescent probe that is not bound to the target, and obtain the target modified by the fluorescent probe;

   Preferably, the target includes IgG;

   Preferably, the mixed mass ratio of the esterified fluorescent probe, target and medium is (0.5-1):(1-2):(2000-4000);

   Preferably, the medium includes any one or a combination of at least two of deionized water, sodium borate solution, sodium bicarbonate solution or PBS buffer;

   Preferably, the reaction time is 1-5 h;

   Preferably, the removal of fluorescent probes that are not bound to the target is carried out through a dialysis bag;

   Preferably, the molecular cut-off of the dialysis bag: the molecular weight of the fluorescent probe<the molecular cut-off<the molecular weight of the target object.
7. According to the preparation method of biosynthesis targeting nano-ultrasound contrast agent according to any one of claims 1-6, it is characterized in that the step of connecting the nano-biological airbag and the target object modified by fluorescent probe comprises:

   (a) mixing the fluorescent probe-modified targeting substance, protective agent, EDC and MES buffer, and stirring to react to obtain a reaction solution;

   (b) Mixing and reacting the nano-biological airbag solution with the reaction solution in step (a), and centrifuging to obtain the biosynthesis targeting nano-ultrasound contrast agent.
8. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to claim 7, characterized in that in step (a), the final concentration of the fluorescent probe-modified targeting substance in the solution after mixing is 0.05-3 mg/mL;

   Preferably, in step (a), the final concentration of the protective agent in the mixed solution is 3-6 mM;

   Preferably, in step (a), the protective agent includes NHS or thio-NHS;

   Preferably, in step (a), the final concentration of EDC in the mixed solution is 2-3mM;

   Preferably, in step (a), the pH of the MES buffer is 5.5-6.5;

   Preferably, in step (a), the rotational speed of the stirring reaction is 100-400 rpm, and the time of the stirring reaction is 15-30 min.
9. The preparation method of biosynthetic targeted nano-ultrasound contrast agent according to claim 7 or 8, characterized in that, in step (b), the nano-bio-balloon solution is a buffer containing nano-bio-balloon;

   Preferably, in step (b), the OD ₅₀₀ of the nano-bio-airbag solution is 10-120, and the pH of the nano-bio-airbag solution is 7.5-8.5;

   Preferably, in step (b), the buffer is an amino-free buffer;

   Preferably, in step (b), the volume ratio of the nano-bio-airbag solution mixed with the reaction solution in step (a) is (1.5-4):(0.5-10);

   Preferably, in step (b), the mixing reaction time is 2-3 h;

   Preferably, in step (b), the centrifugal force of the centrifugation is 200-300 g, and the centrifugation time is 2-4 h;

   Preferably, in step (b), the method for calculating the amount of target labeling of the biosynthesized targeted nano-ultrasound contrast agent includes:

   Labeling amount of target substance = (A _{bubble max} × C _{target substance} ) / (A _{target substance max} × dilution factor × C _bubble )

   in,

   A _{bubble max} : crush the biosynthesized targeted nano-ultrasound contrast agent with an ultrasonic cleaning machine, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

   C _{target object} : the molar concentration of the target object;

   A _{target object max} : Dilute the target object modified by the small molecule fluorescent probe, and measure the absorption value at the absorption peak wavelength of the small molecule fluorescent probe;

   _Bubble C: molar concentration of biosynthesized targeted nano-ultrasound contrast agents.
10. The application of the preparation method of the biosynthetic targeted nano-ultrasound contrast agent described in any one of claims 1-9 in the field of ultrasonic molecular imaging.